Journal articles on the topic 'HPLC troubleshooting'

In compared to prior approaches, HPLC provides far higher resolution, more accurate quantitative results, and quicker analysis durations. As a result, HPLC has developed into a vital instrument in many analytical labs. Troubleshooting is a type of problem solving that is frequently used to fix failing processes or goods. Actually, the term "HPLC" is used to describe a variety of separation methods that employ a liquid mobile phase, or eluent. Understanding the fundamental principles underlying the operation of the instrument and the separation is necessary for troubleshooting HPLC equipment and separations. Component faults (pump, degasser, injector, detector, data system, column) and improper mobile phase or sample preparation are the main causes of HPLC issues. For best results, always prepare food in clean glasses. Cleaning the HPLC with the proper solvent is the primary solution to HPLC issues. It is advised to do a fast visual inspection of the instrument and column if any issue arises. Try to follow the same procedure if a monograph or other protocol is supplied. Troubleshooting is a type of issue resolution that is frequently used to fix failing processes or goods. Alternatively, we advise you to read the complete article to learn some tips that will enable you to prevent issues in the future. We hope that this article will help you detect issues and understand their underlying causes so that you can stop or lessen their occurrence in the future. The most crucial aspect of troubleshooting is the care of the colon and HPLC. We attempted to address all significant troubleshooting issues in this post.

The biochemical measurement of the CoQ status in different tissues can be performed using HPLC with electrochemical detection (ED). Because the production of the electrochemical cells used with the Coulochem series detectors was discontinued, we aimed to standardize a new HPLC-ED method with new equipment. We report all technical aspects, troubleshooting and its performance in different biological samples, including plasma, skeletal muscle homogenates, urine and cultured skin fibroblasts. Analytical variables (intra- and inter-assay precision, linearity, analytical measurement range, limit of quantification, limit of detection and accuracy) were validated in calibrators and plasma samples and displayed adequate results. The comparison of the results of a new ERNDIM external quality control (EQC) scheme for the plasma CoQ determination between HPLC-ED (Lab 1) and LC-MS/MS (Lab 2) methods shows that the results of the latter were slightly higher in most cases, although a good consistency was generally observed. In conclusion, the new method reported here showed a good analytical performance. The global quality of the EQC scheme results among different participants can be improved with the contribution of more laboratories.

OBJECTIVES/SPECIFIC AIMS: This project aims to synthesize an angiogenic decorin mimic (VEGFp-DS-SILY) with varying densities of QK and characterize its angiogenic potential and synergism with VEGF by evaluating (1) endothelial cell (EC) migration and proliferation, (2) EC VEGF receptor activation, (3) EC tubule formation in collagen scaffolds, and (4) angiogenesis from a chick chorioallantoic membrane (CAM assay) growing into the scaffold, reflecting the ability of the collagen scaffold to integrate into existing vasculature. The next main goal is to develop and characterize an MMP-degradable nanoparticle system for controlled release of VEGF. Future work will evaluate in vivo effects of VEGFp-DS-SILY bound to a 3D collagen scaffold on ischemic wound repair in a combined excisional wound/bipedical dorsal skin flap rat model. METHODS/STUDY POPULATION: Peptide hydrazides are conjugated to the free carboxylic acid functional groups on dermatan sulfate using EDC chemistry. We added a 3 amino acid spacer (-Gly-Ser-Gly) to the C-terminus of the established QK sequence before the hydrazide functional group and refer to this modified QK as “VEGFp.” VEGFp, SILY, and N-terminal biotinylated versions were synthesized using standard Fmoc solid-phase peptide synthesis protocols and purified using reverse phase HPLC. Coupling efficiencies of peptides to dermatan sulfate were determined spectroscopically at 280 nm measuring the aromatic residues (Trp or Tyr) using a NanoDrop system. Dermatan sulfate with 1 or 4 VEGFp peptides coupled were termed DSV1 and DSV4, respectively. After further conjugation with SILY, we will blend this VEGFp-DS-SILY with unmodified DS-SILY to a total 10 μM to test increasing densities of VEGFp. To verify that the collagen-binding properties of VEGFp-DS-SILY are not compromised by the addition of VEGFp, we will use a streptavidin-HRP system to detect bound biotinylated VEGFp-DS-SILY on collagen-coated plates by established protocols. DSV1 and DSV4 were tested for their effects on endothelial VEGFR2 phosphorylation using an MSD ELISA-type assay and endothelial proliferation using an MTS assay. Cell migration was monitored using an ORIS assay where cells are grown to confluence around a silicone stopper that is then removed to allow cells to migrate inward. Tubulogenesis was evaluated by examining tubule formation on matrigel. Finally, in vivo angiogenesis will be evaluated using a chorioallantoic membrane assay. For extracellular VEGF release, hollow MMP-degradable thermoresponsive nanoparticles [NIPAM, 5 mol% 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), 1% Acrylic Acid (AAc), 2 mol% MMP-degradable peptide diacrylate, and potassium persulfate initiator] will be synthesized around noncross-linked polymer cores. The cores will then be diffused out through the shell by dialysis prior to drug loading. SILY (and some biotinylated SILY for visualization) will be conjugated with EDC chemistry for targeting nanoparticles to collagen. NPs size and zeta-potential will be measured on a Malvern Zetasizer. VEGF will be loaded into NPs by co-incubating a loading solution of 1 µg/mL VEGF with 1 mg of NPs, incubating overnight at 4°C. VEGF loading and release will be measured by ELISA. Biological activity of the released VEGF from particles will be determined on ECs using assays similar to those outlined previously. RESULTS/ANTICIPATED RESULTS: Preliminary data have verified the synthesis and purification of SILY and VEGFp (QK-Gly-Ser-Gly-hydrazide), as well as an N-terminal biotinylated version, through mass spectrometry and reverse-phase HPLC, respectively. For proof-of-concept, we have verified binding of VEGFp to the VEGF receptor 2 using a ForteBio Blitz interferometry instrument. In addition to support based on published reports showing retained bioactivity of QK after conjugation using other spacers, our preliminary data suggests that VEGFp still binds to VEGF receptor 2, albeit with decreased affinity like QK as compared with VEGF. Circular dichroism also shows that VEGFp has retained its α-helical structure necessary for bioactivity; however it appears that it has some uncoiling when conjugated to dermatan sulfate. We hypothesize that varying densities of VEGFp conjugated to the decorin mimetic (DS-SILY) will modulate the degree of angiogenic activity and synergy with VEGF. We determined that we can achieve ~70% VEGFp conjugation completion to dermatan sulfate after 3.5 hours. We have quantified VEGFR2 phosphorylation after 5 minute treatments by using phospho-specific antibodies and an ELISA-type protocol in a mesoscale discovery system. Preliminary data with human umbilical vein endothelial cells shows that VEGFp exhibits synergism with VEGF at levels similar to QK. DSV1 and DSV4 data suggests synergy with VEGF, although free-peptides and engineered compounds alone did not show effects similar to VEGF in the conditions tested. Prelminary data with 30 minute treatments suggests that the peptides and compounds may require longer exposures to induce activation, as they may have slower binding rates. In contrast, prolonged stimulation with VEGF causes a sharp increase in receptor activation, peaking around 10 minutes and decreasing significantly by 30 minutes. Peptides QK and VEGFp both slightly increased proliferation of dermal microvascular endothelial cells (HMVECs) after 60 hours incubation. However, incubation with dermatan sulfate and DSV caused significant cell death after 24 hours in reduced growth factor media, likely due to sequestering of growth factors. It is possible that VEGFp-DS-SILY may better stimulate proliferation since it would be presented as a surface bound proteoglycan mimic, rather than as a soluble factor. HMVECs migrated farther for all treatment groups (10 µM QK, 10 µM VEGFp, 1 µM DSV4, and 10 µM DSV4) than the 10 ng/mL VEGF positive control, although more cells migrated in response to VEGF. This may be accounted at least in part by the more pronounced proliferation induced by VEGF. Migration will also be tested in 3D culture within a collagen gel. We are currently testing a 2D matrigel system for tubulogenesis. We have found that 10 µM DSV4 forms qualitatively more well-defined tubules than the untreated control on reduced growth factor matrigel. However, we were not able to quantify the improved tubule formation and are still troubleshooting the tubule analysis. After seeding ECs and culturing for 4, 8, and 12 hours, cells will be fluorescently stained with anti-CD31 and imaged for 3D tubule formation. CAM assay angiogenesis growing into a collagen scaffold. In brief, fertilized chicken embryos are incubated for 2 days before exposing the CAM. VEGFp-DS-SILY bound to a collagen gel will be placed onto the CAM. Some treatment groups will receive additional VEGF to investigate synergistic effects. Light microscope images of angiogenesis into the collagen gel coated with VEGFp-DS-SILY, taken every day from days 10 to 13, will reflect the ability of the collagen scaffold to integrate into existing vasculature and 3D angiogenic potential of VEGFp-DS-SILY with or without VEGF. We expect that VEGFp-DS-SILY treatment will increase the number of vessels formed on the CAM. Preliminary data using a Fluoraldehyde assay indicates that loading of ~300 ng VEGF per mg of nanoparticles can be achieved. We expect that using an MMP-degradable peptide diacrylate crosslinker will allow nanoparticles to degrade in protease-rich environments like the chronic wound bed and release VEGF. Adjustments to the formulation, such as crosslinker density, may need to be modified to control the rate of VEGF release. DISCUSSION/SIGNIFICANCE OF IMPACT: We expect that our angiogenic decorin mimetic will lead to a novel treatment to accelerate healing of ischemic diabetic foot ulcers, thereby reducing the need for limb amputation and mortality rate of diabetic patients. We anticipate that the diabetes research and regenerative medicine communities will (1) gain a platform for targeted delivery of growth factors, (2) understand the dependence of vascularization within 3D collagen constructs on VEGFp densities and VEGF receptor activation in controlling the degree of angiogenesis, and (3) gain the benefits of controlled angiogenesis in ischemic diabetic wound healing.

For pharmaceutical industry, HPLC is more than 40 years old. Even after instrumental advancements in HPLC problems still arise, troubleshooting still comes. HPLC is the main stay of analytical section nowadays in pharmaceutical industry whether it is API industry or formulation plant. Best approach for troubleshooting HPLC problems is going with systematic way. At the start of quantitative chromatographic analysis, the first parameter of paramount importance is baseline, especially for measuring the area of any given peak. Baseline usually suffers from these errors, namely, high baseline drift, periodic baseline fluctuation, and spikes. This article aims at suggesting some working methodologies which will resolve as well as avoid cases of such errors and failures.

Sometimes our approach to troubleshooting specific problems has to change in response to changes in high performance liquid chromatography (HPLC) technology over time. In this installment, we discuss changes in technologies for mobile-phase degassing, silica-based stationary phases, and models for reversed-phase selectivity.

The quadrupole mass analyzer is now available to many analytical chemists as a detector for high performance liquid chromatography (HPLC) and gas chromatography (GC), thanks to its increasingly accessible price point. Although it’s not vital to understand the working details of these detectors, insight into their design and operation can help enormously when planning, optimizing, or troubleshooting analytical methods.

Background: High-performance liquid chromatography is one of the most used analytical techniques in quality control in the pharmaceutical industry. Since it is a complex technique, it needs good practices that can contribute to compliance with regulatory requirements. Objetive: This study aims to establish a diagnostic tool for Good Chromatographic Practices (GCP) for the self-assessment of a Quality Control Laboratory (QCL). Methods: The research was carried out on scientific bases, pharmaceutical legislation, as well as guides published by manufacturers. Results: Seven axes of action were identified: implementation, management, and continuous improvement of GCP in the laboratory; GCP in the installation, operationalization, qualification, and validation processes of the equipment and software; GCP in processes related to data management, including guidelines regarding access, generation, integrity, and traceability; GCP related to the management and use of consumables; GCP related to handling, maintenance, analytical and operational troubleshooting; GCP in the processes of preparation, use, and storage of analytical solutions and reagent solutions; and GCP related to the acquisition and processing of standards, samples, and results. These axes resulted in a diagnostic tool with 124 questions. Conclusion: The application of the GCP diagnostic tool provides the mapping of the routine and procedures related to the execution of the HPLC technique for quality control in the pharmaceutical industry, contributing to meeting regulatory requirements.

Authors: Ren-Qi Wang, Teng-Teng Ong, Ke Huang, Weihua Tang & Siu-Choon Ng ### Abstract We described a facile and effective protocol wherein radical copolymerization is employed to covalently bond cationic β-cyclodextrin (β-CD) onto silica particles with extended linkage, resulting in a chiral stationary phase (IMPCSP) that can be used for the enantioseparation of racemic drugs in both high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). Starting from commercially available chemicals, the IMPCSP is prepared in several steps: (i) reaction of β-CD with 1-(p-toluenesulfonyl)-imidazole to afford mono-6A-(p-toluenesulfonyl)-6A-deoxy-β-cyclodextrin (B); (ii) nucleophilic addition between B and 1-vinylimidazole and followed by treatment with anionic-exchange resin to give mono-vinylimidazolium-CD chloride (C); (iii) electrophilic addition between C and phenyl isocyanate to generate 6A-(3-vinylimidazolium)-6-deoxyperphenylcarbamate-β-CD chloride (D); (iv) reaction of silica gel with 3-methacryloxypropyltrimethoxysilane to engender vinylized silica (E); (v) immobilization of C onto vinylized silica via radical copolymerization with 2,3-dimethyl-1,3-butadiene in the presence of 2,2’-azobis(2-methylpropionitrile) (AIBN) to afford the desired chiral stationary phases. The overall IMPCSP preparation and column packing protocol requires ~2 weeks. ### Introduction Chromatographic methodologies have been extensively explored for accurate sample analyses, online monitoring of reaction progress and purifying of synthesized products (1). Especially, modern chromatographic techniques have been developed as powerful tools for chiral separation and preparation of enantiomers, most of which are of biological and pharmaceutical interests. Whatever chiral chromatographic techniques are used, chiral selectors either dissolved as mobile phases or mobilized onto supporting materials as stationary phases are crucial for successful and robust enantioseparations. Among the chiral selectors used so far, cyclodextrins (CDs) and their derivatives have been widely used in chiral chromatography since its first introduction by Armstrong et al. (2). In order to obtain a better solubility, the charged moieties are favorably introduced onto CD rims for capillary electrophoresis (CE). The self-mobility of CD in the ionized form enhances the separation ability when it is opposite to the electrophoretic mobility of the analytes (3). The hydrophobic moieties on the analytes could be included or adsorbed into the chiral cavity of CDs. Meanwhile, the analytes could also interact with the substituents on the CD rims (4,5). The native CDs and their chemically-modified derivatives afford fine-tuned hydrophobicities, charges and cone shapes etc., which ultimately result in different tightness and interactions for CD-analytes complexes selectors and thus chemically-manipulated diving forces for chiral recognitions (6-14). The charged CD-based chiral mobile phases and chiral stationary phases (CSPs) have been extensively explored in drug chiral analyses (15-20). A sulfated β-CD based CSP was developed which enabled great enantioseparations towards 33 racemic drugs in high-perfomace liquid chromatography (HPLC)9. Recently, ionic-liquids featured positively-charged CDs have been covalently bonded onto silica gels to prepare novel CSPs, which exhibited dual selectivity in HPLC for the enantioseparation of both polar and non-polar compounds (18). The additional electrostatic interactions are significant to achieve separations of polar analytes which may interact with the neutral CDs too weakly, where16 aromatic alcohol racemates and 2 drugs were achieved in polar organic mobile phases19. In our previous report, a series of coated CSPs based on fully derivatized cationic β-CD were prepared. These cationic β-CD CSPs have shown strong enantioseparation abilities and moderate retention times towards a series of α-phenyl alcohols in both supercritical fluid chromatography (SFC) and HPLC20. However, polar organic solvents in the mobile phase could cause damage to the coated CSPs. Besides, the efficiencies of the coated CSPs are usually low as the surface thickness of chiral selectors is hard to control (21). Figure-1 (R Wang) Syntheses of Ts-CD (B), compounds C and D. The immobilization of functionalized CD onto silica gel is generally achieved by chemical reactions between highly reactive substituent on CD and the functional groups on silica gel (14,16-18). Tedious synthetic approaches with possible protecting and de-protecting steps are usually required for successful grafting. Comparatively, the facile co-polymerization method is more accessible and reliable, universally employed in producing non-silica fillings for columns applicable for size-exclusion chromatography (SEC) or ion-exchange applications (22). The polymerized materials used for chromatography have wide applicable pH ranges and improved retentions for polar analytes, although lower mechanic strength and efficiency are often observed than the silica-based CSPs. The co-polymerization approach was also explored in the immobilizing of polysaccharide or enantiopure small molecules onto silica gel for the preparation of CSPs (23,24). Figure-2 (R Wang) Preparation of IMPCSP via radical copolymerization. In view of the our continuous and successful research endeavors in developing powerful cationic CDs as chiral selectors for both CE and HPLC (12,25,26), we recently developed the facile copolymerization methodology in preparing novel covalently-bonded cationic β-CD CSPs via co-polymerization approach (see Figs. 1 and 2). These as-prepared CSPs have successfully expanded the enatioseparation windows towards a broader range of chromatographic conditions for both HPLC and SFC application (27,28). The linkage between CD selector and silica-support was built by using diene (ca. 2,3-dimethyl-1,3-butadiene) as the third monomer for the dual copolymerization system with vinyllated CD and silica gel. The extended linkage can effectively improve the surface loading issue of CD onto silica gel, a challenge faced when direct immobilizing CD derivatives onto silica surface with short spacers due to steric hindrance (29). The as-developed CDs CSPs exhibited great potential in drug enantioseparations in both HPLC and SFC applications. The protocol describes herein the synthesis of imidazolium-based IMPCSP. This methodology can also be applied for the synthesis of other ammonium-based cationic CD CSPs (27,28), which may find wide applications for both drug enantioseparations and NOM assessments. ### Reagents 1. β-Cyclodextrin (β-CD; >95%; TCI, cat. no. C0900) - Imidazole (99%; Merck, cat. no. 436151) - Sodium hydroxide (NaOH, 97%; Sigma-Aldrich, cat. no. 138701) - Ammonium chloride (99.5%; Fluka, cat. no. 09725) - p-Toluenesulphonyl chloride (99%; Fluka, cat. no. 89730) - !CAUTION p-toluenesulfonyl chloride is very smelly and highly corrosive. It is recommended that it be weighed in a glovebox and transferred with sealed bottles or directly weighted out in reaction flask. Please refer to the MSD sheet of this compound for safety information. - Dichloromethane (CH2Cl2, 99.6%, ACS reagent; Sigma-Aldrich, cat. no. 443484) - 1-Vinylimidazole (≥ 99%; Sigma-Aldrich, cat. no. 235466) - Ethyl acetate (99.5%, ACS reagent; Sigma-Aldrich, cat. no. 141786) - n-Hexane (98.5%; Sigma-Aldrich, cat. no. 178918) - Phenyl isocyanate (≥ 98%; Sigma-Aldrich, cat. no. 185353) - Amberlite IRA-900 ion-exchange resin (Sigma-Aldrich, cat. no. 216585) - N,N-Dimethylformamide (DMF, 99.8%; Sigma-Aldrich, cat. no. 319937) - Pyridine (99.5%, Extra dry, Fisher, cat. no. AC33942) - ! CAUTION Pyridine is very harmful to eyes and skin. Goggle and gloves must be worn in handling pyridine and conduct experiments in well ventilated fumehood to avoid inhalation. - Chloroform (99.8%, ACS reagent; Fisher, cat. no. AC40463) - Magnesium sulphate (≥ 97%, anhydrous, reagent grade; Sigma-Aldrich, cat. no. 208094) - Nitrogen gas (ALPHAGAZ™; SOXAL) - Liquid nitrogen (SOXAL) - Paraffin oil (puriss.; Sigma-Aldrich, cat. no. 18512) - Silica gel (5 μm, Kromasil) - 3-Methacryloxypropyltri-methoxysilane (98%; Sigma-Aldrich, cat. no. 440159) - 2,2’-Azobis(2-methyl-propionitrile) (AIBN, ≥ 98%, purum; Sigma-Aldrich, cat. no. 11630) - Toluene (99.8%, anhydrous, reagent grade; Sigma-Aldrich, cat. no. 244511) - 2,3-Dimethyl-1,3-butadiene (98%; Sigma-Aldrich, cat. no. 145491) ### Equipment 1. Magnetic stirrer with thermal and speed controller (Heidolph) - Rotary evaporator (Büchi, R205) - Teflon-coated magnetic stirring bars - Vacuum pump - Balance - Round-bottomed flask - Conical flask - Pressure-equalizing addition funnel - Büchner funnel - Soxhlet extractor - Liebig condenser - Dewar dish - Glass and plastic syringes (polypropylene) - Disposable hypodermic syringe needles - NMR spectrometer (300 MHz; Brüker, cat. no. ACF300) - FTIR spectrometer (FTS165) - Vario EL universal CHNOS elemental analyzer - MALDI-TOF-MS (Shimadzu, AXIMA Confidence) - Stainless steel HPLC column (15 cm length, 2.1 mm in inner diameter; Isolation Technologies) - HPLC pump (LabAlliance-Scientific) - Filter membrane used for syringe (0.45 μm pore size; Millipore) - Membrane Filter (0.45 μm pore size; Millipore) - Agilent HPLC (HP 1100) equipped with a variable-wavelength detector (190–300 nm) - Jasco SFC (SF 2000) equipped with a variable-wavelength detector (190–900 nm) and a back pressure regulator (0-30 MPa) ### Procedure - **Synthesis of compound C** 1. Fit a 100-ml double-necked round-bottomed flask containing a Teflon-coated magnetic stir bar with a rubber septum and a Liebig condenser. Fit the condenser a rubber septum with inlet of dry N2 and an outlet towards a bubbler containing paraffin oil, in order to prevent the ingress of moisture and air. - Weigh out 6A-toluenesulfonyl-β-CD B 12.91 g (0.01 mol) into the flask. - Turn on the circulating water in the condenser. - Add 20 ml DMF into the flask and switch on the magnetic stirrer and heater. - Inject 1-vinylimidazole 3 ml (0.03 mol) into the flask though a plastic syringe. ? TROUBLESHOOTING - Allow the reaction to proceed at 90°C for 48 h under reflux. Cool down to room temperature. - Precipitate the product in acetone (200 ml). Collect the precipitate by filtration. Wash the raw product with acetone. - Dissolve the solid into water/methanol (200 ml/50 ml) - ! CAUTION The solution could be heated towards 50°C to afford a clear solution. - Fill a column (I.D. 30 × 250 mm) with Amberlite IRA-900 ion-exchange resin and washed with MilliQ water till the effluent pH going neutral. - Transfer the solution from step 8 into the column and let it hold for 1 h. Subsequently, collect the effluent drop by drop. Flush the column with equal volume MilliQ water and collect the effluent. - Distill off water on rotary evaporator to yield C as a light yellow solid (9.7 g, 78% yield) - PAUSE POINT Compound C can be stored in oven at 80°C for several weeks. - **Synthesis of compound D** - Fit a 250-ml double-necked, round-bottomed flask containing a Teflon-coated magnetic stir bar with a rubber septum and a Liebig condenser. Fit the condenser a rubber septum with inlet of dry N2 and an outlet towards a bubbler containing paraffin oil, in order to prevent the ingress of moisture and air. - Weigh out compound C 2.15 g (1.72 mmol) into the flask. - Add 20 ml dried pyridine into the flask and switch on the magnetic stirrer and heater. - ! CAUTION Pyridine is highly toxic solvent. All experiments dealing with pyridine should be operated in fumehood. Goggles, gloves and mask should be worn. - Inject phenyl isocyanate 12 ml (110.32 mmol) into the flask though a plastic syringe. - ! CAUTION Phenyl isocyanate has acute toxicity. It may cause severe skin burns and eye damage. It may cause allergy or asthma symptoms or breathing difficulties if inhaled. Adding 12 ml phenyl isocyanate as a whole would cause large extent of side reaction to produce triphenyl isocyanurate as a by-product. It is strongly recommended to add phenyl isocyanate dropwise with pressure equalizing funnel. Goggles, gloves and mask should be worn before experimental operation in fumehood. - ▲ CRITICAL STEP Phenyl isocyanate should be added with four equal portions. Add 12 ml as a whole would cause large extent of side reaction to produce triphenyl isocyanurate as a by-product. Add phenyl isocyanate drop by drop with pressure equalizing funnel would end up with its transformation in the funnel and the liquid colour changes to light yellow. - Allow the reaction to proceed at 85 °C for 20 h. Set up the vacuum distillation pipeline. - Distill off pyridine under reduced pressure at 85 °C. ? TROUBLESHOOTING - Dissolve the residue with chloroform 15 ml. - Add the solution into silica column. Flush the impurities with n-hexane/ethyl acetate (70:30 v:v). - ▲ CRITICAL STEP The ratio of n-hexane/ethyl acetate was determined by TLC analyses. The flash column separation progress was also monitored by TLC analyses. Lowering the ratio would result in an increase of the amount of solvents used for eluting the impurities completely. - Flush the product out of the column with MeOH. - Remove the solvent on rotary evaporator to yield D as a dark yellow solid (6.2 g, 66% yield). - PAUSE POINT Compound D can be stored in oven at 80 °C for several weeks. - **Synthesis of vinylized silica E** - Dry spherical silica gel particles (5 μm, 5g) in vacuum (10 mm Hg) at 150 °C for 24 h. Cool down to room temperature. - Fit a 250-ml double-necked, round-bottomed flask containing a Teflon-coated magnetic stir bar with a rubber septum and a Liebig condenser. Fit the condenser a rubber septum with inlet of dry N2 and an outlet towards a bubbler containing paraffin oil, in order to prevent the ingress of moisture and air. - Add dry toluene 100 ml into the flask. Switch on the magnetic stirrer and heater. - Inject 3-methacryloxypropyltrimethoxysilane (2.3 ml) into the flask. - Add dried silica gel from step 22 into the flask. ? TROUBLESHOOTING - Allow the reaction to stand at 90 °C for 18 h. Product was collected by filtration through 0.45 μm pore size membrane and washed with MeOH in Soxhlet apparatus overnight. - Collect the product by filtration through 0.45 μm pore size membrane and washed with MeOH in Soxhlet extractor overnight. - Dry the product overnight in an oven at 60°C in vacco to afford the vinylized silica E. - PAUSE POINT Vinylized silica E can be stored at room temperature for several months. - **Co-polymerization for preparation of IMPCSP** - Dissolve compound D (0.7 g) in chloroform (30 ml). Filter the solution through 0.45 μm pore size membrane. - Transfer the filtrate and drop onto vinylized silica gel E (1.4 g) evenly with a glass syringe. - ! CAUTION A glass syringe was preferred to avoid any introduction of siloxal impurities from plastic syringes. Chloroform could corrode the rubber piston of the plastic syringe. - Dry the colloid-like mixture in vacuum (10 mm Hg) at 25 °C. - Fit a 100-ml double-necked, round-bottomed flask containing a Teflon-coated magnetic stir bar with two rubber septums. - Add the solid from step 32 into the flask. Add AIBN (3 mg) into the flask. - ▲ CRITICAL STEP The amount of AIBN should be controlled. A less amount of AIBN would lead to slow reaction rate but a great amount resulted in rapid radical reactions and short chain growth. In both conditions, successful immobilized CD amounts were low. - Inject anhydrous toluene (20 ml) and 2,3-dimethyl-1,3-butadiene (2.6 ml) into the flask. - ▲ CRITICAL STEP 2,3-Dimethyl-1,3-butadiene should be added before heating started. It was initiated together with the vinyl groups in compound D and vinylized silica E. Therein, 2,3-dimethyl-1,3-butadiene could act as a interlink. A delayed addition led to a lower CD amount on IMPCSP, since the radicals formed on D and E could have been annihilated before 2,3-dimethyl-1,3-butadiene was added. - Freeze the mixture in liquid N2 in a Dewar dish and degas the reaction system in vacuum (10 mm Hg) for 0.5 h. - ! CAUTION Liquid N2 - Take the flask out of the Dewar dish and thaw at room temperature. - Repeat step 36 and 37 for three times. ? TROUBLESHOOTING - Fit the flask with a Liebig condenser. Fit the condenser a rubber septum with inlet of dry N2 and an outlet towards a bubbler containing paraffin oil, in order to prevent the ingress of moisture and air. - Switch on the magnetic stirrer and heater. Leave the reaction to proceed at 60 °C for 18 h. - Cool down the reaction mixture and collect the product by filtration through 0.45 μm pore size membrane. - Wrap the filter cake with filter paper. Extract the solid in a Soxhlet extractor overnight. - Stop heating and collect the silica from the Soxhlet extractor. Dry the product in an oven at 60 °C for 24 h. - Pack IMPCSP into an empty stainless steel column with MeOH at 8,000 psi for 30 min. - PAUSE POINT IMPCSP can be stored at room temperature for several months. ### Timing - Synthesis of B: ~40 h include synthesis of A. - Steps 1-11 Synthesis of C: Steps 1-5, 1 h; Step 6, 48 h; Steps 7-8, 1 h; Steps 9-10, 1 h; Step 11, 12 h. - Steps 12-21 Synthesis of D: Steps 12-14, 1 h; Step 15, 2 h; Step 16, 20 h; Step 17, 5 h; Steps 18-20, 12 h; Step 21, 12 h. - Steps 22-29 Preparation of E: Step 22, 12 h; Steps 23-25, 4 h; Steps 26-27, 18 h; Steps 28-29, 20 h. - Steps 30-44 Preparation of IMPCSP: Steps 30-32, 6 h; Steps 33-35, 1 h; Steps 36-38, 1 h; Steps 39-40, 20 h; Steps 41-42, 20 h; Step 43, 24 h; Step 44, 2 h. ### Troubleshooting **? TROUBLESHOOTING** Troubleshooting advices can be found in Table 1. ### Anticipated Results The success of immobilizing CD onto silica gel could be characterized by typical FT-IR vibration bands of phenyl-groups in phenylcarbamate substituents in CSP. The amount of immobilized CD derivatives on silica gel could be calculated based on elemental analyses results (%N exclusively from CD derivative). The representative analytical data of organic compounds: C, D, E and IMPCSP are given below. **Organic synthesis**: Compound C: m.p. 254-268°C. 1H NMR (300 MHz, DMSO-d6, δ ppm) 2.73 (m, 1H H-2), 2.88 (m, 1H, H-4), 3.00-3.15 (m,1H, H-5), 3.32-3.45 (overlap with solvent peak, 12H, H-2,4), 3.20-3.80 (overlap with solvent peak, 27H, H-3,5,6)， 4.30-4.40 (m, 1H, OH-6), 4.48-4.59 (m, 6H, OH-6, =CH2vinyl) 4.84-4.86 (m, 6H, H-1) 5.00 (d, 1H, H-1) 5.40-5.50 (m, 1H, -CHvinyl) 5.64-5.84 (m, 13H, OH-2,3) 5.95-6.10 (d, 1H, OH-2), 7.87 (s, 1H, =CH-4im) 8.18 (s, 1H, CH-5im) 9.42 (s, 1H, =CH-2im) Compound D: m.p. 197-199°C. MALDI-TOF-MS [M+]: (expected) 3592.16; (found) 3592.07. 1H NMR (300 MHz, CDCl3, δ ppm) 3.00-6.00 (m, 52H, H-CD, H-vinyl) 6.00-7.80 (m, 100H, H-phenyl). Microanalysis for C187H175ClN22O54 (expected) C: 61.87%, H: 4.86%, N: 8.49%, (found) C: 60.25%, H: 5.13%, N: 9.11%. Surface modified silica gel E: Obvious vibration bands in FT-IR spectrum of 2964, 2855 cm-1 (C-H) 1705 cm-1 (C=O) 1635 cm-1 (C=C) and 1130 cm-1 (C-O and Si-O) represent the successful surface modification with mathacryloyl-groups. Microanalyses data give the surface double bond loading of 5 μm silica as 2.16 μmol/m2 based on the carbon content (Table 2) (30). **Prepared IMPCSP** The characteristic peaks in FT-IR spectrum at 1720 cm-1 (C=O), 1647, 1558, 1458 cm-1 (C=C phenyl group) and 1130 cm-1 (C-O and Si-O) show the CD derivative has been successfully bonded onto silica surface. The cyclodextrin derivatives’ grafting coverage was calculated based on the nitrogen content (%N), to be 0.09 μmol m-2 (Table 2)30. **Chromatographic separation results**: The packed column with IMPCSP was applied for enantiomeric separations in RP-LC, NP-LC and SFC respectively. The cationic β-CD exhibited good enantioselectivity and stability towards four representative racemic analytes in Figure 3. ### References 1. Ryan, J.F. *Chromatography: creating a central science*. (American Chemical Society, 2001). - Armstrong, D.W. et al. Separation of drug stereoisomers by the formation of beta-cyclodextrin inclusion complexes. *Science* 232, 1132-1135 (1986). - Amini, A. Recent developments in chiral capillary electrophoresis and applications of this technique to pharmaceutical and biomedical analysis. *Electrophoresis* 22, 3107-3130 (2001). - Gübitz, G. & Schmid, M.G. *Chiral separations: methods and protocols*. (Humana Press, Totowa, USA, 2004). - Cox, G.B. *Preparative enantioselective chromatography*. (Blackwell Pub., Oxford, UK, 2005). - Hinze, W.L. et al. Liquid chromatographic separation of enantiomers using a chiral beta-cyclodextrin-bonded stationary phase and conventional aqueous-organic mobile phases. *Anal. Chem*. 57, 237-242 (1985). - Lubda, D. et al. Monolithic silica columns with chemically bonded β-cyclodextrin as a stationary phase for enantiomer separations of chiral pharmaceuticals. *Anal. Bioanal. Chem*. 377, 892-901 (2003). - Guo, Z.M. et al. Synthesis, chromatographic evaluation and hydrophilic interaction/reversed-phase mixed-mode behavior of a “Click beta-cyclodextrin” stationary phase. *J. Chromatogr. A* 1216, 257-263 (2009). - Stalcup, A.M. & Gahm, K.H. A sulfated cyclodextrin chiral stationary phase for high-performance liquid chromatography. *Anal. Chem*. 68, 1369-1374 (1996). - Lai, X.H., Tang, W.H. & Ng, S.-C. Novel cyclodextrin chiral stationary phases for high performance liquid chromatography enantioseparation: effect of cyclodextrin type, *J. Chromatogr. A*, 1218, 5597-5601 (2011). - Lai, X.H., Tang, W.H. & Ng, S.-C. Novel -cyclodextrin chiral stationary phases with different length spacer for normal-phase high performance liquid chromatography enantioseparation, *J. Chromatogr. A*, 1218, 3496-3501 (2011). - Wang, Y. et al. Preparation of cyclodextrin chiral stationary phases by organic soluble catalytic ‘click’ chemistry. *Nat. Protoc*. 6, 935-942 (2011). - Poon, Y.F. et al. Synthesis and application of mono-2(A)-azido-2(A)-deoxyperphenyl-carbamoylated b-cyclodextrin and mono-2(A)-azido-2(A)-deoxyperacetylated beta-cyclodextrin as chiral stationary phases for high-performance liquid chromatography. *J. Chromatogr. A* 1101, 185-197 (2006). - Lai, X.H. & Ng, S.C. Enantioseparation on mono(6A-N-allylamino-6A-deoxy)permethylated -cyclodextrin covalently bonded silica gel. *J. Chromatogr. A* 1101, 53-59 (2004). - Cherkaoui, S. & Veuthey, J.L. Use of negatively charged cyclodextrins for the simultaneous enantioseparation of selected anesthetic drugs by capillary electrophoresis–mass spectrometry. *J. Pharm. Biomed. Anal*. 27, 615-626 (2002). - Wang, R.Q. et al. . Recent advances in pharmaceutical separations with supercritical fluid chromatography and chiral columns, *TrAC Trends Anal. Chem*., in press, DOI: 10.1016/j.trac.2012.02.012 (2012). - Zukowski, J., De Biasi, V. & Berthod, A. Chiral separation of basic drugs by capillary electrophoresis with carboxymethylcyclodextrins. *J. Chromatogr. A* 948, 331-342 (2002). - Armstrong, D.W. et al. Examination of ionic liquids and their interaction with molecules, when used as stationary phases in gas chromatography. *Anal. Chem*. 71, 3873-3876 (1999). - Zhou, Z. et al. Synthesis of ionic liquids functionalized β-cyclodextrin-bonded chiral stationary phases and their applications in high-performance liquid chromatography. *Anal. Chim. Acta* 678, 208-214 (2010). - Wang, R.Q. et al. Synthesis of cationic [beta]-cyclodextrin derivatives and their applications as chiral stationary phases for high-performance liquid chromatography and supercritical fluid chromatography. *J. Chromatogr. A* 1203, 185-192 (2008). - Glenn, K.M. & Lucy, C.A. Stability of surfactant coated columns for ion chromatography. *Analyst* 133, 1581-1586 (2008). - Walsh, G. *Pharmaceutical biotechnology: concepts and applications*. (John Wiley & Sons, Chichester, UK, 2007). - Chen, X.M. et al. Synthesis of chiral stationary phases with radical polymerization reaction of cellulose phenylcarbamate derivatives and vinylized silica gel. *J. Chromatogr. A* 1034, 109-116 (2004). - Gasparrini, F. et al. New hybrid polymeric liquid chromatography chiral stationary phase prepared by surface-initiated polymerization. *J. Chromatogr. A* 1064, 25-38 (2005). - Tang, W.H. & Ng, S.C. Synthesis of cationic single-isomer cyclodextrins for the chiral separation of amino acids and anionic pharmaceuticals. *Nat. Protoc*. 2, 3195-3200 (2007). - Tang, W.H. & Ng, S.C. Facile synthesis of mono-6-amino-6-deoxy-α-, -, γ-cyclodextrin hydrochlorides for molecular recognition, chiral separation and drug delivery, *Nat. Protoc*. 3, 691-697 (2008). - Wang, R.Q. et al. Cationic cyclodextrins chemically-bonded chiral stationary phases for high-performance liquid chromatography, *Anal. Chim. Acta* 718, 121-129 (2012). - Wang, R.Q. et al. Chemically bonded cationic -cyclodextrin derivatives and their applications in supercritical fluid chromatography, *J. Chromatogr. A* 1224, 97-203 (2012). - Liu, M. et al. Study on the preparation method and performance of a new β-cyclodextrin bonded silica stationary phase for liquid chromatography. *Anal. Chim. Acta* 533, 89-95 (2005). - Siles, B.A. et al. Retention and selectivity of flavanones on homopolypeptide-bonded stationary phases in both normal- and reversed-phase liquid chromatography. *J. Chromatogr. A* 704, 289-305 (1995). ### Acknowledgements We acknowledge funding from the A*STAR (SERC Grant No.: 0921010056) in support of this project. R.-Q.W. is grateful for the award of a research scholarship by NTU and helpful discussions with Dr A. Rajendran of NTU SCBE. ### Figures **Figure 1: Figure-1 (R Wang) Syntheses of Ts-CD (B), compounds C and D**.  **Figure 2: Figure-2 (R Wang) Preparation of IMPCSP via radical copolymerization**.  **Figure 3: Figure-3 Enantioseparations of racemic drugs using IMPCSP with multiple channel UV detector detection at 254 nm**.  *Flow rate is 0.4 ml min-1 in normal-phase HPLC (NP-LC), 0.5 ml min-1 in reverse-phase HPLC (RP-LC) and 1.0 ml min-1 in SFC. Separation conditions are as follows: (a) 7-methoxyflavanone, reverse-phase HPLC buffer (0.1% TEAA pH 4.3)/MeOH (30/70); NP-LC n-hexane/2-propanol (97/3, v/v); SFC CO2/2-propanol (99/1, v/v); (b) 4’-hydroxyflavanone, RP-LC buffer (0.1% TEAA pH 4.3)/MeOH (40/60); NP-LC n-hexane/2-propanol (97/3, v/v); SFC CO2/2-propanol (99/1, v/v); (c) bendroflumethiazide, NP-LC n-hexane/2-propanol (85/15, v/v); SFC CO2/2-propanol (70/30, v/v); (d) althiazide, NP-LC n-hexane/2-propanol (70/30, v/v); SFC CO2/2-propanol (70/30, v/v)*. **Table 1: Troubleshooting table** [Download Table 1](http://www.nature.com/protocolexchange/system/uploads/2170/original/Table_1.pdf?1338951762) **Table 2: Microanalysis results** [Download Table 2](http://www.nature.com/protocolexchange/system/uploads/2171/original/Table_2.pdf?1338951848) **Full corrected version of the protocol: Copolymerization preparation of cationic cyclodextrin chiral stationary phases for drug enantioseparation in chromatography** [Download Full corrected version of the protocol](http://www.nature.com/protocolexchange/system/uploads/2178/original/protex.2012.023_-_full_text.doc?1339152323) ### Associated Publications 1. **Cationic cyclodextrins chemically-bonded chiral stationary phases for high-performance liquid chromatography**. Ren-Qi Wang, Teng-Teng Ong, Weihua Tang, and Siu-Choon Ng. *Analytica Chimica Acta* 718 () 121 - 129 [doi:10.1016/j.aca.2011.12.063](http://dx.doi.org/10.1016/j.aca.2011.12.063) - **Chemically bonded cationic β-cyclodextrin derivatives as chiral stationary phases for enantioseparation applications**. Ren-Qi Wang, Teng-Teng Ong, and Siu-Choon Ng. *Tetrahedron Letters* 53 (18) 2312 - 2315 [doi:10.1016/j.tetlet.2012.02.105](http://dx.doi.org/10.1016/j.tetlet.2012.02.105) - **Chemically bonded cationic β-cyclodextrin derivatives and their applications in supercritical fluid chromatography**. Ren-Qi Wang, Teng-Teng Ong, and Siu-Choon Ng. *Journal of Chromatography A* 1224 () 97 - 103 [doi:10.1016/j.chroma.2011.12.053](http://dx.doi.org/10.1016/j.chroma.2011.12.053) ### Author information **Ren-Qi Wang, Teng-Teng Ong, Ke Huang & Siu-Choon Ng**, Division of Chemical and Biomolecular Engineering, College of Engineering, Nanyang Technological University, 16 Nanyang Drive, Singapore 637722, Singapore **Weihua Tang**, Key Laboratory of Soft Chemistry and Functional Materials (Ministry of Education of China), Nanjing University of Science and Technology, Nanjing 210094, People’s Republic of China Correspondence to: Ren-Qi Wang (wangrenqi@gmail.com) Siu-Choon Ng (ngsc@ntu.edu.sg) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2409) (2012) doi:10.1038/protex.2012.023. Originally published online 6 June 2012*.

Authors: Pierre Brat, Jean Christophe Valmalette, Christian Mertz, George de Sousa, Aviv Dombrovsky, Maria Capovilla & Alain Robichon ### Abstract Carotenoids are compounds synthesized in plants, bacteria and fungi, closely associated to the chlorophyll to perform photosynthesis. A spectacular evolutionary achievement allowed the aphid to produce carotenoids obviously by lateral transfer of genes from fungi. We have recently documented that these molecules are involved in photo conditioning of metabolism such ATP synthesis in this insect model. The carotenoid synthesis in aphid was directly determined in living insects without extraction procedure by Raman imaging technique and the analysis was compared between insects raised in distinct environments. In contrast with the Raman spectrum that provides information on molecular motifs recognition (conjugated double bonds) but does not discriminate between isomers and ester forms, the mass spectrometry technology allowed us to analyze finely the variations in carotenoid composition. In parallel to these spectral analysis and chemistry determination, series of dosage of ATP were performed in order to correlate carotenoid synthesis and metabolism in aphid. ### Introduction Carotenoids are constituted of five joined isoprenoid units. These highly unsaturated and lipophilic molecules are very unstable, due to their susceptibility to isomerize and/or to oxidize rapidly during the analytical procedures. Thus, precautionary measures to avoid formation of artifacts and quantitative losses should be standard practice in the laboratory. Therefore, the major focus should be to complete the analysis within the shortest timing, to avoid reaction with oxygen and acids, to protect samples from light and high temperature. The use of high purity solvents that are free from harmful impurities is a strong recommendation (Rodriguez-Amaya, 2001). Oxygen, especially in combination with light and heat, is highly destructive for carotenoid molecules. Exposure to sunlight and/or ultraviolet light induces trans/cis photoisomerization and photo-cleavage of carotenoids. Thus, the experimental work must be done under red light and/or in dark. The frequent errors and drawbacks commonly associated with the extraction, identification and quantification of carotenoids are carefully pinpointed in the Procedures in the next section. To allow the reliability of the results, each analysis was performed in triplicate. More precisely, the following points should be considered: - The amount of extracted matrix must be representative of the biological material. - Before extraction, the matrix is stored at -80°C but not the extract which is very sensible to oxidation (when the extracted samples are thawed/frozen repeatedly). - The efficiency of extraction should be complete (see Troubleshooting section). - The amount of loss during the different steps of extraction should be reduced by cautious handling of samples. - The chromatographic separation must be adapted to the specific carotenoid distribution. - The mass spectrometry data and literature information should be combined to limit the failure of identification. In parallel with this analytical procedure aimed to identify the repertoire of the carotenoid family, a direct molecular imaging is performed using the Raman imaging spectrometry and living animals without previous extraction. Although this does not allow us to identify specifically each isomer of the carotenoid repertoire, a global quantification can be done based on common molecular motifs generating specific Raman profiles. This procedure is non-destructive and allows researchers to visualize the molecules of interest in their cellular and/or organ context. ### Reagents All solvents were of HPLC grade, purchased from Carlo Erba (Val de Reuil, France) or Sigma-Aldrich (Steinheim, Germany). ATP dosage was performed with the kit FLASC purchased from Sigma Aldrich, based on quantification of emitted visible light proportional to the concentration of ATP in the reaction medium. List of molecules and organic solvents used for the mass spectrometry analysis (LC-MS): 1. Ethanol RPE-ACS (Carlo Erba, cat. n° 4146312) - Methanol RPE-ACS (Carlo Erba, cat. n° 414816) - Hexane RPE-ACS (Carlo Erba, cat. n° 446903) - Dichloromethane RS HPLC stabilized with ethanol (Carlo Erba, cat. n° 412662) - Methyl tert-butyl ether (MTBE) (Carlo Erba, cat. n° 432032) - Ammonium acetate RPE-ACS (Carlo Erba, cat. n° 418776) - Magnesium carbonate RPE (MgCO3) (Carlo Erba, cat. n° 459285) - Butylated hydroxytoluene (BHT) (Aldrich, cat. n° B1378) - Sodium chloride ≥ 99.8% (Sigma-Aldrich, cat. n° 31434) - Anhydrous sulphate sodium ≥ 99.0% (Sigma-Aldrich, cat. n° 31481) - β-carotene standard ≥ 98% (Extrasynthese, cat. n° 0303 S) ### Equipment For mass spectrometry analysis: 1. Pipetman (Gilson, P-20, P-200, P-1000) and pipette tips - Filter funnel porosity n°2 (vol. 50 mL) - Glass cotton - Separatory funnel (vol. 100 mL) - Rotary evaporator (T°C bath = 40°C) - HPLC apparatus coupled with a photodiode array detector (UV-visible) - HPLC apparatus coupled with a LCQ mass spectrometer fitted with an electrospray interface - C30 column (250 X 4.6 mm, 5 µm particle size) (YMC EUROP, GmbH). For Raman imaging analysis, the equipment was a spectrophotometer Labram HR800 Horiba Jobin-Yvon. An ion laser beam was focused on the immobilized aphids (legs were cut or alternatively fixed with glue) on a glass slide by using a 100x objective (NA 0.9) and/or a 50x LWD objective (NA=0.45) depending on the depth of the analysis of the biological sample. Raman back scattered light is collected by the same objectives. Then, we estimated the analyzed area to about 1 μm square with a 100x objective and about 10 μm square with a 50x objective. ### Procedure **1) Mass spectrometry analysis**. The β-carotene standard amount (Extrasynthese, cat n° 0303 S) is very low (~ 1 mg). To overcome these limitations, the concentration of the initial solution was determined by measuring its specific absorbance in hexane solution at 451 nm and using the extinction coefficient of β-carotene (ε1%) in hexane (= 2592). The successive dilutions of the initial solution were then prepared in hexane and injected into HPLC. The β-carotene chromatographic profiles were used to standardize the concentrations. The initial β-carotene solution was kept in amber vial under nitrogen atmosphere at -80°C for a maximum of one week. - CRITICAL STEP 1: For each carotenoid analysis, one gram of aphids was hand-milled using liquid nitrogen. Aphids were weighed before milling and then the resulting powder was carefully recovered. We notice that the water condensation on the aphid cuticle after defrosting tends to bias the weighing. - CRITICAL STEP 2: Carotenoids are light sensible. Consequently all the extractions and standard solution preparations are performed under red-light. Furthermore, these compounds being easily oxidized, the extracts are injected immediately into HPLC column. - CRITICAL STEP 3: Carotenoid extracts dissolved in dichloromethane / [MTBE/methanol], v/v) (see below: Step S9) were not filtrated before HPLC analysis. The solvent mixture is extremely volatile and filtration would considerably reduce the volume of extract. - CRITICAL STEP 4: Carotenoids are also highly heat sensitive. The temperature along the full process and particularly the concentration step in the water bath should not exceed 40°C. - CRITICAL STEP 5: Carotenoids being easily oxidized particularly when extracted out of the matrix, we advice to keep these molecules under their native form in insects stored at -80°C instead of freezing the extract. *GENERAL EXTRACTION AND ANALYTICAL PROCEDURE*. The procedure was adapted from the article published by Taungbodhitham et al. (1998), Dhuique-Mayer et al. (2005) and Mertz et al. (2010). Each analysis was made in triplicate. The major points of carotenoid analysis are the sampling and sample preparation, extraction, partition/solubilization in appropriate solvents depending on their polar/apolar properties, washing, evaporation of solvents, chromatographic separation, and finally at the end of this process, identification and quantification of the carotenoid compounds. *1st Step: extraction and solvent preparation*. - Prepare 15 mL of solvent A: ethanol (20 mL)/hexane (15 mL) containing 35 mg of BHT as antioxidant. - Prepare 80 mL of 10% sodium chloride. *2nd Step: extraction and purification steps*. This entire step was conducted under red light until transfer in an amber vial. Figure 1 presents the main steps of this procedure. - S1: weight 1 g of aphids (balance precision ± 0.01 mg). - S2: carefully milled the aphids in a mortar pestle with liquid nitrogen (very low T°C breaks easily the cuticle of the insects). - S3: in a 50 mL beaker, add 15 mL of solvent A and 80 mg of MgCO3 to neutralize the acidity of the mixture and stir for 5 min. - S4: filtrate the mixture on a filter funnel n°2 and wash the precipitate successively with 15 mL of solvent A, 15 mL of ethanol, then with 15 mL of hexane to recover most of lipophilic compounds. - S5: transfer the solvent mixture in a separatory funnel and washed: - S5-1: once with 40 mL of 10% sodium chloride (salt enhances the ionic strength of the solution. The hydrophilic compounds are concentrated in the water phase). - S5-2: twice with 40 mL of distilled water to rinse the eventual salty traces. - S6: recover the hexanic phase in a beaker. - S7: dry the phase with 1 g of sodium sulphate and filtrate on a cotton glass in a 100 mL conical ball. - S8: evaporate the dried organic phase using a rotavapor with a T°C of the water bath not exceeding 40°C. - S9: the residue is recovered with 250 µL of dichloromethane and 250 µL of MTBE/methanol (80:20, v/v). These solvents being very volatile, it is important to pipette immediately in an amber vial. - S10: the extract is injected quickly on HPLC for analysis purpose. *HPLC-MS ANALYSIS OF CAROTENOIDS*. - Carotenoids separation with a C30 column (250 X 4.6 mm, 5 µm particle size). - Gradient solvent program is presented Table 1. Injection volume: 10 µL, UV-vis. Detection: 250- 600 nm. Acetate ammonium (20 mM) was added in solvent A and B to favor ionization of the molecules in the ESI chamber. - In order to get simultaneously UV-visible and MS data, after passing through the flow cell of the diode array detector, the column eluate was split and 0.5 ml was directed to the ion trap of the LCQ mass spectrometer. - MS experiments in (+) ion mode, scan range: 100–2000 amu, scan rate: 1 scan/s and temperature for dissolving: 250 °C. *HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY QUANTIFICATION OF CAROTENOIDS*. - Column and gradient conditions (Table 1, below) were the same as used in mass spectrometry analysis. Ammonium acetate was not added. - Injection volume: 20 µL, absorbance followed at 450 nm. - All carotenoids concentrations are expressed in β-carotene equivalent (standard solutions injected at 450 nm). These data are summarized in figure 1. **2) Raman imaging spectrometry**. Raman spectrometry provides a fingerprint of molecules specific of its global structure and/or some of its motifs (Roman et al. (2010). The intensity of the peaks is proportional to the quantity of molecules which generate them and can be determined in cell context without destructive extraction process. Raman imaging spectrometry can be also used for a spatial visualization of molecules in tissue allowing the analysis of molecular dynamics. Briefly, υ(C=C) 1500 – 1900 cm-1 and υ(C≅C) 2100 – 2250 cm-1 and υ(C−Η) 2800 – 3000 cm-1 give strong Raman signal. Three peaks specify carotenoid molecules in Raman analysis: C-CH3 (1000 cm-1), C-C (1150 cm-1), C=C(1500 cm-1). Different ratios were determined: the ratio C-C/C=C; C=C (torulene/carotene); C-C / C=C; C-CH3 / ½ (C-C + C=C; ); C-CH3/C-C and C-CH3/C=C. Considering that the Raman peaks result from inelastic scattering involving vibrational levels of molecules, the baseline is related to radiative relaxation processes giving rise to fluorescence. The latter phenomenon strongly depends on the excitation wavelength and the interaction of molecules with its environment (Figure 3). **3) ATP dosage of individual aphid extract in a complex population**. The dosage was conducted according to the recommendation of the company and is based on the visible light production obtained with an equal amount of protein in tested samples (Figure 4). **4) Maintenance and propagation of aphids: selection of aphid variants synthesizing carotenoid molecules**. The pea aphid Acyrthosiphon pisum (Homoptera order, Aphididae family) are raised on the Vicia faba plant. Aphids were maintained in cages in a Sayo incubator at about 22˚C ± 3˚C and/or at 8°C ± 1°C, humidity 60% and with a photoperiodicity of 16/8 hours light/dark. Three colored phenotypes are the orange, the white and the green. Basically the white phenotype emerged when the plants are declining and food resources are rare. The orange phenotype is dominant in optimal temperature and food resources. Finally the green phenotype was selected over 5 months by placing 10 orange adults each day at 8°C ± 1°C. The orange phenotype was not viable in these conditions (orange larvae died at the stage 3 or 4), but the selected green variant turned out to be robust at this temperature. Placing back the green variant at room temperature results in the fast fading of the green pigment in each individual and its disappearance in approximately two days (aphids become orange again). ### Timing - Mass spectrum analysis: two days work. - Raman analysis: about one minute for single point analysis and a few hours for mapping. - ATP dosage: few hours. ### References 1. Dhuique-Mayer, C., Caris-Veyrat, C., Ollitraut, P., Curk, F., and Amiot, M. J. Varietal and interspecific influence on micronutrient contents in citrus from the mediterranean area. *J Agric Food Chem* 53, 2140-5 (2005). - Mertz C., Brat, P., Caris-Veyrat C., and Gunata Z. Characterization and thermal lability of carotenoids and vitamin C of tamarillo fruit (Solanum betaceum Cav.). *Food Chem* 119, 653-9 (2010). - Taungbodhitham, A. K., Jones, G. P., Walhlqvist, M. L., and Briggs, D. R. Evaluation of method for the analysis of carotenoids in fruits and vegetables. *Food Chem* 63, 577-84 (1998). - Rodriguez-Amaya D. B. *A guide to carotenoids analysis in foods*. ILSI Press International Life Sciences Institute. Washington USA (2001). - Romann, J., Valmalette, J.C., Chevallier, V., and Merlen, V. Surface Interactions between Molecules and Nanocrystals in Copper Oxalate Nanostructures, *J. Phys. Chem. C*. 114, 24, 10677 (2010). ### Figures **Figure 1 : Scheme to identify carotenoid compounds**.  *The scheme represents the rationale of the procedure detailed in the text in order to identify molecular components of the carotenoid family extracted from aphids*. **Figure 2: Microscopic photographs of aphids realized with the Raman equipment.**  *A, B and C represent an adult eye of aphid, an aptere and winged adult orange aphid, and embryos after dissection of the abdomen of a parthenogenetic mother, respectively. 1, 2 and 3 represent the microscopic photographs realized with the Raman equipment (Horiba scientific) that were obtained with an extract of a crashed adult aphid (we see lipidic droplets and crystals). The laser beam of the Ramam spectrometer is focused on each droplet and/or crystals. 4, 5 and 6 represent a germarium, an ovariole and detailed embryos on which the laser beam is focused, respectively. 7 represents the eyes of an embryo. These photographs, except for B and C, were realized with the microscope of Raman spectrometer to guide the laser beam.* **Figure 3: Raman spectrum of a crystal of carotenoid compounds**.  *The green spectrum was obtained with the laser beam focused on a crystal (spectrum obtained with the excitation in the absorption band of carotene – 488 nm). The other spectra were obtained with other parts of the aphid extract (droplets, germarium, young embryos, etc…)*. **Figure 4: Heterogeneity in a population of aphids and correlation with ATP amounts**.  *A and B represent a random analysis of distinct individuals represented in C and D, respectively. This protocol highlights the heterogeneity in a population of aphids obtained on the same plant and in the same conditions. ATP amounts correlate with the intensity of the orange color. The scheme in E summarizes the interaction between the environmental stress (red arrow) and the emergence of an induced phenotype two generations later (red stars). Clonal aphids propagate in telescopic generations with the first and second generations of embryos in the same mother like russian dolls The carotenoid content in individual aphids depends on this genetic scenario*. **Table 1: Gradient program of carotenoids elution**.  *HPLC analysis of carotenoid molecules. (a) For HPLC-MS analysis, ammonium acetate at 20 mM is added to the solvent*. **Troubleshooting: Troubleshooting in mass spectrum and Raman analysis**.  ### Associated Publications **Light- induced electron transfer and ATP synthesis in a carotene synthesizing insect**. Jean Christophe Valmalette, Aviv Dombrovsky, Pierre Brat, Christian Mertz, Maria Capovilla, and Alain Robichon. *Scientific Reports* 2 () 16/08/2012 [doi:10.1038/srep00579](http://dx.doi.org/10.1038/srep00579) ### Author information **Pierre Brat & Christian Mertz**, UMR QualiSud, CIRAD Monptellier, Montpellier, France **Jean Christophe Valmalette**, UMR7334 CNRS, Université du Sud-Toulon-Var, Toulon, France **George de Sousa, Maria Capovilla & Alain Robichon**, UMR7254 INRA/CNRS/UNS, Institut Sophia Agrobiotech, Sophia Antipolis, France **Aviv Dombrovsky**, The Volcani Center Institute, Bet Dagan, Israel Correspondence to: Pierre Brat (brat@cirad.fr) Alain Robichon (Alain.Robichon@sophia.inra.fr) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2474) (2012) doi:10.1038/protex.2012.047. Originally published online 10 October 2012*.

Authors: Afzal Hussain, Iqbal Hussain, Mohamed F. Al-Ajmi & Imran Ali ### Abstract Small peptides (di-, tri-, tetra- penta- hexa etc. and peptides) control many chemical and biological processes. The biological importance of stereomers of peptides is of great value. The stereo-separations of peptides are gaining importance in biological and medicinal sciences and pharmaceutical industries. There is a great need of experimental protocols of stereo-separations of peptides. The various chiral selector used were polysaccharides, cyclodextrins, Pirkle’s types, macrocyclic antibiotics, crown ethers, ligand exchange, etc. The attempts have been made to develop stereo-separations protocols for peptides using capillary electrophoretic and chromatographic techniques. In addition to these, the optimization strategies of stereo-separations were also discussed in the details. The efforts are also made to discuss the future perspectives of peptides stereo-separations. ### Introduction Its 21st century scientists are attempting to provide the best lives to the society. Medication is one of the most important aspects in our lives. Chirality in the drugs is a complex phenomenon creating confusion in medications. The demand of chiral drugs is increasing constantly due to different pahramaceutical activities of drugs enantiomers. One enantiomers may be active while the other inactive, toxic or ballast; leading to various side effects and problems [1]. It is because of chiral nature of our biological systems. Mostly biological reactions are stereo-selective because of different enantioselective distribution rates, metabolisms, excretion and clearances of enantiomers. Due to these facts, scientists, clinicians, industrialists, academicians and government authorities are asking data for optically active drugs and other biological important molecules. US FDA, Health Canada, European Committee for Proprietary Medicinal Products and Pharmaceutical and Medical Devices Agencies of Japan, have banned the marketing of all racemic drugs [2-4]. Small peptides (monomers n < 6) are of great importance because of contribution in various biological processes. The biological activities of small peptides include protein synthesis, fertility, neurotransmission, inflammation process, pathogenic microorganisms activities and other functions of human beings. These functions made peptide vital molecules in drug development and health care [5-7]. Besides, these peptides are also being used as biological markers in the biological systems [8]. The small peptides are also considered as important molecules in food and nutrition industries. For examples, aspartame, carnosine, etc. are being prepared at industrial scale [7]. It is important to mention here that biological functions of peptides are stereoselective; especially related to enzymatic reactions. In view of these facts, stereo-separation of small peptides is very important in drugs development and health care. Stereo-separation of peptides may be achieved by capillary electrophoresis and chromatography. Literature has many papers on stereo-separation of peptides [9-12]. Recently, Ali et al. [13] reviewed stereo-separations of small peptides by capillary electrophoresis and chromatography techniques. It was observed that all these papers contain sufficient information on stereo-separations of peptides but no one describes the experimental procedures, methods development and optimization strategies in details, which are urgently required at laboratory level globally. Therefore, the attempts have been made to describe a protocol for stereo-separations of peptides by capillary electrophoresis and chromatography. The present article describes the state-of-the-art of stereo-separations of peptides using capillary electrophoresis, chromatography. The efforts have been made to discuss optimization strategies and future perspectives of stereo-separations of peptides. ### Materials 1. All solvents and reagents should be of HPLC and AR grades - Optically active pure and racemic peptides standards - Deionised water - Acetonitrile - Methanol - Reagents for the preparation of phosphate, acetate and borate buffers. - Required chiral selectors - Acids and bases for pH adjustment ### Equipment 1. Capillary Electrophoresis Instrument - Personal computer (PC) for data acquisition - Fused silica capillaries (~ 50 cm effective length with 50 or 75 μm inner diameter) - Special capillary cutting blade - pH meter - UV–Vis. Spectrometer - Degasification unit - Filtration unit - Micro balance ### Procedure 1. Prepare stock solutions of peptides (optically active pure and racemic) in water (0.1 mg/mL). - Prepare required BGE and dissolve suitable and appropriate amount of chiral selector in it. - Protocols given in Figure 2 should be used before selecting and preparing BGE. - Filter through 0.45 μm membrane and degas by sonication. - Rinse the capillary for 5 min. with 0.5 M NaOH followed by 10 min with deionize water. - Fill BGE in CE reservoirs and dip the ends of capillary into them. - Rinse capillary for 5 min with BGE. - Inject racemic peptides sample solutions. - Apply appropriate potential and run CE instrument. - Among the measurements, rinse capillary for 2 min with BGE from time to time. - Optimize the stereo-separations. - Identify the resolved enantiomers by running standard pure optically active stereomers. - Wash capillary by deionised water before stopping HPLC instrument. - Calculate capillary electrophoretic parameters using standard equations [14]. - Determine the qualitative and quantitative stereo-separations. ### Timing - Rinse the capillary for 5 min. with 0.5 M NaOH followed by 10 min with deionize water. - Rinse capillary for 5 min with BGE - Among the measurements, rinse capillary for 2 min with BGE from time to time. ### Troubleshooting 1. CE is gaining importance in stereo-separations of peptides. - CE instrument has two injection modes i.e. electrokinetic and pressure injection modes. - pH meter should be calibrated using pH 4.0 and pH 10.0 standards. - First filter about 10 mL of deionized water in order to remove any impurities from filtration unit. - pH of electrolyte is a crucial parameter and should be adjusted as per the requirements. - The addition of organic modifiers improves the stereo-separations. - Generally, organic modifiers are toxic to health. - Care should be taken to avoid skin contact, inhalation and swallowing. - Organic modifiers should be handled with cautions using gloves, glasses, etc. - These organic modifiers should be stored in cool, dry and well ventilated places. - Both ends of capillary should be sealed by heating; if instrument is kept for long time. ### Anticipated Results Stereo-separation of small peptides is a growing research area since early 1990s. The metabolism of D-amino acid containing peptides stimulated stereo-separations of peptides The stereo-separations methods are gaining advancement with respect to time. For example, switching from indirect methods to direct stereo-separations and a gradual replacement of protocols requiring derivatization of samples to the analysis of underivatized peptides are the advancements. The cyclodextrines and macrocyclic antibiotics are the most commonly used chiral selectors. However, in recent time polysaccharides and other chiral selectors have been used. During last few years, hyphenation of CE and HPLC with MS detectors, 2D-LC [57] and the development of miniaturized analytical devices are other developments [58]. Reducing analysis time and complication of analyzed samples have economic impact [59]. Fast speed UPLC instrument has not been used in chiral chromatograph1 of peptides. But UPLC is used for stereo-separations of derivatized amino acids [60]. Therefore, it is expected that UPLC may acquire a good position in stereoseparations of peptide. The stereo-separation of peptides diastereomers is important in physiological researches and industries. This is due to the fact that multi-components, multistereoisomer mixtures are found in the food and pharmaceutical industries. Such situation will expand industries with respect to enantiopure peptides. Keeping these facts into consideration, the stereo-separations of peptides at preparative scale is the requirement of today. Literature survey indicates only one report at preparative scale [61]. Therefore, there is a great need for stereo-separation of peptides at preparative scale. Really, optically active pure di- and tri-peptides can be obtained by chiral synthetic methods but not larger peptides. This is a niche for preparative chromatographic methods. It is assumed that more attention will be drawn to the area in pharmaceutical industries for peptide stereo-separations. ### References 1. Aboul-Enein, H.Y., & Ali, I., *Chiral separations by liquid chromatography and related technologies* (Marcel Dekker, Inc., New York, USA, 2003). - Simonyi, M., Fitos, I., Visy, J., Chirality of bioactive agents in protein binding storage and transport process. *Tips*, 7, 112-116. (1986). - FDA policy statement for the development of new stereoisomeric drugs, Publication Date: 05/01/1992. - Ali, I., Gaitonde, V.D., Aboul-Enein, H.Y. Hussain, A., Chiral separation of β-adrenergic blockers on cellucoat column by HPLC. *Talanta* 78, 458-463 (2009). - Humphrey, M.J., Ringrose, P.S., Peptides and related drugs: A review of their adsorption, metabolism, and excretion. *Drug Met. Rev*. 17, 283-310 (1986). - Prasad, C. Bioactive cyclic dipetides. *Pept*. 16, 151-164 (1995). - Yagasaki, M., Hashimoto, S. Synthesis and application of dipeptides; current status and perspectives. *Appl. Microbiol. Biotechnol*. 81, 13-22 (2008). - Soga, T., Sugimoto, M., Honma, M., Mori, M., Igarashi, K., Kashikura, K., Ikeda, S., Hirayama, A., Yamamoto, T., Yoshida, H., Otsuka, M., Tsuji, S., Yatomi,Y., Sakuragawa, T., Watanabe, H., Nihei, K., Saito, T., Kawata, S., Suzuki, H., Tomita, M., Suematsu, M. Serum metabolomics reveals γ-glutamyl dipeptides as biomarkers for discrimination among different forms of liver disease. *J. Hepatol*. 55, 896-905 (2011). - Scriba, G.K.E. Recent advances in enantioseparations of peptides by capillary electrophoresis. *Electrophoresis* 24, 4063-4077 (2003). - Czerwenka, C., Lindner, W. Stereoselective peptide analysis. *Anal. Bioanal. Chem*. 382, 599-638 (2005). - Schmid, M.G. Chiral metal-ion complexes for enantioseparation by capillary electrophoresis and capillary electrochromatography: A selective review. *J. Chromatogr. A* 1267, 10-16 (2012) - Kasicka Kašička, V. Recent developments in CE and CEC of peptides (2009-2011). *Electrophoresis* 33, 48-73 (2012). - Imran Ali, Zeid A. Al-Othman, Abdulrahman Al-Warthan, Leonid Asnin, Alexander Chudinov, Advances in chiral separations of small peptides by capillary electrophoresis and chromatography, *J. Sepn. Sci*. In Press (2014). - Imran Ali and Hassan Y. Aboul-Enein, *Chiral pollutants: Distribution, toxicity and analysis by chromatography and capillary electrophoresis* (J. Wiley & Sons, Chichester, UK, 2004). - Neue, U.D. Peak capacity in unidimensional chromatography. *J. Chromatogr. A* 1184, 107- 130 (2008). - Czerwenka, Ch., Lämmerhofer, M., Lindner, W. Structure- enantioselectivity relationships for the study of chiral recognition in peptide enantiomer separation on cinchona alkaloid-based chiral stationary phases by HPLC: Influence of the N-terminal protecting group. *J. Sep. Sci*. 26, 1499-1508 (2003). - Hirayama, C., Hirayama, H., Ihara, K., Tanaka Chromatographic resolution of dipeptide enantiomers and diastereomers on chiral stationary phases from poly-(L-leucine) or poly(Lphenylalanine) *J. Chromatogr*. 450, 271-276 (1988). - Chen, S. High performance liquid chromatographic enantioseparation of benzoyl amino acids, dipeptides and tripeptide on β-cyclodextrin bonded stationary phase using polarorganic acetonitrile as the mobile phase. *J. Chin. Chem. Soc*. 46, 239-244 (1999). - Tonoi, T., Nishikawa, A., Yajima, T., Nagano, H., Mikami, K. Fluorous substituent-based enantiomer and diastereomer separation: Orthogonal use of HPLC columns for the synthesis of nonproteinogenic polyfluoro amino acids and peptides. *Eur. J. Org. hem*. 1331-1335 (2008). - Wan, H., Blomberg, L.G. Enantioseparation of amino acids and dipeptides using vancomycin as chiral selector in capillary electrophoresis. *Electrophoresis* 17, 1938-1944 (1996). - Wan, H., Blomberg, L.G. Chiral separation of DL-peptides and enantioselective interactions between teicoplanin and D-peptides in apillary electrophoresis. *Electrophoresis* 18, 943-949 (1997). - Sabah, S., Scriba, G.K.E. Electrophoretic stereoisomer separation of aspartyl dipeptides and tripeptides in untreated fused-silica and polyacrylamide-coated capillaries using charged cyclodextrins. *J. Chromatogr. A* 822, 137-145 (1998). - Suß, F., Poppitz, W., Sanger-van de Griend, C.E., Scriba, G.K.E. Influence of the amino acid sequence and nature of the cyclodextrin on the separation of small peptide enantiomers by capillary electrophoresis using randomly substituted and single isomer sulfated and sulfonated cyclodextrins. *Electrophoresis* 22, 2416-2423 (2001). - Sabah, S., Scriba, G.K.E. Resolution of aspartyl dipeptide and tripeptide stereoisomers by capillary electrophoresis. *J. Microcolumn* Sep 10, 255-258 (1998). - Sabbah, S., Scriba, G.K.E. Separation of dipeptide and tripeptide enantiomers in capillary electrophoresis using carboxymethyl-β-cyclodextrin and succinyl-β-cyclodextrin: Influence of the amino acid sequence, nature of the cyclodextrin and pH. *Electrophoresis* 22, 1385- 1393 (2001). - Verleysen, K., Sabah, S., Scriba, G.K.E., Chen, A., Sandra, A. Evaluation of the enantioselective possibilities of sulfated cyclodextrins for the separation of aspartyl di- and tripeptides in capillary electrophoresis. *J. Chromatogr. A* 824, 91-97 (1998). - Verleysen, K., Sabah, S., Scriba, G., Sandra, P. Enantioseparation of aspartyl dipeptides by CE: Comparison between 18-crown-6-tetracarboxylic acid and carboxymethyl-β-cyclodextrin as chiral selector. *Chromatographia* 49, 215-218 (1999). - Sidamonidze, N., Süß, F., Poppitz, W., Scriba, G.K.E. Influence of the amino acid sequence and nature of the cyclodextrin on the separation of small peptide enantiomers by capillary electrophoresis using α-, β-, and γ-cyclodextrin and the corresponding hydroxypropyl derivatives. *J. Sep. Sci*. 24, 777-783 (2001). - Sänger van de Griend, C.E. Enantiomeric separation of alanyl and leucyl dipeptides by capillary electrophoresis with cyclodextrins as chiral selectors. *Electrophoresis* 21, 2397-2404 (2000). - Sungthong, B., Ivanyi R., Bunz, S.C., Neusu, C., Scriba, G.K.E. CE-MS characterization of negatively charged α-, β- and γ-CD derivatives and their application to the separation of dipeptide and tripeptide enantiomers by CE. *Electrophoresis* 31, 1498-1505 (2010). - Wan, H., Blomberg, L.G. Enantiomeric separation of small chiral peptides by capillary electrophoresis. *J. Chromatogr. A* 758, 303-311 (1997). - Scriba, G.K.E. Recent developments in peptide tereoisomer separations by capillary electromigration techniques. *Electrophoresis* 30, S222- S228 (2009). - Dubsky, P., Svobodová, J., Tesařová, E., Gaš, B. Enhanced selectivity in CZE multi-chiral selector enantioseparation systems: Proposed separation mechanism. *Electrophoresis* 31,1435-1441 (2010). - Gong, X.Y., Dobrunz, D., Kümin, M., Wiesner, M., Revell, J.D., Wennemers, H., Hauser P.C. Separating stereoisomers of di-, tri-, and tetrapeptides using capillary electrophoresis with contactless conductivity detection. *J. Sep. Sci*. 31, 565-573 (2008). - Wernisch S, Lindner W. Versatility of cinchona-based zwitterionic chiral stationary phases: Enantiomer and diastereomer separations of non-protected oligopeptides utilizing a multimodal chiral recognition mechanism. *J Chromatogr A* 1269, 297-307 (2012). - Francotte, E.R. Enantioselective chromatography as a powerful alternative for the preparation of drug enantiomers. *J. Chromatogr. A* 906, 379-397 (2001). - Fornstedt, T., Sajonz, P., Guiochon, G. A closer study of chiral retention mechanisms. *Chirality* 10, 375-381(1998). - Vespalec, R., Bocek, P. Chiral Separations in Capillary Electrophoresis. *Chem. Rev*. 100, 3715-3754 (2000). - Wren, S.A.C., Rowe, R.C. Theoretical aspects of chiral separation in capillary electrophoresis. I. Initial evaluation of a model. *J. Chromatogr. A* 603, 235-241 (1992). - Rawjee, Y.Y, Staerk, D.U., Vigh, G. Capillary electrophoretic chiral separations with cyclodextrin additives: I. acids: chiral selectivity as a function of pH and the concentration of β-cyclodextrin for fenoprofen and ibuprofen. *J. Chromatogr. A* 635, 291-306 (1993). - Xia, S., Zhang, L., Lu, M., Qiu, B., Chi,Y., Chen, G. Enantiomeric separation of chiral dipeptides by CE-ESI-MS employing a partial filling technique with chiral crown ether. *Electrophoresis* 30, 2837-2844 (2009). - Wan, H., Blomberg, L.G. Enantiomeric separation of small chiral peptides by capillary electrophoresis. *J. Chromatogr. A*. 792, 393-400 (1997). - Xu, W., Nakagama, T., chiyama, K., Hobo,T. Enantioseparation of aromatic dipeptides using carboxymethyl-β-cyclodextrin polymer as chiral selector by capillary electrophoresis. *Anal. Lett*. 33, 1147-1165 (2000). - Scriba, G.K.E. *Differentiation of enantiomers by capillary electrophoresis* (Springer Berlin Heidelberg, 2013). - Hammitzsch-Wiedemann, M., Scriba, G.K.E. Mathematical Approach by a Selectivity Model for Rationalization of pH- and Selector Concentration-Dependent Reversal of the Enantiomer Migration Order in Capillary Electrophoresis. *Anal. Chem*. 81, 8765-8773 (2009) - Schmid, M.G., Gübitz, G. Capillary zone electrophoretic separation of the enantiomers of dipeptides based on host-guest complexation with a chiral crown ether. *J. Chromatogr. A*. 709, 81-88 (1995). - Salami, M., Jira, T., Otto, H.H., Capillary electrophoretic separation of enantiomers of amino acids and amino acid derivatives using crown ether and cyclodextrin. *Pharmazie*. 60, 181-185 (2005). - Waibel, B., Scheiber, J., Meier, C., Hammitzsch, M., Baumann, K., Scriba, G.K.E., Holzgrabe, U. Comparison of Cyclodextrin-Dipeptide Inclusion Complexes in the Absence and Presence of Urea by Means of Capillary Electrophoresis, Nuclear Magnetic Resonance and Molecular Modeling. *Eur. J. Org. Chem*. 2007, 2921-2930 (2007). - Uwe Conrad, Bezhan Chankvetadze, Gerhard K. E. Scriba, High performance liquid chromatographic separation of dipeptide and tripeptide enantiomers using a chiral crown ether stationary phase, *J. Sep. Sci*. 28, 2275-2281 (2005). - Ekborg-Ott, K.H., Liu,Y., Armstrong, D.W., Highly enantioselective HPLC separations using the covalently bonded macrocyclic antibiotic, ristocetin, a chiral stationary phase, *Chirality* 10, 434 (1998). - Asnin, L., Sharma, K., Park, S.W., Chromatographic retention and thermodynamics of adsorption of dipeptides on a chiral crown ether stationary phase. *J. Sep. Sci*. 34, 3136-3144 (2011). - Schmid, M.G., Schreiner, K., Reisinger, D., Gubitz, G., Fast chiral separation by ligandexchange HPLC using a dynamically coated monolithic column. *J. Sep. Sci*. 29, 1470-1475 (2006). - Schmid, M.G., Hölbling, M., Schnedlitz, N., Gübitz, G., Enantioseparation of dipeptides and tripeptides by micro-HPLC comparing teicoplanin and teicoplanin aglycone as chiral selectors. *J. Biochem. Biophys. Methods* 61, 1-10 (2004). - Czerwenka, Ch., Lämmerhofer, M., Lindner,W., Micro-HPLC and standard-size HPLC for the separation of peptide stereoisomers employing an ion-exchange principle. *J. Pharm. Biomed. Anal*. 30, 1789-1800 (2003). - Bai, L., Sheeley, S., Sweedler, J.V. Analysis of endogenous D-amino acid-containing peptides in Metazoa. *Bioanal. Rev*. 1, 7-24 (2009). - Winn, M., Goss, R.J.M., Kimura, K., Bugg, T.D.H. Antimicrobial nucleoside antibiotics targeting cell wall assembly: Recent advances in structure–function studies and nucleoside biosynthesis. *Nat. Prod. Rep*. 27, 279-304 (2010). - Czerwenka Ch., Maver, N.M., Linder, W. Liquid chromatographic–mass spectrometric separation of oligoalanine peptide stereoisomers: influence of absolute configuration on enantioselectivity and two-dimensional separation of diastereomers and enantiomers. *J., Chromatogr*. A 1038, 85-95 (2004). - Ali, I, Aboul-Enein, H.Y. & Gupta, V.K. *Nano Chromatography and Capillary Electrophoresis: Pharmaceutical and Environmental Analyses* (Wiley & Sons, Hoboken, USA, 2009). - Bazylak, G. Reversed-phase high-performance liquid chromatography of the stereoisomers of some sweetener peptides with a helical nickel(II) chelate in the mobile phase. *J. Chromatogr*. A 668, 519-527 (1994). - Xiao, Y., Tan, T.T.Y., Ng, S.C. Enantioseparation of dansyl amino acids by ultra-high pressure liquid chromatography using cationic β-cyclodextrins as chiral additives. *Analyst* 136, 1433-1439 (2011). - Florance, J., Galdes, A., Konteatis, Z., Kosarych, Z., Langer, K., Martucci, C. Improvement of chemical analysis of antibiotics : XIII. Systematic simultaneous analysis of residual tetracyclines in animal tissues using thin-layer and high-performance liquid chromatography. *J. Chromatogr*. 414, 313-322 (1987). ### Acknowledgements The author would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University, Riyadh, Saudi Arabia for its funding this research group No. RGP-150. **Figure 1: Diastereomers of N-(α-Aspartyl)-phenylalanine. LL-aspartame is sweet**.  **Figure 2: Protocol for development and optimization of CE conditions for chiral resolution**.  **Figure 3: Protocol for development and optimization of mobile phases on polysaccharides CSPs under normal phase mode.**  **Figure 4: Protocol for development and optimization of mobile phases on polysaccharides CSPs under reversed phase mode.**  **Figure 5: Protocol for development and optimization of normal mobile phases on CDs based CSPs under normal phase mode.** ![Figure 5](http://i.imgur.com/bf95iHz.png Figure 5") **Figure 6: The Protocol for development and optimization of normal mobile phases on CDs based CSPs under reversed phase mode.**  **Figure 7: The protocol for the development and optimization of mobile phases on CDs based CSPs under polar organic phase mode.**  ### Author Information Afzal Hussain, College of Pharmacy, King Saud University Iqbal Hussain & Mohamed F. Al-Ajmi, Unaffiliated Imran Ali, Department of Chemistry, Jamia Millia Islamia, New Delhi, India Correspondence to: Imran Ali ([drimran_ali@yahoo.com, drimran.chiral@gmail.com](drimran_ali@yahoo.com, drimran.chiral@gmail.com)) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/3455). Originally published online 31 October 2014*.

Authors: David Hevia, Aida Rodriguez-Garcia, Marta Alonso-Gervós, Isabel Quirós-González, Henar M Cimadevilla, Carmen Gómez-Cordovés, Rosa M Sainz & Juan C Mayo ### Abstract The protocol reported here describes a simple, easy, fast and reproducible method aimed to know the geometric parameters of living cells based on confocal laser scanning microscopy combined with 3D reconstruction software. Briefly, the method is based on intrinsic fluorescence properties of acridine orange (AO), a molecule taken up by living adherent cells. Dual binding of AO to either DNA or RNA allows complete staining. When combined with confocal microscopy, 3D software can be used for in vivo living cell reconstruction. Beside the purpose that we intend here, a fast and easy system for cell volume determination, the protocol is an easy approach to study changes in morphology during cellular processes such as cell differentiation. Novel therapeutic approaches would require some knowledge about how these drugs enter into cells/tissues. For this purpose fast and accurate in vivo cell volume determinations such as the method reported here, in combination with analytical methods, would allow estimating intracellular concentrations of compounds and might be further employed for finding out whether any new drug can reach the effective concentration inside its cellular target. Furthermore this protocol with minimal adjustments will permit the determination of morphometric parameters in vivo in different types of adherent cells. ### Introduction It is widely accepted that confocal laser scanning microscopy (CLSM) and other derivative techniques (e.g. FRET) are currently the state-of-the-art image technologies in microscopy. Because of the top quality and high definition of cell images provided, CLSM is therefore used for many applications including immunocytochemical detection, nuclear or other organelle localization of target proteins, FRET or any other fluorophore-based technique for different purposes in which high resolution images of cells are required, even in living cells and tissues (1,2). Nonetheless in addition to its cell image capabilities, this powerful technique has opened a broad range of new image-related possibilities to solve biological questions (3). Therefore confocal microscopy is a great tool in order to obtain three-dimensional object images using two-dimensional optical sectioning plus 3D reconstruction (4). For this purpose, image processing software has evolved at such a high speed during the last few years that it has made possible to manage, combine and process digital images in a very short time. Accurate cell volume determination is extremely useful for many Cell Biology techniques, including morphometric studies, physiological studies (5) or estimation of intracellular concentration of substances (6). Interestingly there are not many simple and straightforward methodological approaches for the estimation of volume especially in living cells. Furthermore many of those methods often requires very specific technology (7), use of isotopes (8) or non-friendly software that requires a time-consuming, long-learning curve. This sometimes discourages users from employing such methods for morphometric analysis. As we suggest here, internalization of targeted drugs could be easily monitored and quantified using both a simple protocol for volume determination as well as parallel analytical methods for quantification of substances inside cells. Therefore combination of these powerful techniques, confocal microscopy and HPLC, which are available for most of laboratories, is the key for these types of pharmacokinetic studies. With this goal in mind, the aim was to develop an easy protocol to obtain cell images for volume determination but maintaining them in their own growth media so osmotic pressure and therefore potential morphological changes are minimized. Consequently cell volume estimated under this paradigm is more realistic since cell manipulation is minimized. **ADVANTAGES AND DISADVANTAGES OF OUR METHOD** Flow cytometry can also be employed to determine with accuracy cell volume using fluorescent latex particles with a known diameter as standards (9). Even though this method appears as a good choice in some cases, cells processed for flow cytometry are submitted to some handling prior to injection (i.e. centrifugation) which can alter the normal volume that they exhibit in cell culture plates, so real values may differ. Furthermore prior to flow cytometer analysis cells are commonly detached from substrate and centrifuged which always induces round shape morphology with changes in real cell volume. However, the method we propose here, using CLSM combined with 3D image processing software and cell cultures, is very simple and fast for measuring geometric parameters of cells and would open new possibilities of knowledge. ### Reagents **REAGENTS** 1. NaCl (Sigma, Cat. N°. S3014) - KCl (Sigma, Cat. N°. P4504) - Na2HPO4 (Sigma, Cat. N°. S5136) - KH2PO4(Sigma , Cat. N°. P5655) - HCl (Sigma, Cat. N°. H1758) - HEPES (Sigma, Cat. N°. H4034) - Ultraglutamine 1, 200 mM in 0,85% NaCl solution (Lonza, Cat. N°. BE17-605E/U1) - Antibiotic-antimycotic 100x (GIBCO, Invitrogen, Cat. N°. 15240) - Ampicillin sodium salt (SIGMA- ALDRICH, Cat. N°. A9518-56) - Kanamycin B sulfate salt (Sigma, Cat. N°. B5264-250M6) - Trypsin, 0.25% (1X) with EDTA (Invitrogen, Cat. N°. 25200-072) - Fetal Bovine Serum E.C. Approved (GIBCO, Invitrogen, Cat. N°. 10106) - Acridine Orange (Calbiochem, MERK, Cat. N°. 113000-1GM) - Mounting medium for microscope preparation EUKITT (Aname, Cat. N°. RT15320) - Inmersion liquid, type F (Leica microsystem, GmbH, Cat. N°. 11513859 **Cell culture** 1. Corning 75 cm2 Flask, Canted Neck (BD Biosciences, Cat. N°. H108CO2836) - Cell Culture Plate, 6-well, (BD Falcon™, Cat. N°. 353046) - Cell Culture Coverslip, Sterile, Thermanox plastic, 13 mm diameter (NUNC™ Brand Products, Cat. N°. 174950) - Microscope Slides, 76×26 mm (Menzel-Gläser, Cat. N°. AA00000112E) - Microscope Cover Slips, 24×36 mm (Menzel-Gläser, Cat. N°. BB024036A1) - Counting Chamber Improved Neubauer (BRAND, Cat. N°. 717805) - Sterile Pipette 10 ml (APL, Cat. N°. PN10E1), 1 ml (APL, Cat. N°. PN1E1) - Micropipette PIPETMAN P1000 (Gilson, Cat. N°. F123602), P100 (Gilson, Cat. N°. F123615), P10 (Gilson, Cat. N°. F144802) - Syringe Filter, Acrodisc (PAAL corporation , Cat. N°. PN 4433) - Syringe 5ml, luer slip (BD Discardit , Cat. N°. 309050) - Universal yellow tip 5-200 μl, blue tip 100-1000 μl, tip 1-5 ml (Daslab, Cat. - N°. 162001, 162222, 162005) and tip 0,1-10 μl (Deltalab, Cat. N°. 200024) - Microtubes MCT-150-C 1,5 ml (Axygen Quality, Cat. N°. 311-08-051 ) - Conical tube 15 ml (17×120 mm) (BD Falcon, Cat. N°. 352096) - Conical tube 50 ml (30×115 mm) (BD Falcon, Cat. N°. 352070) - Pasteur Pipettes – soda glass (230mm) (Deltalab, Cat. N°. 702) - Manual Counter cash. 4 digits (Quirumed, Cat. N°. 052-63047001) - Tweezers, style 62A (ANAME, Cat. N°. 78350-62A) **REAGENTS SETUP** 1. Ampicillin stock 500x: Dissolve 500 mg of ampicillin powder in milli-Q H2O to a final volume of 100 ml. Filter-sterilized and store 1 ml aliquots at – 20ºC. - Kanamicyn stock 500x: Dissolve 500 mg of kanamicyn powder in milli-Q H2O to a final volume of 100 ml. Filter-sterilized and store 1 ml aliquots at -20ºC - HEPES stock 100x: Dissolve 26.029 g of HEPES and adjust to a final volume of 500 ml with milli-Q H2O and filter sterilize. Store at 4°C. - Acridine Orange solution: Dissolve 20 mg of acridine orange in 20 ml of PBS and then filter it and keep in the dark to avoid light. Before using, dilute it until 40 μg/ml in PBS. In order to obtain final concentration 10 μl of this solution is added to cultured medium. - Phosphate saline buffer (PBS): Dissolve 0.2 g of KCl , 0.2 g of KH2PO4, 8 g of NaCl , 1.15 g of Na2HPO4 in 1 L of milli-Q H2O and filter sterilize. **Cell lines** 1. Mouse embryonic fibroblast cell line (NIH3T3) (ATCC, CRL-1658) - Normal human prostate epithelium immortalized with SV40 cells (PNT1A) (Sigma 95012614) - Chinese hamster ovary cell line (CHO) (ATCC, CCL-61) - Androgen-sensitive human prostate adenocarcinoma cell line (LNCaP) (Sigma-Aldrich, 89110211) - Human prostate carcinoma cell line (DU 145) (ATCC , HTB-81) - Androgen-insensitive human adenocarcinoma cell line (PC3) (ATCC, CRL-1435) - Human breast adenocarcinoma cells (MCF7) (ATCC, HTB-22) - Human epithelial cervical carcinoma cell line (HeLa) (Sigma Aldrich, 93021013) - Murine melanoma cell line (B16-F10) (ATCC, CRL-6475) - Mouse leukemic monocyte macrophage cell line (RAW 264.7) (ATCC , TIB-71) - Rat glioma cell line (C6) (ATCC, CCL-107). **Growth media and supplements** 1. RPMI 1640 (Lonza, Cat. N°.BE12-167F sterile filtered) supplemented with 10% - Fetal Bovine Serum (FBS), 2 mM Ultraglutamine, 0,1% Ampicillin, 0,1% kanamycin and 15 mM HEPES. - DMEM (Dulbecco´s Modified Eagle´s Medium with 1 g/L Glucose, without L-Glutamine)(Lonza, Cat. N°.BER-707F) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM Ultraglutamine, 15 mM HEPES and 1% GIBCO antibiotic-antimycotic cocktail. - DMEM/F12 (Dulbecco´s Modified Eagle´s Medium, Ham´s F-12 1:1 mix with 15 mM Hepes and L-Glutamine) (Lonza, Cat. N°.BE12-719F) supplemented with 10% Fetal Bovine Serum (FBS) and 1% GIBCO antibiotic-antimycotic cocktail. ### Equipment **EQUIPMENT SETUP** 1. Cell culture incubator with both, temperature and gas composition controls, was set at 37°C and 5% CO2, (New Brunswick and Eppendorf Company, Cat. N°. 00170S-230-1000) - Biosafety cabinet suitable for cell culture and equipped with UV light for decontamination (Polaris, Cat. N°. 11339) - Vacuum pump ROCKER 300 (Rocker, Cat. N°. 167300-22) - Thermostatic bath Model IDL-AG12 (Labolan, Cat. N°. 506012) - Inverted contrasting microscope for living cell applications Leica DM IL (Leica) - Centrifuge 5810 R, bench top centrifuge, without rotor, refrigerated (Eppendorf, Cat. N°. 5811 000.010) Fixed-angle rotor F-34-6-38 for Centrifuges 5804/5804 R and Centrifuges 5810/5810 R (Eppendorf, Cat. N°. 5804727.002). Swing-bucket rotor A-4-62 for Centrifuges 5810/5810 R (Eppendorf , Cat. N°. 5810 709.008). - Confocal microscopy (Leica TCS AOBS SP2)-40x oil immersion objective (NA 1.25-0.75) was used and images were acquired using a 496 nm argon/krypton ion laser. The acridine orange signals were detected at 506-590 nm. Z-series profiles of an average of 20 optical sections were collected at intervals of 1 μm with a line average of 2 to reduce noise. ### Procedure - **Culture of cells-TIMING 30 min** 1. Harvest cells growing in monolayer in T-75 ml cell culture flasks. Add 5.0 ml of trypsin-EDTA to each plate and incubate at room temperature for 4 min. Add 5.0 ml of RPMI 1640 with 10% FBS and 25 mM HEPES cell culture medium and transfer cell suspension into a 15 ml conical tube. - Pellet suspended cells in 15 ml conical tubes using a centrifuge for 5 min at 500g. Re-suspend each cell pellet in 2-3 ml of media and perform cell counts. - Place a coverslip inside culture plates (6 well-plate) with sterilized tweezers. - CRITICAL STEP. It is very important that all coverslips used for growing cells must be carefully handled to avoid contamination. Caution should also be taken when place the growing side (usually only one side is treated for cell attachment) of the coverslip correctly. - Complete media is then added to culture plates (2 ml) without dropping media over the coverslips to ensure that cells keep attached to the surface. - Cells are seeded at the appropriate final density (50,000 cells/ml but may vary depending on cell type/size). After adding cells, plates are slowly shaken to homogenize cell distribution. **? TROUBLESHOOTING** - Cells were left growing for 48 hours inside cell incubator. - **Staining with fluorescence molecule- TIMING 20 minutes** - Add 2 ml of fresh RPMI medium with 0.2% FBS in new 6 well plates. Three of the wells are used for washing (PBS-glucose) while the other can be used employed for staining. - Add 20 µl of AO solution to RPMI medium of three plates and shake to homogenize. - Using sterilized tweezers place each one of the coverslips containing cells in one of the wells with washing solution for 1 minute and then take coverslips into wells for staining during 10 minutes at RT. - After this time pipet 100 µl of RPMI medium with 0.2% FBS on a clean slide and then place coverslips with cells over medium upside-down. Then mounting medium is used as adhesive, using it only in the external limits of coverslip to avoid auto-fluorescence. - Immediately after mounting place slide with one drop of immersion oil under the confocal microscopy. **? TROUBLESHOOTING** - POINT WITHOUT PAUSE- It is strongly required to start collecting images from confocal microscope immediately after mounting. Therefore we do recommend mounting slides in the same room where confocal analysis is done. - **Capturing images with confocal microscopy** - Run the software with the setting up indicated above (see equipment setup). - Search area of interest to include as many individual cells as possible. - Indicate the coordinates to make the slices for analyzing. - Establish 1 µm depth slices and store images for further processing - To ensure reproducibility, repeat the process described in steps 12-14 until a number over 100 cells is reached. - **Image data preparation** - Run the ‘Imaris’ program for processing images. - In the menu ‘File’, select ‘Open’ and click in the first section image, so the software will open all section images taken. - In the new window that pops-up, select ‘volume’, ‘surface’ and then click on the `blend’ button . In the keyboard press ‘Ctrl+D’ and a new window pops-up. In this new window select “auto blend” and then close this window. - Now, select ‘surface’ and in the flag of ‘create’ press two times blue bottom of play. - In this step it is possible to modify the absolute intensity but we rather recommend to click the ‘automatic mode’ so we minimize manipulation of images. - Click again on the ‘play’ button and a new window will pops-up filled with several lines corresponding to the different cell volumes. You must move the threshold line up in order to eliminate those objects that correspond to incomplete cells with too low volumes or “cut-off” cells on both sides of image selected. Then click again on the blue button to finish these steps. - Select tab labeled as ‘pencil’ and click on ‘select’ for selecting all those incomplete cells that were impossible to eliminate in the previous step. For eliminating those cells just select them and then ‘delete’. **? TROUBLESHOOTING** - When working with specific cell types, many of them grow either in clusters or they form groups in which many of the cells remain bound one to each other. When this happens, it is necessary to cut between two or more cells. However it is quite easy to identify the binding area because usually it appears as a “valley” just between adjacent cells thus indicating the joining point. Consequently user has to choose these points and make the appropriate cuttings for obtaining accurate measurements. **? TROUBLESHOOTING** - Once user has completed this part of the software image processing of all cells, all data have been acquired by computer. To obtain data, select tab labeled as ‘statistic’ and then ‘detailed’ and choose the desired parameter such as ‘volume’, ‘surface’ or ‘sphericity’. A table containing data will then appear. Just click on any of the data displayed and then the corresponding cell image will appear, so user can easily correlate any of the data with the cellular source. - Data can be easily exported to an excel datasheet by selecting in the same tab the ‘save’ command. A new window will then appear with the corresponding data. - *OPTIONAL STEP*; It is recommended to save a copy of image using the ‘snapshot’ command. This will save pictures as a ‘tiff’ file which can be further used under any other program for treating images (i.e. Photoshop, Paint Shop, etc…). This would allow users to compare and correlate data with cells using other programs with a more intuitive interface. ### Timing - Culture of cells-TIMING 30 min-48h (Steps 1-6) - Staining with fluorescence molecule-TIMING 20 min (Steps 7-11) - Capturing images with confocal microscopy -TIMING 15 min (Steps 12-16). Without repetition - Image data preparation -TIMING 20 min (Steps 17-27. Without repetition. ### Troubleshooting - PROBLEM: Step 5 and 24-Density of cultured cells. Too many/few cells appear at final magnification. - SOLUTION: The density of cells is crucial at this point since the main objective is the real morphology of cells and therefore the treatment of images coming from real cells. To do so it is highly recommended to seed a few cells initially so image handling is easier when cell density is much below confluence. If cell density is near 90-100% it becomes difficult to isolate cells without cutting joints between cells. Otherwise it would be time consuming and not easy to obtain clean images and reproducibility is also lower. - PROBLEM: Step 11-Speed too low while manipulating coverslips and confocal microscopy. - SOLUTION: At this step when confocal microscopy is ready if operator is quick enough it is then feasible to scan more than five fields in the same coverslip in a minimum time. Otherwise, if all these steps are performed too slowly, only 2-3 fields can be analyzed and more coverslip are required to scan to obtain optimal results. The stopping point is indicated by the presence of bubbles or any other visual artifact, as this is an indicator that cells are not kept under normal conditions. - PROBLEM: Step 14 and 23-Height of cells out of range. - SOLUTION: When choosing low and high limits in z-axis for cell screening (step 14), some of the cells are placed too up or down and consequently the whole cell is not contained in the gap. In these cases cells should not be considered for quantifying and operator must eliminate them manually at step 23. - PROBLEM: Step 17 to 27-Treatment of images1. - SOLUTION: Although it might be deduced that these steps depend highly on operator, following a few indications ensures that the whole process becomes more reproducible. By comparing two independent observers here we demonstrate that differences obtained in cells parameters after the whole process is completed are below 1%. To this aim it is very important that all steps are set in automatic mode. If operator changes intensity manually in the software, care should be taken so intensity must be changed accordingly for all the measurements, otherwise process is subjective. On the contrary, setting mode to ‘automatic’, measurements are performed equally in all the cells. All these steps are further explained in a tutorial video shown as supplementary material video 1. ### Anticipated Results For several researchers it may be very important to know exactly the intracellular concentrations of exogenous and endogenous compounds, proteins, DNA, RNA or any others molecules of interest. Currently there are several analytical applications for estimating the presence and the amount of virtually any molecule inside cells (11). On the contrary, scientists do not have simple tools to estimate cell volume. To fill this gap, the primordial objective in this work was to create an easy, reproducible and accurate method to calculate volume of cells. Thus, in combination with HPLC or any other analytical method, the protocol shown here will allow estimation of intracellular concentration of molecules. Results with data of volumes from different cell types using the protocol reported here are shown in table 1. As it can be seen in table 1, it is remarkable the differences in cell volumes measured in different cell types. In some cases, differences of volume can be observed at microscopy level because cell diameter is the major source of variation. However, in other cell types diameter do not vary significantly and differences observed are rather due to cell height so our protocol would be especially useful for the latter. Protocol is easy to perform with most cells. Other cell types like murine macrophages RAW 264.7 cells offer particular difficulties. RAW 264.7 macrophages are easily activated by LPS or other factors including high cell density. When cells are activated they change dramatically in morphology which can be easily followed up under microscope. This particular feature was also identified using our method as it can be seen in table 2. In this case we observed that when RAW 264.7 cells were cultured at a density near 70% a high error in cell volume was noticed. This was due to both morphology and volume changes. When those “activated” cells are identified and processed apart (table 2, see RAWc), the volume of the rest cells (RAW^b ) are virtually the same that in other tests (see table 1 for comparing). Furthermore with this data we can assure that morphology changes can be identified and even quantified. Furthermore, although we have mainly emphasized the use of this method for cell volume determination, it is possible to quantify other geometric parameters such as surface area or sphericity. In the latter case, sphericity, it can be used for quantifying morphological changes in which exogenous stimuli trigger changes in morphology or for quantifying differences of morphology between cell types. Figure 3 shows an example of how the protocol may illustrate and quantify the differences in cell morphology by comparing the 3D image reconstruction. For this purpose, we have provided videos 2, 3, 4 and 5 as supplementary material, where differences in cell morphology between cell types can be observed. Furthermore video 6 shows a 360° rotation of PC3 cell around other cells. All data and supplementary material shown here demonstrates that the protocol shown here is a simple, fast and accurate tool for measuring different cell parameters in several cell types using a common CLSM which is accessible to most laboratories. More over in combination with other techniques available for many researchers, intracellular concentration of substances or any other comparative analysis of morphological changes can be easily performed. Currently many therapeutic approaches of how new drugs can be useful either in cancer, neurodegeneration or many other pathologies usually require some knowledge about how these substances enter into cells/tissues and what is the real concentration reached in those tagets. In fact the intracellular concentration truly discriminates between successful new drugs and the rest. However most researchers do not approach the possibility of establishing intracellular concentration of molecules because of the intrinsic difficulties in obtaining cell volume measurements. Therefore, the method proposed here may fill this gap and offers an easy and inexpensive way to overcome those difficulties. ### References 1. Conn, P. M. *Confocal microscopy*. (Academic Press, 1999). - Matsumoto, B. *Cell biological applications of confocal microscopy*. 2nd edn, (Academic Press, 2002). - Ntziachristos, V. Going deeper than microscopy: the optical imaging frontier in biology. *Nat Methods* 7, 603-614, (2010). - Luzzati, F., Fasolo, A. & Peretto, P. Combining confocal laser scanning microscopy with serial section reconstruction in the study of adult neurogenesis. *Front Neurosci* 5, 70, (2011). - Kiehl, T. R., Shen, D., Khattak, S. F., Jian Li, Z. & Sharfstein, S. T. Observations of cell size dynamics under osmotic stress. *Cytometry A*, (2011). - Hevia, D., Mayo, J. C., Quiros, I., Gomez-Cordoves, C. & Sainz, R. M. Monitoring intracellular melatonin levels in human prostate normal and cancer cells by HPLC. *Anal Bioanal Chem* 397, 1235-1244, (2010). - Korchev, Y. E. et al. Cell volume measurement using scanning ion conductance microscopy. *Biophys J* 78, 451-457, (2000). - Bennett, B. D., Yuan, J., Kimball, E. H. & Rabinowitz, J. D. Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach. *Nat Protoc* 3, 1299-1311, (2008). - Stewart, C. C. & Steinkamp, J. A. Quantitation of cell concentration using the flow cytometer. *Cytometry* 2, 238-243, (1982). - Krolenko, S. A., Adamyan, S. Y., Belyaeva, T. N. & Mozhenok, T. P. Acridine orange accumulation in acid organelles of normal and vacuolated frog skeletal muscle fibres. *Cell Biol Int* 30, 933-939, (2006). - Stadheim, T. A., Li, H., Kett, W., Burnina, I. N. & Gerngross, T. U. Use of high-performance anion exchange chromatography with pulsed amperometric detection for O-glycan determination in yeast. *Nat Protoc* 3, 1026-1031, (2008). ### Acknowledgements This work was supported by a grant from “Fondo de Investigación Sanitaria” (FISS), Instituto de Salud Carlos III (PS09/02204). D.H. acknowledges fellowship from JAE-DOC program (CSIC). A.R-G. is supported by a pre-doctoral fellowship from “Severo Ochoa” program (PCTI, Asturias). H.R-C. acknowledges support from “Manuel de la Oya” program (“Centro de Información Cerveza y Salud”). IUOPA is grateful for the support from “Obra Social Cajastur”. ### Figures **Figure 1: Test for potential error sources in the CLSM with 3D image processing method for determining geometric parameters in living cells** [Download Figure 1](http://www.nature.com/protocolexchange/system/uploads/2012/original/Figure_1.tif?1322829342) *A, Comparative fluorescence in PC3 cells stained with AO between scans 1 and 30 (top micrographs, x400) and the resulting graph comparing number of cells per field showing changes in surface area in each slide between -3.5% and 3.5% of total. B, PC-3 cell volume data from 30 PC3 cells using different width of slides (0.4-1.0 µm). C, Analysis of PC3 cells (n=4) when different objectives are used. D, Cell volume data from two cell lines, HeLa and C6 glioma cells, when the whole protocol is performed independently by two observers*. **Figure 2: Outline of method** [Download Figure 2](http://www.nature.com/protocolexchange/system/uploads/2024/original/Figure_2.tif?1323097526) *Diagram showing the simple workflow process for the method described here, from cell seeding to data processing and the corresponding timing*. **Figure 3: 3-D reconstruction images from PNT1A (A) and PC-3 (B) cells using the protocol reported here**. [Download Figure 3](http://www.nature.com/protocolexchange/system/uploads/2014/original/Figure_3.tif?1322829761) **Table 1: Cell volume** [Download Table 1](http://www.nature.com/protocolexchange/system/uploads/2015/original/Table_1.tif?1323095833) *Cell volume (µm3) as estimated by the protocol reported here, using several common cell lines. Mean volume, SEM and the number of cells used for each determination are shown*. **Table 2: Differences observed in cell volume data from the same cell line**. [Download Table 2](http://www.nature.com/protocolexchange/system/uploads/2016/original/Table_2.tif?1323095909) - *a, whole RAW 264.7 cell population*. - *b, subpopulation of not-activated RAW 264.7 cells*. - *c, subpopulation of activated RAW 264.7 cells only* **Video 1: Image data preparation** [Download Video 1](http://www.nature.com/protocolexchange/system/uploads/2017/original/Video_1.wmv?1323096743) *Steps for processing imaging (steps 17-26)* **Video 2: LNCaP cell** [Download Video 2](http://www.nature.com/protocolexchange/system/uploads/2018/original/Video_2-LNCaP_cell.mov?1323096848) **Video 3: PC3 cell** [Download Video 3](http://www.nature.com/protocolexchange/system/uploads/2019/original/Video_3-PC3_cell.mov?1323096933) **Video 4: CHO cell** [Download Video 4](http://www.nature.com/protocolexchange/system/uploads/2020/original/Video_4-CHO_cell.mov?1323096993) **Video 5: PNT1A cell** [Download Video 5](http://www.nature.com/protocolexchange/system/uploads/2021/original/Video_5-PNT1A_cell.mov?1323097070) **Video 6: PC3 cells** [Download Video 6](http://www.nature.com/protocolexchange/system/uploads/2023/original/Video_6-PC3_cells.mov?1323097319) *Video 6 shows a 360° rotation of PC3 cell around other cells* ### Author information **David Hevia, Aida Rodriguez-Garcia, Marta Alonso-Gervós, Isabel Quirós-González, Henar M Cimadevilla, Carmen Gómez-Cordovés, Rosa M Sainz & Juan C Mayo**, Nutraceuticals and cancer Correspondence to: David Hevia (heviadavid@ifi.csic.es), Juan C Mayo (mayojuan@uniovi.es) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2264) (2011) doi:10.1038/protex.2011.272. Originally published online 8 December 2011*.

Authors: Anne-Leila Meistertzheim, Isabelle Calves, Sébastien Artigaud, Carolyn S. Friedman, Christine Paillard, Jean Laroche & Claude Ferec ### Abstract This protocol permits the mutation scanning of PCR products by high-resolution DNA melting analysis requiring the inclusion of a saturating intercalating dye in the PCR mix without labelled probe. During a scanning process, fluorescent melting curves of PCR amplicons are analyzed. Mutations modifying melting curve shapes, are allowed to be further characterized by sequencing because melting is not destructive. The method detects on small amplicons (120pb), 100% of heterozygous and 75% of homozygous variants in a single step. Homozygous variants are detected in a second time at 100% by adding wild-type reference DNA in the tube. For mitochondrial haplotypes, homozygous variants are discriminated by increased the size of the amplicons (700pb). HRMA can be conducted without knowledge on number of mutations, small insertions or deletions of the studied sequence in non-model species. The method accomplishes simultaneous gene scanning in a fraction of the time required when using traditional methods, while maintaining a closed-tube environment. The PCR requires <1h20 (96- or 384-well plates) and melting acquisition takes 10 min per plate. ### Introduction Melting curve analysis in combination with real-time PCR was introduced in 1997 (1,2) and is a natural extension of continuously monitored PCR within each cycle (3). During High resolution DNA melting analysis (HRM or HRMA), melting curves are produced using dyes that fluoresce in the presence of double-stranded DNA (dsDNA). Using specialized instruments designed to monitor fluorescence during heating; as the temperature increases, the fluorescence decreases, producing a characteristic melting profile (4). Each PCR product is identified by its temperature melting (Tm) corresponding to the temperature at which half of the double strand DNA is denatured. When the PCR product sequence is altered, duplex stability is changed, leading to different melting behavior. When the change is homozygous, a shift in melting temperature is usually observed (5). When the change is heterozygous, four duplexes are formed following PCR: two heteroduplexes and two homoduplexes. Each duplex will have differing stabilities, the sum of which can be observed by high-resolution melting analysis (see Fig. 1), enabling sequence variations to be detected. HRM analysis detects single nucleotide polymorphisms (SNPs) and small insertions or deletions in a fragment of amplified DNA by comparing the fluorescence as a function of the temperature. Several years after its first introduction, HRMA appears as a rapid method for genotyping known variants or scanning unknown variants (6,7), with its applications recently reviewed (8-11). Variations of HRMA have been developed to enhance resolution in small amplicons, the 3’ ends of a primer set placed at a very short distance from the informative SNP (12). Alternatively, unlabelled probe can be used when multiple informative SNPs are present within a longer stretch of sequence, but also when the GC content is low or when the presence of polymorphisms prevents the placement of HRMA primers for small amplicons. In this latter case, the Tm of the unlabelled probe, and not of the entire amplicon, is used for genotyping (13-15). However, the software available on HRMA instruments is an important element determining the ultimate sensitivity achieved (16), and not all packages yet facilitate the use of temperature calibration probes. HRMA presents a specificity of 98.8% with an overall sensitivity of 99.3% of the samples with one or more heterozygous loci distinguished from wild type10. Although HRMA heterozygote detection is excellent with a rate of 100% (17), successful discrimination between homozygous variants that melt in a single domain depends mostly on their Tm with an average detection rate of 75% from 6 prior studies using constitutional human variants (8,17,18). The ability to use absolute temperatures differences for genotyping depends on the temperature precision of the instrument (19) and the consistency (ionic strength) of compared samples (20). Interestingly, best homozygous variants detection rates of 93% and 96.5% were reported for human BRCA1 and mitochondrial DNA (21,22). In both cases, PCR products were longer than 500 pb. These results suggest that longer PCR products may be preferred for homozygote detection, while for heterozygote detection shorter PCR products showed better results (10,11). Another technique consists in creating an artificial heteroduplex by mixing wild type DNA with homozygous variants, converting homozygous variants into heterozygotes with an overall detection sensitivity of 96.9% (10,23). HRMA is simple, rapid, and inexpensive but depends strongly on the quality of the PCR, instruments and dyes. Different DNA isolation methods did not influence scanning accuracy (21). Although different reconstitution buffers can affect absolute Tms (20) also modified by salt, MgCl2 and dye concentrations used for the amplification. DNA concentrations could vary at least 4-fold without affecting melting results (20), but identical concentrations must be favored. Saturating DNA dyes are not required for some HRMA applications like methylation analysis (24). However, scanning and genotyping are entirely dependent on heteroduplex identification and different dyes are variably effective. For example, LCGreen® Plus detects heterozygotes better than SYTO® 9, which is better than EvaGreen®, which is in turn better than SYBR Green I (8). Pricing of the dyes differs significantly; all have slightly different characteristics, and they often demand slightly different PCR buffers and conditions. Best PCR product length was also studied on the HRTM-1(25) and LightScanner® Instruments (11), revealing more errors as the length increases above 400 bp. However the optimal number of melting domains remains controversial between authors who argue between one domain melting (18) and two or more domains (22). As the precision of qPCR and melting further improves with time, the number of replicates required decreases, making the approach even more accessible. Simultaneous scanning and genotyping allows better differentiation of multiple variants, the sequencing burden concerning only the different variants in large-scale screening projects. Firstly confined to clinical and diagnostic studies (10), this method appeared recently in wild population studies of fish populations (26,27), symbionts from the genus Symbiodinium (28) or Wolbachia using reference genotypes (29). As a closed-tube system, HRMA represents a sensitive inexpensive and fast technique comparatively to new generation sequencing (30) or other modern gene scanning methods such as single-strand conformational polymorphism analysis in capillary sequencer (31), denaturing high-performance liquid chromatography (32) or temperature gradient capillary electrophoresis (33). Each of these methods requires application of the PCR products onto a matrix to separate and detect the heteroduplexes. In contrast, closed-tube systems eliminate the need for automation, greatly decrease the risk of laboratory contamination from open PCR product and significantly reduce analysis time. Based on all these attributes, a wide use of HRMA would be expected; however, a literature review as of May 2012 revealed no hits of this technique in non-model species despite the increasing importance of SNPs in this field, except on four recent populations studies focused on a a salmon (27), swordfish (26), a dipter (34) and a plant (35). Here we used HRMA to genotype alleles in fish and shellfish populations. The entire procedure we propose of HRMA in combination with qPCR is completed within 1h20 as a single closed-tube assay in non-model species without SNPs knowledge required. In the same time this procedure allows the real-time monitoring of PCR quality and the scanning of 384 or 96 samples on 384- or 96-well plates on a LightCycler® 480 Instrument. Screened polymorphism of different genes is compared between the best dyes LCGreen® Plus dye in LightScanner® Instrument previsouly described (15) with the new Resolight® in LightCycler® 480 Instrument on two different marine species: an invertebrate, the abalone Haliotis tuberculata and a vertebrate, the fish Platichthys flesus. Experimental design There are several experimental design considerations that should be considered before initiating mutation screening using HRM, explained further below. DNA extraction. The DNA extraction method should be similar for all the DNA templates, as salts in the DNA template can influence the melting curve in the HRM analysis. This can be overcome to some extent by adding unlabelled probe as internal temperature standards (12), but this is both a laborious and to some degree expensive step. Regarding the quality and quantity of DNA, it is also better to add the exact amount of template DNA to the PCR before amplification, as the dynamic range of HRMA is closed (see FIRST RESULTS). The quantity of the DNA can be measured by OD measurements or by using commercially available kits for measuring double-stranded DNA (e.g., PICOgreen) or directly by Nanodrop technology. The quality of DNA must be assessed by Agilent or migration on 1% agarose gel. The DNA samples were further diluted with PCR grade water to a concentration of 15 ng/μL for use in qPCR. PCR design and optimization. For nuclear gene, PCR primers should be designed to amplify fragments of 150–400 bp and each amplicon should ideally span an exon with one or more melting domains identified by software such as Poland server (36). To PCR-amplify larger exons, it may be necessary to design primers that amplify several overlapping amplicons. As a significant loss of sensitivity has been shown for heterozygous variants detection when using long PCR amplicons, we therefore do not recommend the amplification of PCR products longer than 400 bp. Long products commonly have multiple melting domains and the ability to detect all the variants decrease with the number of domains. Short PCR products have usually only one domain and homozygous variants result in little if any shape change. However, we find it critical for high sensitivity that the melt profile contains not more than one or two melt domains. When assays are designed to type specific variants (SNP typing) we recommend fragment sizes of 80–100 bp. On the contrary, for mitochondrial genome screening, we recommend amplicons longer than 600 bp to identify homozygous variants (22) containing more than one domain and with mutations located at different parts of melt domains. The primers should hybridize a region without any known SNP (use appropriate SNP databases) and the subsequent PCR amplicon could include more than one SNP. Primers were designed to flank the coding regions, without second structure (hairpin or dimer formation) and to be annealed at 60°C using Primer Express software (Applied Biosystems, Foster City, CA). All primers should be HPLC-purified before use to ensure the correct size and quality of the primers. PCRs should be optimized using a temperature gradient to determinate the optimal annealing temperature for primer sets at which a specific and robust amplicon is obtained. As the primer Tms are around 60, first PCR should consist on a touchdown temperature from 65 to 53°C. Controls. At least one normal control must be used for comparison of melting curve patterns when unknown samples are analyzed (e.g., if a specimen is screened for mutations in several exons of a gene, one wild sample should be screened in the same exons for comparison). It should be noted that when larger series of sample are screened for the presence of different mutations, we recommend using representative known samples as references in each PCR to standardize the calibration between samples. Each sample with a peak pattern that differs from the normal controls should be sequenced to verify the presence of a mutation and to determine the exact nucleotide change. A negative ‘no-template’ control may be included in the PCR to detect potential DNA contamination. However, false-positive or false-negative results due to PCR carryover are usually not a problem in this assay, because each sample contains same amount of genomic DNA as starting material. Instrumentation and dyes. Different sets of fluorescent dyes are commercially available, al in the same spectral emission: SYTO® 9, LC Green® and more recently the Resolight® included in HRM Master Mix (Roche, Indianapolis, IN). This protocol describes HRM analysis using two different types of dyes and their corresponding instrument base procedures. The LC Green® on the LightScanner® Instrument (Roche, Indianapolis, IN) and the Resolight® on real time PCR, the LightCycler® 480 Instrument (Roche, Indianapolis, IN). These Instruments generate fluorescence data from 45–95°C at a temperature transition rate of 0.1 to 0.02°C/sec, and 22 to 25 acquisitions per °C for the LightScanner® and the LightCycler® 480 Instruments, respectively. The uses of these two dyes are slightly different. Resolight® presents the advantage to be less concentrated than LCGreen® PLUS dye that prevents the inhibitory effect of the intercalating fluorescent dye during PCR amplification. Thus, LightCycler® 480 Instrument allowed the visualization of the real-time amplification additionally to the melting curve analysis. In principle, only thermocycler intended for real-time PCR with more than 22 images per second can be used. Some alterations may be necessary when adapting the protocol to a different instrument. Data analysis. This is the most time-consuming part of the procedure and also the part that requires some practice. Data can be analyzed using a program designed for melting curves analysis which depends on the instrument used: data from LightCycler® 480 Instrument can be analyzed by LightCycler® 480 Gene Scanning Software SW 1.5.0 and also LightScanner® Software version 2.0 Call-IT (v2.0.0.1331;Idaho Technology) using the melt calibration module, previously adapted only for LightScanner® Instrument data. The normalization of the melting curve required to choose two ranges of temperatures corresponding to 100% and 0 % values of fluorescence (Fig. 2 A). In the area chosen, parallel curves must be observed. Another horizontal calibration called temperature shift can be done between all the samples (threshold of 5 per default, Fig. 2 B1). This normalization is made to compensate the dispersion between samples from same group and improve homogeneity of the group (Fig. 2 C1). Differential efficiency of the amplification between the samples in the 96-well plate could create a Tm shift of 1.5 °C between same variants. In the case of all the samples are similar (same DNA concentration and quality) and similar PCR amplification efficiency, this normalization is not required because disturbed the analysis of homozygous variants corresponding to peaks at different Tms. However, to use the temperature shift without loss of information identified homozygote samples can be mixed with wild type reference in a second time. Data are then compared to reference samples identified as standards and then converted to difference plots (6) (Fig. 2 C2). The temperature window for those specific genotyping tests is ranged from 50°C to 95°C. ### Reagents **DNA extraction** 1. Sodium chloride (NaCl; Merck, cat. no. 567441) - Cethyl trimethyl amonium bromide (CTAB; Amersco, cat. no. 0833) - Chloroform:isoamyl alcohol (24:1) (Uptima, cat. no. UP899255) ! CAUTION Irritant - Tris-HCl (Uptima, cat. no. UP091549) ! CAUTION Irritant - Tris Biotech.Grade (Uptima, cat. no. 031657) - Boric acid (Uptima, cat. co. UP070440) - EDTA (Sigma, cat. no. ED4SS) ! CAUTION Irritant. - Sodium acetate (NaOAC; Merck, cat. no. 567418) - Ethanol (Merck, cat. no. 1.00983.1000) - proteinase K, 10 % (wt/vol) (Sigma, T2308 500mg) - β-Mercaptoethanol (Merck, cat. no. 444203) ! CAUTION Hazardous. **DNA migration electrophoresis and purification for HRM by LightScannerTM products** 1. Ethidium bromide (Roth, cat. no. 2218.1) ! CAUTION Mutagenic properties. DNA ladder 100pb with loading buffer (Gena Biosciences, cat. no. M-214) - Agarose (Interchim, cat. no. 31292L) - 50X TAE buffer (Eppendorf, cat. no. 955155335) **High Resolution Melting** 1. HPLC-purified PCR primers - (1) LightScanner® Master Mix (Idaho Technology, cat. no. HRLS-ASY-003) and Mineral Oil Light (Sigma, cat. no. M5904) - or (2) LightCycler®480 High Resolution Melting Master (Roche, cat. no. 04 909 631 001) **REAGENT SETUP** - Δ CRITICAL. Prepare solutions in DNase-free glassware using autoclaved - DNA extraction buffer Mix 100mM Tris-HCl ph8, 20 mM EDTA pH8, 1.4 M NaCl, 2% CTAB, stable up to 1 month at RT. - Agarose gel Mix 1%(wt/vol) agarose dissolved in 0.6 X electrophoresis buffer. - Electrophoresis buffer (TBE 10X) Mix 89 mM Tris base, 89 mM Boric acid and 2 mM EDTA (pH 8.3); store at RT. ### Equipment 1. 2ml microcentrifuge tubes - Pipetman (Gilson, P-20, P-200 and P1000) - Pipette tips (Rainin) - Bath incubator - Thermo-fast ® 96, semi-skirted (Thermo scientific, cat. no. AB0990) - Thermocycler Gene Amp PCR System 7500 (ABI) - Gel tank for electrophoresis (Maxicell® EC360M) - UV light, camera (Vilber Lourmat) - LightScanner® Instrument (Roche) - LightCycler® 480 Instrument (Roche) - Nanodrop (MD-1000, Spectrophotometer) - Centrifuge with rotor for microtiter plates (e.g., Eppendorf 5804) ### Procedure **DNA extraction** ● TIMING 45 min to 4 h (depending on the number of samples) 1.Extract DNA from the tissue/organism of interest (e.g., muscle, gills or fin) using an appropriate DNA extraction method. We routinely use the CTAB (37) method for marine mollusks muscle, and the CCDB (38) method for the fish. - Δ CRITICAL STEP Any other kits/standard methods of DNA extraction could alternatively be used (see Experimental design). Nevertheless, when samples have to be compared with each other or with a control, all of them should be extracted using the same extraction method. 2.Resuspend the DNA in water. Determine DNA quantity and quality by the A260/A280 ratio using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE). For reliable results, and following the HRM manufacturer´s recommendations, a ratio of 1.8-2 was required. Adjust concentrations of DNA to 15ng/µL. **PCR amplification and HRM analysis** ● TIMING ~ 2 h 3.Perform PCR amplification using any conventional PCR amplification protocol appropriate for the chosen primer pair according to manufacture protocols. Include a negative control where no template DNA is added (optional) and positive control with known genotype. In the case of homozygous variants, template DNA is constituted by half of wild type reference and half of the unknown samples. For example, as in a standard PCR protocol for amplicons in the size range between 150-400 bps (10 µl reaction), combine the following reagents using option A or option B depending on the instrument used: 3.A.Using LightScanner Instrument: 2µL template DNA, 4 µL of 2.5X LightScanner® Master Mix, 10 pmol of each primer (HPLC-purified) and water up to 10 µL. Load PCR-Plate with 15µL mineral oil, seal with film and centrifuge briefly 1min at 2500 g. Use the following amplification protocol: 95°C 2 min, 40-45 cycles of 94°C 30 s, 60 °C 30 s, 72°C 40 s and a terminal cycle for heteroduplex formation 94°C 30s, 25-28°C 30s. - PAUSE POINT Products may be stored at this point at -20°C for at least 2 weeks. If plates are refrigerated, spin for 30 s at 1600g before analysis. Following PCRs, insert the 96-well plate into a LightScanner® Instrument to melt the samples with a continuous increase temperature starting at 50°C to 95°C with a continuous acquisition mode. Following the melt, use the LightScanner 2.0 software to manage and analyze the data. 3.B.Using LightCycler®480 Instrument To obtain LightCycler® 480 data: 2µL template DNA, 2.5 µL of 2X LightCycler® 480 High Resolution Melting Master, 1µL of primer mix at 4µM (HPLC-purified), 3mM MgCl2 and water up to 10 µL. Seal with film and centrifuge briefly at 2500 rpm. Use the following amplification protocol: 95°C 10 min, 45 cycles of 95°C 15 s, 60°C (or 65°C – 0.5°C per cycle until 53°C) 30 s, 72°C 30 s. High resolution melting protocol consists of heteroduplex formation 95°C 1 min, 40°C 1min following by a continuous increase temperature starting at 50°C to 95°C with a continuous acquisition mode. - PAUSE POINT Products may be stored at this point at -20°C for at least 2 weeks before sequencing. Following the melt, use the LightCycler® 480 Software release 1.5.0. with the Gene Scanning module to manage and analyze the data. ? TROUBLESHOOTING **Melting analysis** ● TIMING ~ 2-10 min 4.Verify the amplification efficiency and removed of the data any sample without amplification before analysis. For LightCycler® 480 data, samples presenting unusual or abnormal amplification curve can also be removed (Fig. 3). Standard amplification curves presents crossing point (Cp) or cycle threshold, Cp < 30 cycles or Cpsample =+/- 1,5 Cpwild type. ? TROUBLESHOOTING 5.Calibration (see Fig. 2) : Select two ranges of 1°C temperatures corresponding to 100% and 0 % fluorescence values when curves of all samples are parallel, before and after the fall curves (Fig. 2 A). Temperature shift: let the threshold per default at 5 (Fig. 2 B1). 6.Compare samples data to reference samples identified as standards (Fig. 2 C1) then convert to differences plots (Fig. 2 C2). Different resolution levels can be used to group the different variants: modify the level starting per default at 0.3. Melting peaks allowed visualizing different Tms of homozygous variants with the temperature shift engaged (Fig. 2 B2). For nuclear gene, homozygous variants can be discriminated from each other by adding wild type reference DNA to sample DNA in a second step and start over the HRMA (step 3). For mitochondrial genes, large amplicons can be directly discriminated by their melting peaks when ΔTm>0.2°C. ? THROUBLESHOOTING **Agarose gel electrophoresis (optional for LightCycler® 480 treatment)** ● TIMING ~ 1 h 7.Prepare agarose gel (1% (w/v) agarose in 1X TAE buffer). Add one drop of ethidium bromide 0.625 mg. mL-1 for every 50 ml gel. 8.Perform agarose gel electrophoresis to verify successful PCR amplification as follows: mix 5 µl PCR product with 1 µl 6X loading buffer and perform the electrophoresis for 20 min at 120 V. Visualize the DNA bands using a UV-transilluminator. Distinct single bands should be visible for each PCR amplification. - PAUSE POINT Products may be stored at this point at -20°C for at least 2 weeks. ? THROUBLESHOOTING **Purification (and/or cloning) of PCR products for variants sequencing** 9.To determine the exact nature of the genetic variation, purify PCR products directly after PCR for LightCycler® 480 procedure or from DNA bands after electrophoresis of the products for LightScanner procedure and subsequently perform DNA double pass-sequencing analysis for mitochondrial gene. For nuclear marker, purify qPCR products, tail with the *Escherichia coli* poly A polymerase (New England Biolabs, Ipswich, MA, USA) for LightCycler® 480 procedure, clone and perform DNA sequencing analysis of several clones to separate alleles. Alleles can also be separated by a Single Strand Conformation Polymorphism (SSCP) starting from PCR products. ### Troubleshooting Troubleshooting advice can be found in Table3. ### Anticipated Results Examples of results used for a genetic study of marine populations39 from a standard HRM assay using the LightScannerTM Instrument and the LightCycler® 480 Instruments are shown in Figure 3 and 4. Variants of nuclear genes like exon 1 of gene encoding ferritin from the abalone *Haliotis tuberculata* correspond to small size amplicons (A-B), whereas variants of exon 2 of gene encoding myoadenylate deaminase from the teleost fish *Platichthys flesus* correspond to large sequences with multiple melting domains (C-D). Melting peaks allow visual differentiations of curves comforted with normalized and temp-shifted difference plots representing the data normalized by reference samples. Homozygote sample usually produces only one narrow melting peak corresponding to only one double-stranded DNA fragment (see Fig. 3 A1-B1), and single narrow peaks at different Tms could correspond to homozygous variants (green and blue melting peaks, Fig. 3 A1-B1). In contrast, heterozygous variants display broad double melting peaks (red line, Fig. 3 A1-B1), similar to heteroduplex forms in the case of mixed homozygote with wild type DNA. During scanning process 100% of heterozygous and 75% of homozygous variants are expected to be detected. Homozygous variants are detected in a second time at 100% by adding wild type reference DNA in the tube. The peak patterns of mixed of DNA amplification (homozygote with wild type) are reproducible. The location of more than one SNP within the same PCR product usually displays a complex variation of peak patterns across a series of samples (see Fig. 3 C1-D1). However, some distinct peaks display very close Tms (<0.2°C) that could correspond to only one mutation (A/T; Table 1). Peak heights depend on the concentration of the DNA used for the PCR. Variable amplification efficiencies of a single sequence modify the Tm of the peaks in a range of 0.5°C, a level which discriminated supplementary variants for a single SNP (blue and yellow melting peaks Fig. 3 A1-B1). Homogeneity among variant groups on small size amplicons is similar between LightCycler® 480 and LightScanner data, whereas more advantages clearly appear when mutation scanning is realized using LightCycler® 480 Instrument and chemistry (see Fig. 3). Firstly, the Resolight© dye allows the amplification of amplicons with more than two melting-domains compared to the LC Green©. Amplification of particular long sequences with several SNPs permits to differentiate mitochondrial sequences without heteroduplexes formation as observed for a 689 bp region of the Cytochrome C Oxidase sub-unit 1 (CO1) gene with 3 SNPs in P. flesus (see Fig. 4)39. This chemistry increased also the Δfluorescence between the normalized and Temp-shifted difference plots (Fig. 3 A2-B2). On LightCycler® 480 data, plots representing relative signal differences lower than 4 could be grouped (Fig. 3 B2). Secondly, primer-dimers or unspecific priming in the PCR may occasionally result in additional background peaks which are reduced when using Resolight©. Thirdly, amplification and HRMA steps are performed on a single step, without opening the tube. Finally on the LightCycler® 480 Instrument, variable amplification efficiencies between variants are directly assessed during the real-time PCR and negative samples are identified (Fig. 5). We recommend considering two samples represented by their melting peaks with Tm shift of 0.2°C as two distinct variants, whatever the instrument and chemistry. All variants identified in 96-well plates have to be sequenced to determine the exact nature of the genetic variation. We also recommend sequencing samples with aberrant peaks. In marine populations, number of sequences is reduced from 250 and 290 samples to only 10 and 18 variants per marker for a vertebrate and invertebrate species, respectively. In conclusion, HRMA allows the efficient scanning of 96 (up to 384 for the LightCycler® 480 Instrument) samples and reduces drastically the number of samples to sequence in a cheap, fast and reproducible manner compared to other modern gene scanning methods (Table 2). ### References 1. Ririe, K.M., Rasmussen, R.P., and Wittwer, C.T. Product Differentiation by Analysis of DNA Melting Curves during the Polymerase Chain Reaction. *Anal. Biochem*. 245 (2), 154-160 (1997). - Lay, M.J. & Wittwer, C.T. Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. *Clin. Chem*. 43 (12), 2262-2267 (1997). - Wittwer, C.T. et al. The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. *BioTechniques* 22 (1), 176 (1997). - Montgomery, J.L., Sanford, L.N., and Wittwer, C.T. High-resolution DNA melting analysis in clinical research and diagnostics. *Expert Rev. Mol. Diagn*. 10 (2), 219-240 (2010). - Palais, R.A., Liew, M.A., and Wittwer, C.T. Quantitative heteroduplex analysis for single nucleotide polymorphism genotyping. *Anal. Biochem*. 346 (1), 167-175 (2005). - Wittwer, C.T. et al. High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen. *Clin. Chem*. 49 (6), 853-860 (2003). - Gundry, C.N. et al. Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes. *Clin. Chem*. 49 (3), 396-406 (2003). - Farrar, J.S., Reed, G.H., and Wittwer, C.T., in *Molecular Diagnostics*, (eds. Ansorge W Patrinos GP , editors. (2nd Ed. Burlington: Elsevier, 2009) - Reed, G.H., Kent, J.O., and Wittwer, C.T. High-resolution DNA melting analysis for simple and efficient molecular diagnostics. *Pharmacogenomics* 8 (6), 597-608 (2007). - Wittwer, C.T. High-resolution DNA melting analysis: advancements and limitations. *Hum. Mutat*. 30 (6), 857-859 (2009). - McKinney, J.T. et al., in *Handbook of Plant Mutation Screening* (eds.) 149-163. (2009) - Gundry, C.N. et al. Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons. *Nucleic Acids Res*. 36 (10), 3401 (2008). - Liew, M. et al. Closed-Tube SNP Genotyping Without Labeled Probes. *Am. J. Clin. Pathol*. 127 (3), 341-348 (2007). - Poulson, M.D. & Wittwer, C.T. Closed-tube genotyping of apolipoprotein E by isolated-probe PCR with multiple unlabeled probes and high-resolution DNA melting analysis. *BioTechniques* 43 (1), 87-91 (2007). - Montgomery, J., Wittwer, C.T., Palais, R., and Zhou, L. Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis. *Nat. Protocols* 2 (1), 59-66 (2007). - Herrmann, M.G., Durtschi, J.D., Wittwer, C.T., and Voelkerding, K.V. Expanded Instrument Comparison of Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping. *Clin. Chem*. 53 (8), 1544-1548 (2007). - Audrezet, M.P., Dabricot, A., Le Marechal, C., and Ferec, C. Validation of High-Resolution DNA Melting Analysis for Mutation Scanning of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) *Gene. J. Mol. Diagn*. 10 (5), 424-434 (2008). - Tindall, E.A. et al. Assessing high-resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments. *Hum. Mutat*. 30 (6), 876-883 (2009). - Herrmann, M.G. et al. Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping: Cross-Platform Comparison of Instruments and Dyes. *Clin. Chem*. 52 (3), 494-503 (2006). - Seipp, M.T. et al. Unlabeled oligonucleotides as internal temperature controls for genotyping by amplicon melting. *J. Mol. Diagn*. 9 (3), 284-289 (2007). - Van der Stoep, N. et al. Diagnostic guidelines for high-resolution melting curve (HRM) analysis: An interlaboratory validation of BRCA1 mutation scanning using the 96-well LightScannerTM. *Hum. Mutat*. 30 (6), 899-909 (2009). - Dobrowolski, S.F., Gray, J., Miller, T., and Sears, M. Identifying sequence variants in the human mitochondrial genome using high-resolution melt (HRM) profiling. *Hum. Mutat*. 30 (6), 891-898 (2009). - Erali, M. & Wittwer, C.T. High resolution melting analysis for gene scanning. *Methods* 50 (4), 250-261 (2010). - Wojdacz, T.K., Dobrovic, A., and Hansen, L.L. Methylation-sensitive high-resolution melting. *Nat. Protocols* 3 (12), 1903-1908 (2008). - Reed, G.H. & Wittwer, C.T. Sensitivity and Specificity of Single-Nucleotide Polymorphism Scanning by High-Resolution Melting Analysis. *Clin. Chem*. 50 (10), 1748-1754 (2004). - Smith, B.L., Lu, C.P., and Alvarado Bremer, J.R. High-resolution melting analysis (HRMA): a highly sensitive inexpensive genotyping alternative for population studies. *Mol. Ecol. Res*. 10 (1), 193-196 (2010). - Seeb, J.E. et al. Transcriptome sequencing and high-resolution melt analysis advance single nucleotide polymorphism discovery in duplicated salmonids. *Mol. Ecol. Res*. 11 (2), 335-348 (2012). - Granados-Cifuentes, C. & Rodriguez-Lanetty, M. The use of high-resolution melting analysis for genotyping Symbiodinium strains: a sensitive and fast approach. *Mol. Ecol. Res*. 11 (2), 394-399 (2012). - Henri, H. & Mouton, L. High-Resolution Melting technology: a new tool for studying the Wolbachia endosymbiont diversity in the field. *Mol. Ecol. Res*. 12 (1), 75-81 (2012). - Metzker, M.L. Sequencing technologies: the next generation. *Nat Rev Genet* 11 (1), 31-46 (2010). - Larsen, L.A., Jespersgaard, C., and Andersen, P.S. Single-strand conformation polymorphism analysis using capillary array electrophoresis for large-scale mutation detection. *Nat. Protocols* 2 (6), 1458-1466 (2007). - Xiao, W. & Oefner, P.J. Denaturing high-performance liquid chromatography: a review. *Hum. Mutat*. 17 (6), 439-474 (2001). - Li, Q., Liu, Z., Monroe, H., and Culiat, C.T. Integrated platform for detection of DNA sequence variants using capillary array electrophoresis. *Electrophoresis* (Weinheim, Fed. Repub. Ger.) 23 (10), 1499-1511 (2002). - Malewski, T. et al. Identification of forensically important blowfly species (Diptera: Calliphoridae) by high-resolution melting PCR analysis. *Int. J. Legal Med*. 124 (4), 277-285 (2010). - Mader, E., Lohwasser, U., Borner, A., and Novak, J. Population structures of genebank accessions of Salvia officinalis L. (Lamiaceae) revealed by high resolution melting analysis. *Biochem. Syst. Ecol*. 38 (2), 178-186 (2010). - Poland. Biophysics Department University, Duesseldorf, available at http://www.biophys.uni-duesseldorf.de/local/POLAND/poland.html. - Doyle, J.J. & Doyle, J.L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical bulletin 19 (11) (1987). - Ivanova, N.V., Dewaard, J.R., and Hebert, P.D.N. An inexpensive, automation-friendly protocol for recovering high-quality DNA. *Mol. Ecol*. Notes 6 (4), 998-1002 (2006). - Calves, I. et al. Genetic structure of the European flounder (Platichthys flesus) considering the southern limit of the species’ range and the potential impact of chemical stress. *Mar. Ecol. Prog. Ser*. Doi:10.3354/meps09797 (2012). - Venter, J.C. et al. The Sequence of the Human Genome. *Science* 291 (5507), 1304-1351 (2001). ### Acknowledgements The authors declare no competing financial interests. This research program was financially supported by (1) the European program SUDEVAB (Grant Agreement no 222 156), (2) a CPER – FEDER program for the sequencing part, and (3) by the EVOLFISH Program (financed by the National Agency for Research-VMCS, Paris, France). ### Figures **Figure 1.: Schematic principle of HRMA**.  *A. DNA fragments are amplified using fluorescently intercalating DNA dye, heat-denatured and cooled. Heterozygote (W/U) variant formed after denaturation and rehybridization, two homoduplexes (W/W and U/U) and two heteroduplexes (W/U). B. The results are illustrated as denaturation melting curves (fluorescence normalized or derivative fluorescence in function of temperature). HRMA detects mutations in DNA fragments due to temperature shift of the melting curve caused by variation of the amplicon Tms or variation of the curve shapes in heteroduplexes presence*. **Figure 2.: Step by step of the HRMA data analysis**.  *A. HRMA Melting curves of heterozygous (red) and homozygous variants (blue, yellow and green) are obtained after a continuous denaturation of amplicons with a progressive liberation of the intercalated fluorescent dyes. B1. Normalized melting curves are obtained after determination of 100% and 0% fluorescence values. B2. Normalized melting peaks are the derivative of the normalized melting curves. C1. Normalized and shifted melting curves were obtained according to a horizontal normalization by the temperature shift at a threshold of 3 (red line in B1). C2. Normalized and Temp-shifted difference plots are obtained by comparison of the curves to a reference sample (blue line). He. Heterozygote. Ho. Homozygote* **Figure 3.: Examples of HRMA results on nuclear marker**.  *HRMA results obtained by LightScanner® (A-C) and LightCycler® 480 (B-D) Instruments on exon 1 of ferritin gene from the abalone Haliotis tuberculata (A-B) and exon 2 of myoadenylate deaminase gene from the teleost fish Platichthys flesus (C-D). Normalized and Temp-shifted difference plots are obtained by comparison of the melting curves to reference one. Homozygous genotypes are distinguished by Tm, whereas heterozygous by melting curve shape. Details and localizations of SNPs are presented for each variant regrouped by color*. **Figure 4.: Examples of HRMA on mitochondrial marker**.  *HRMA results obtained by LightCycler® 480 Instruments on partial mitochondrial gene (689 pb) coding for Cytochrome C Oxidase sub-unit 1 (CO1) from the teleost fish Platichthys flesus. Temp-shifted difference plots are obtained by comparison of the melting curves to reference one. Homozygous genotypes are distinguished by Tm and melting curve shape. Details and localizations of SNPs are presented for each variant regrouped by color*. **Figure 5.: Amplification curves obtained only on LightCycler® 480 Instrument**.  *Amplification of positive samples is observed before 30 cycles. Samples with negative or abnormal amplification can be directly identified and removed of the analysis*. **Table 1: SNP classes as defined in the human genome** [Download Table 1](http://www.nature.com/protocolexchange/system/uploads/2125/original/Table_1.doc?1337031099) **Table 2. : Comparison of High Resolution Melting Analysis to some other modern gene scanning methods** [Download Table 2.](http://www.nature.com/protocolexchange/system/uploads/2126/original/Table_2.doc?1337031153) **Table 3. : Troubleshooting table**. [Download Table 3](http://www.nature.com/protocolexchange/system/uploads/2127/original/Table_3.doc?1337031233). ### Associated Publications [Genetic structure of the European flounder (Platichthys flesus) considering the southern limit of the species’ range and the potential impact of chemical stress](http://www.nature.com/protocolexchange/protocols/2383/publications/1290) ### Author information **Anne-Leila Meistertzheim**, Centre de Formation et de Recherche sur les Environnements Méditerranéens (CEFREM) **Isabelle Calves, Sébastien Artigaud, Christine Paillard & Jean Laroche**, Laboratoire des Sciences de l'Environnement Marin, UMR CNRS/UBO/IRD 6539, Institut Universitaire Européen de la Mer, Université de Bretagne Occidentale, 29280 Plouzané, France **Carolyn S. Friedman**, School of Aquatic and Fishery Sciences, University of Washington, Box 355020, Seattle, 98195 Washington, United States **Claude Ferec**, Laboratoire de génétique moléculaire et d'histocompatibilité. CHU Morvan UBO, EFS-Bretagne BP 454 29275 Brest Cedex, France Correspondence to: Anne-Leila Meistertzheim (leila.meistertzheim@gmail.com) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2383) (2012) doi:10.1038/protex.2012.015. Originally published online 15 May 2012*.

Authors: Bevin Gangadharan & Nicole Zitzmann ### Abstract Two-dimensional gel electrophoresis (2-DE) is a protein separation technique often used to separate plasma or serum proteins in an attempt to identify novel biomarkers. This protocol describes how to run 2-DE gels using narrow pH 3-5.6 immobilised pH gradient strips to separate 2 mg of serum proteins. pH 3-6 ampholytes are used to enhance the solubility of proteins in this pH range before the serum proteins are separated in the first dimension by isoelectric point (isoelectric focusing) followed by molecular weight (SDS-PAGE). This approach using the pH 3-5.6 range differs from pH ranges more commonly used for serum or plasma biomarker discovery which span three or more pH units (e.g. pH 3-10 and 4-7), and has the advantage that the pH range lies outside the range of three highly abundant proteins and therefore improves separation and representation of low abundance features. The protocol described takes approximately 8 days.  ### Introduction There is much interest in discovering biomarkers to assess the pathological states of disease, and blood is the most common sample taken from patients to determine disease severity. (1) In hospital laboratories, plasma or serum is obtained from these blood samples and the levels of the biomarkers are determined using automated immunoanalysers or mass spectrometry. However, for many diseases there are no reliable serum/plasma biomarkers available and as a result occasionally invasive approaches such as biopsies are necessary. Not all biomarkers currently used for clinical diagnoses are reliable (e.g. they may show large variation in marker levels between different individuals with the same pathological state). There is an urgent need for reliable and novel biomarkers for many diseases in order to aid both patients and clinicians for diagnosis as well as for monitoring disease and therapeutic regimens. Two-dimensional gel electrophoresis (2-DE) separates proteins and is often used to search for novel biomarkers in serum or plasma. This technique separates proteins in the first dimension by isoelectric point (isoelectric focusing) followed by molecular weight (SDS-PAGE). Commonly used pH ranges for isoelectric focusing are pH 3-10 and pH 4-7 which have been used by us and others to successfully discover novel disease biomarkers. (2,3) However, plasma and serum contain high abundant proteins in these pH ranges. These high abundant proteins restrict the amount of protein that can be loaded onto 2-DE gels and therefore decreases the chances of identifying low abundance proteins which potentially could serve as biomarkers. In the protocol described here we use 2-DE over a narrow pH 3-5.6 range since this lies outside the range of the highly abundant proteins albumin, transferrin and immunoglobulins. The lack of highly abundant proteins in this pH range allows four times more serum or plasma to be loaded compared to a 2-DE gel using a wide pH 3-10 range. Here we present for the first time the detailed protocol for using pH 3-5.6 immobilised pH gradient strips to separate serum and plasma by 2-DE gels. (4,5) Protocol development included analysis of various narrow pH ranges for 2-DE and their comparison to the more commonly used wide pH range. The use of a narrow pH 3-5.6 range led to an improved separation and visualisation of low abundance proteins. In Gangadharan et al. (4) for example, we demonstrate that using the pH 3−5.6 range with a load of 2 mg 262 additional protein features were detected compared to a pH 3−10 gel across the same pH 3−5.6 range. These extra features are potentially new disease biomarkers which are missed when using 2-DE gels with a wide pH range. This novel approach described here not only helps in identifying new biomarkers in serum/plasma but would also be of great benefit in the separation and analysis of other samples where high abundant proteins like albumin may pose a problem such as cerebrospinal fluid or urine. A work flow diagram to show how the experimental steps fit together and examples of equipment which can be used is shown in Fig. 1. This work flow could also be adapted for use in 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE). ### Reagents 1. Blood collection tubes such as P100, plasma or serum tubes (BD). - Bicinchoninic acid (BCA) assay kit, 3-([3-Cholamidopropyl]dimethylammonio)-1-propanesulphonate (CHAPS), tributyl phosphine, iodoacetamide, pooled human serum (Sigma). - pH 3-6 SERVALYT® carrier ampholytes (SERVA). - Urea, dithiothreitol (DTT), γ-methacryloxy-propyl-trimethoxysilane (Bind-Silane), Repel-Silane ES, dry strip cover mineral oil, Immobiline pH 3-5.6 NL IPG DryStrips (18 cm, 3 mm wide), electrode wicks (GE Healthcare). - Sodium dodecyl sulphate (SDS), bromophenol blue, ammonium bicarbonate (NH4HCO3) (Fluka). - HPLC grade water, absolute ethanol, hydrochloric acid (HCl), glycine (BDH). - 2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris), agarose, sequencing grade bovine trypsin (Roche). - Thiourea, glycerol (Fisher Scientific). - Dimethylbenzylammonium propane sulphonate: non-detergent sulphobetaine-256 – NDSB-256 (Calbiochem). - Acetic acid, acetonitrile (Riedel-de Haën). - SYPRO Ruby (Invitrogen). - 96 well reaction plate (Intavis). - 96 well collection plate – non-skirted 200 μl PCR plate (ABgene). **REAGENT SETUP** 1. Rehydration sample buffer (5 M urea, 2 M thiourea, 4% (w/v) CHAPS, 65 mM DTT, 2 mM tributyl phosphine, 150 mM NDSB-256, and 0.0012% (w/v) bromophenol blue). - Equilibration solution (4 M urea, 2 M thiourea, 50 mM Tris HCl (pH 6.8), 30% (v/v) glycerol, 2% (w/v) SDS, 130 mM DTT, 0.002% (w/v) bromophenol blue). - Laemmli reservoir buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS). ### Equipment 1. Reswelling tray, Multiphor II, EPS 3500XL power supply, DALT gradient maker, Hoefer DALT running tank (GE Healthcare) - LAS1000Pro Intelligent Dark Box II CCD camera (Fuji). - Melanie image analysis software (Genebio). - Robotic gel excisor – GCM instrument (Horizon Instruments). - Dark Reader light box (Clare Chemical Research). - Savant SpeedVac and vacuum vaporiser (Thermo Electron). - Automated DigestPro workstation (ABiMED). ### Procedure To avoid keratin contamination of samples it is recommended, although not essential, that all steps below are carried out in clean room conditions using a hair cap bouffant, face mask / beard cover, extended cuff gloves and a non-shedding labcoat. - **Sample preparation** 1. Collect blood from patients using P100 tubes according to the manufacturer’s recommendation or in other serum/plasma tubes as previously described for proteomics. (6,7) - PAUSE POINT Serum / plasma samples can be stored at – 80 °C until required. - Determine the total protein concentration of the sample using a protein assay such as BCA protein assay according to the manufacturer’s recommendation. - Mix 2.4 mg of serum / plasma protein with rehydration sample buffer up to a total volume of 442 μl. - CRITICAL STEP Never heat the samples after adding urea. Elevated temperatures can result in urea hydrolysing to isocyanate. This can cause protein modification by carbamylation leading to artifactual trains of protein features on 2-DE gels. - ! CAUTION Tributyl phosphine in the rehydration sample buffer is spontaneously inflammable in air. - Add 8 μl of pH 3-6 carrier ampholytes (final ampholyte concentration = 1.8%) and vortex mix for 2 minutes. Leave the samples at room temperature for 30 minutes to ensure complete denaturation and solubilisation. Spin samples at 16,000 g for 15 min. - Pipette 375 μl of supernatant (containing 2 mg serum/plasma) into separate lanes of a reswelling tray. Place Immobiline pH 3-5.6 NL IPG DryStrips face down onto the protein-containing samples in each lane of the reswelling tray and overlay with 2 ml of dry strip cover mineral oil. Leave to rehydrate for at least 16 h at room temperature. - CRITICAL STEP To ensure successful rehydration, the gel should be swollen to 1 mm thick and the bromophenol blue dye should be stained across the full length of the strip. - **Isoelectric focusing (IEF)** - Drain off excess mineral oil and transfer to the sample tray of Multiphor II apparatus with the gel facing upwards. - For each IPG DryStrip, cut two electrode wicks 2 cm in length. Soak electrode wicks with 100 μl HPLC grade water and blot with a cleanwipe to ensure that the wicks are damp but not excessively wet. Place damp wicks on either end of the IPG strips and fix electrode bars onto the wicks at either end of the IPG strips. Pour mineral oil into the Multiphor sample tray until the strips are immersed. Prod wicks gently using tweezers to remove air bubbles and ensure good contact with the IPG gel. - Carry out IEF at 300 V for 2 h, a gradient increase to 3500 V over 3 h and then maintain at 3500 V up to 70 kVh using an EPS 3500XL power supply. For all stages, set the current limit to 10 mA for 12 gels, and the power limit to 5 W. Maintain the temperature of the Multiphor at 17 °C using a recycling thermostatic water bath. - PAUSE POINT Strips can be snap frozen on dry ice and then stored at – 80 °C until required. - **Two dimensional polyacrylamide gel electrophoresis (2D-PAGE)** - Gels can be poured using a gradient gel casting machine such as the DALT Gradient maker connected to a peristaltic pump. Pour gels according to the manufacturer’s recommendation. Precast gels can also be used such as the ExcelGel system (GE Healthcare). Gels can be either of fixed percentage such as 12% or of a gradient such as 9-16% the latter of which gives better separation for serum / plasma. Gels must be able to accommodate 18 cm IPG strips. - Optional step: If pouring gels, the plates can be treated so that the gel covalently binds to one of the glass plates in the gel cassette. This makes it easier to manipulate the gels for fixing, staining, scanning, storage and cutting. One plate needs to be treated with Bind-Silane and the other with Repel-Silane ES according to the manufacturer’s recommendations. - Immediately post IEF, incubate the IPG strips in 2 ml of reducing equilibration solution for 15 min at room temperature. Drain strips of equilibration solution. Overlay the strips onto the second dimension gels and seal in place with 90 °C, 0.5% (w/v) agarose in Laemmli reservoir buffer. Use the flat end of a spatula to aid placement of the IPG strip. - Perform second dimension electrophoresis with Laemmli reservoir buffer using a Hoefer DALT running tank or any other equivalent electrophoresis running tank capable of running large 18 cm by 18 cm gels. Set the current to 20 mA per gel for 1 h, followed by 40 mA per gel for approximately 4 h. Set the power limit to 150 W for a tank containing 6 gels and the voltage limit to 600 V throughout the run. Maintain the temperature at 10 °C using a recycling thermostatic water bath. Terminate electrophoresis once the bromophenol blue tracking dye has reached the bottom of the gel. - Remove gels from the running tank and open the glass plates. Discard the IPG strip and the overlay agarose. Wash gels briefly in ultrapure water to remove running buffer and then place into a staining tank such as the Dodeca stainer (BioRad) or equivalent. Fix the proteins on the gels in 40% (v/v) ethanol, 10% (v/v) acetic acid overnight. - Gels can then be stained. For highest sensitivity and greatest compatibility by mass spectrometry, fluorescent stains such as SYPRO Ruby are recommended.8 Perform staining according to the manufacturer’s recommendations. - Image gels using a scanner/camera appropriate for the stain used. Scanners/cameras are available capable of imaging multiple stains such as the Fuji LAS1000Pro CCD camera which can image either fluorescent, silver or Coomassie stained gels. In the case of SYPRO Ruby stained gels, set the parameters to fluorescence and cool the CCD camera to -25 °C prior to capturing images. Place the gel onto the imaging tray and acquire images over different exposure times (typically between 0.5 to 2 min) until the optimum image has been produced. - Place scanned gels in a plastic bag with approximately 10 ml of 40% (v/v) ethanol, 10% (v/v) acetic acid and seal the bag with a bag sealer. Store at 4 °C until required for excising protein spots. - **Differential image analysis and spot excision** - Perform differential image analysis of the gels. This can be carried out using various commerically available software such as the Medical ELectrophoresis ANalysis Interactive Expert (Melanie) software. Perform differential image analysis according to the manufacturer’s recommendations. An optional step is to calibrate all gels internally for pI and molecular weight using the E. coli proteome as a standard (9), typically using 10-15 calibrated landmarks on each gel. Landmarks allow the software to warp the gels so that they can be superimposed onto each other to aid with image analysis. All features displayed as differentially expressed by the software must be validated further by visualising the features across all gels in a montage format. - Differentially expressed features can be excised from the gel manually using a clean scalpel or using any software-driven robot. To aid manual excision gels can be visualised in a dark room using either a white light box (for silver or Coomassie stained gels) or a Dark Reader light box (for fluorescent stained gels). For excision of features using a robotic gel excisor (such as a GCM instrument), the co-ordinates of each differentially expressed protein feature is sent to the robot as a list of commands which will programme x and y movements of the robot arms and direct the cutting head to cut and remove features. To avoid contamination between spots, use a new cutter tip on the cutting head for each gel feature. The robot excises gel features by shearing and aspirating actions. Eject the cutter tips with the isolated gel pieces into separate wells of a 96 well reaction plate with laser made holes on the bottom. - **In-gel trypsin digestion** - In-gel trypsin digestion can then be performed either manually (see Supporting Information) or using an automated workstation such as the DigestPro as described below (all steps at room temperature unless stated otherwise). The DigestPro workstation adds solutions to the 96 well plate containing the gel pieces using needles and removes liquid through laser made holes on the bottom of the plate by applying nitrogen pressure as previously described. (10) Prepare 18 ng/μl bovine trypsin and place inside the automated robot in its inactive form by storing in acidic conditions (10% (v/v) acetonitrile, 1 mM HCl). - Wash gels with 50 μl acetonitrile and 50 μl 50 mM NH4HCO3 for 15 min. Remove supernatant. Dehydrate gels with 100 μl acetonitrile for 10 min. Remove acetonitrile. - Reduce proteins in the gel with 30 μl 10 mM DTT in 25 mM NH4HCO3 for 10 min at 60 °C. Remove supernatant once the samples have cooled (20 min). - Alkylate gel proteins with 30 μl 50 mM iodoacetamide in 25 mM NH4HCO3 for 15 min. Remove iodoacetamide solution. - Wash gel pieces with 50 μl 50 mM NH4HCO3 for 15 min. Remove 50 mM NH4HCO3. Dehydrate gels twice with 50 μl acetonitrile for 15 min. Remove acetonitrile. Pause workstation for 10 min to allow drying. - Activate trypsin by diluting it 2-fold with 25 mM NH4HCO3. Add 15 μl of this 9 ng/ml trypsin solution to each gel piece and leave for 10 min to allow gel swelling. Incubate gel pieces at 37 °C for 2 h. Add 10 μl water to compensate for any water loss. Incubate gel pieces at 37 °C for a further 2 h. - Add 10 μl 25 mM NH4HCO3 to each gel piece and incubate for 10 min. Add 20 μl acetonitrile and leave for 10 min to dehydrate the gel. Transfer the supernatant to a 96 well collection plate. - Add 20 μl 10% (v/v) formic acid to the gels and leave for 10 min to extract the peptides. Add supernatant to the collection plate. Dehydrate gels with 30 μl acetonitrile for 15 min. Add supernatant to the collection plate. - Dry pooled extracts completely in a SpeedVac and reconstitute peptides by dissolving in 0.1% (v/v) formic acid. Perform mass spectrometric analysis as previously described. (11) ### Timing Collection of samples from patients (Step 1) can take several weeks to months after which 2-DE can be performed with the following timings: - Day 1: Step 2, 2 h; Step 3, 15 min; Step 4, 1 h; Step 5, 30 min (+16 h overnight incubation). - Day 2: Step 6, 10 min; Step 7, 5 min; Step 8, > 23 h overnight run; Step 9, no waiting time (precast gels) or 1-2 days in advance (poured gels), Step 10: 2 h. - Day 3: Step 11, 30-45 min; Step 12, 5 h; Step 13, 30 min (+ 16 h overnight incubation). - Day 4: Step 14, 1 h to overnight (depending on staining method used); Step 15, 1 h; Step 16, 15 min. - Day 5: Step 17, 1 day (can take several days – timing depends on software used and experience of user). - Day 6: Step 18, 1 h; Step 19, 15 min; Step 20, 35 min; Step 21, 40 min; Step 22, 20 min; Step 23, 50 min; Step 24, 4-5 h; Step 25, 30 min; Step 26, 35 min - Days 7 and 8: Step 27, 1-2 days. ### Troubleshooting Table 1: Troubleshooting table.   ### Anticipated Results Typically 500 to 600 features are seen when using 2 mg of serum / plasma protein on a 2-DE gel over the pH 3-5.6 range. Fig. 2 shows a typical image when running a gel following the conditions described using normal human serum from Sigma. Anticipated results can also be referred to in our publications. (4,5) ### References 1. Hanash, S. M. et al. Mining the plasma proteome for cancer biomarkers. *Nature*. 452, 571-579 (2008). - Gangadharan, B. et al. Novel serum biomarker candidates for liver fibrosis in hepatitis C patients. *Clin Chem*. 53, 1792-1799 (2007). - Steel, L. F. et al. A strategy for the comparative analysis of serum proteomes for the discovery of biomarkers for hepatocellular carcinoma. *Proteomics*. 3, 601-609 (2003). - Gangadharan, B. et al. New approaches for biomarker discovery: the search for liver fibrosis markers in hepatitis C patients. *J Proteome Res*. 10, 2643-2650 (2011). - Gangadharan, B. et al. Clinical diagnosis of hepatic fibrosis using a novel panel of low abundant human plasma protein biomarkers, Patent US20100291602, 2010. - Randall, S. A. et al. Evaluation of blood collection tubes using selected reaction monitoring MS: implications for proteomic biomarker studies. *Proteomics*. 10, 2050-2056 (2010). - Hsieh, S. Y. et al. Systematical evaluation of the effects of sample collection procedures on low-molecular-weight serum/plasma proteome profiling. *Proteomics*. 6, 3189-3198 (2006). - White, I. R. et al. A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis. *Electrophoresis*. 25, 3048-3054 (2004). - Tonella, L. et al. ‘98 Escherichia coli SWISS-2DPAGE database update. *Electrophoresis*. 19, 1960-1971 (1998). - Houthaeve, T. et al. Automated protein preparation techniques using a digest robot. *J Protein Chem*. 16, 343-348 (1997). - Terry, D. E. et al. Optimized sample-processing time and peptide recovery for the mass spectrometric analysis of protein digests. *J Am Soc Mass Spectrom*. 15, 784-794 (2004). Other related publication ePoster at EASL: [DISCOVERY OF NOVEL BIOMARKER CANDIDATES FOR LIVER FIBROSIS IN HEPATITIS C PATIENTS USING PROTEOMICS](http://www.multiwebcast.com/easl/2011/nice/6848/) ### Acknowledgements The authors wish to thank Professor Raymond Dwek for his valuable support and advice. This work was supported by the Oxford Glycobiology Endowment and a ‘Blue Skies’ research grant from United Therapeutics Corp. B. G. and N. Z. were supported by the Oxford Glycobiology Institute. N. Z. is a Senior Research Fellow of Linacre College, Oxford. ### Figures **Figure 1: The work flow used to run 2-DE gels using the pH 3-5.6 range**  *(a) Serum/plasma samples are initially denatured, solubilised, reduced and prepared with pH 3-6 ampholytes. (b) pH 3-5.6 NL IPG strips are overlaid onto samples in separate lanes of a re-swelling tray. (c) Proteins are separated by charge on a Multiphor using isoelectric focusing. (d) pH 3-5.6 NL strips are transferred onto SDS-PAGE gels and proteins are further separated by molecular weight with a Hoefer DALT running tank. (e) The proteins on the gel are stained and scanned using a Fuji LAS1000Pro camera. (f) Gel images are analysed using the Melanie computer-aided software to identify differences in feature intensity. Features on a pH 3-5.6 serum gel are outlined in red and 3D view of the feature intensity is shown for a selected feature. (g) Features of interest are excised from the gel using a GCM robotic gel excisor. (h) Gel pieces are digested with trypsin using the automated DigestPro workstation. (i) The resulting peptides are analysed by mass spectrometry to identify the proteins of interest*. **Figure 2: Anticipated results**  *Two milligrams of normal human serum (Sigma) run on a 2-DE gel using the pH 3-5.6 range*. **Table 1: Troubleshooting table** [Download Table 1](http://www.nature.com/protocolexchange/system/uploads/1942/original/Table_1.doc?1318256650) **Supporting Information: Manual in-gel digestion** [Download Supporting Information](http://www.nature.com/protocolexchange/system/uploads/1943/original/Supporting_Information.doc?1318256699) **Two dimensional gel electrophoresis using narrow pH 3-5.6 immobilised pH gradient stripss** [Download Two dimensional gel electrophoresis using narrow pH 3-5.6 immobilised pH gradient strips](http://www.nature.com/protocolexchange/system/uploads/1966/original/Bevin_Gangadharan_et_al_2011.pdf?1319545443) ### Associated Publications 1. **New Approaches for Biomarker Discovery: The Search for Liver Fibrosis Markers in Hepatitis C Patients**. Bevin Gangadharan, Robin Antrobus, David Chittenden, Jan Rossa, Manisha Bapat, Paul Klenerman, Eleanor Barnes, Raymond A. Dwek, and Nicole Zitzmann. *Journal of Proteome Research* 10 (5) 2643 - 2650 06/05/2011 [doi:10.1021/pr101077c](http://dx.doi.org/10.1021/pr101077c) - **Novel Serum Biomarker Candidates for Liver Fibrosis in Hepatitis C Patients**. B. Gangadharan, R. Antrobus, R. A. Dwek, and N. Zitzmann. *Clinical Chemistry*. 53 (10) 1792 - 1799 01/10/2007 [doi:10.1373/clinchem.2007.089144](http://dx.doi.org/10.1373/clinchem.2007.089144) - **OP15 Discovery of new liver fibrosis markers in hepatitis C patients using proteomics**. B. Gangadharan, M. Bapat, J. Rossa, R. Antrobus, D. Chittenden, B. Kampa, E. Barnes, R. A. Dwek, and N. Zitzmann. *Gut* 60 (Suppl 2) A56 - A56 01/09/2011 [doi:10.1136/gutjnl-2011-300857b.15](http://dx.doi.org/10.1136/gutjnl-2011-300857b.15) ### Author information **Bevin Gangadharan & Nicole Zitzmann**, Oxford Antiviral Drug Discovery, Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford Correspondence to: Bevin Gangadharan (Bevin.Gangadharan@bioch.ox.ac.uk) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2215) (2011) doi:10.1038/protex.2011.261. Originally published online 11 October 2011*.

Authors: Yalin Tang, Qian Shang, Junfeng Xiang, Qianfan Yang, Qiuju Zhou, Lin Li, Hong Zhang, Qian Li, Hongxia Sun, Aijiao Guan, Wei Jiang & Wei Gai ### Abstract This protocol presents the screening of ligand(s) from medicinal plant extracts based on target recognition by using NMR spectroscopy. A detailed description of sample preparation and analysis process is provided. NMR spectroscopies described here are 1H NMR, diffusion-ordered spectroscopy (DOSY), relaxation-edited NMR, 1H–13C HSQC and HMBC. This method includes three steps: First, investigate the NMR spectroscopy properties of target and choose the suitable spectral editing NMR method; second, judge the existence of ligand(s) and determine the proton signals of ligand(s) in the extracts; third, verify the structure(s) of the ligand(s). This method allows the direct structural identification of ligand(s) from medicinal plant extracts without separation and purification. Thus it provides a very promising strategy for the fast screening of lead components based on target recognition. ### Introduction Plant-derived agents, owing to their diversity in structure and bioactivity1-6, play critical roles in pharmaceutical research. They provide lead compounds for biopharmaceutical technology and supply an economical ways for the discovery of new drugs. The drugs, which are used widely in modern medicine, mostly derived from natural compounds and their derivatives. For example, aspirin was originated from salicylic acid in the bark of the willow tree (Salix species), which is used traditionally to treat fever and inflammation in many cultures worldwide7. The successes of the early “blockbuster” drugs set the stage for ongoing drug discovery efforts from medicinal plants. The screening of bioactive ingredients based on targets recognition attracts researchers’ attention8-10. Nevertheless, these methods can hardly be applied in direct screening of bioactive ingredients from plants extracts which compose of various compounds with different contents. Currently screening from plant extracts against specific targets always follows the strategy of “Isolation  Structure identificationActivity confirmation”. Yagura et al.11 isolated isosalipurposide for anticarcinogenic compounds in the Uzbek medicinal plant by bioactivity-directed fractionation. Bai et al.12 screen the G-quadruplex ligands from Kalopanax septemlobus (Thunb) Koidz extract by high performance liquid chromatography (HPLC). Compared with these methods, our method will accelerate the screening process of lead components greatly by providing the structural information of bioactive ingredients without isolating the ligand(s) molecules experimentally. Thus it provides chemists and pharmacologists with useful info to optimize lead compound for the final drug in a more rapid way. The implementation of our goal benefits from the powerful NMR techniques. NMR has now become an important tool in drug discovery13-19, owing to its advantage in structure identification. But the complexity and overlapping of signals in complex systems bring difficulties in the recognition of spectra. With the development of spectral editing NMR techniques, selected useful information also can be obtained. Among these techniques, diffusion-ordered spectroscopy (DOSY) and relaxation-edited methods are two successful examples20-23, which could provide means for signal filtering and selecting, and consequently achieve “virtual separation”. In this protocol, DOSY and relaxation-edited NMR techniques are used to screen the bioactive ingredients interacting with target in medicinal plant extracts. Our method has drawn a profound attention on screening bioactive ingredients from medicinal plants and there are many reviews commenting on it24, 25. The reviewer of Angewandte Chemie International Edition pointed out that the predominance of this method was integration screening and structure identification, which carried out the fast screening and structure identification of bioactive ingredients in natural plant extracts. Nature China highlighted this method and commented on it with Screening Methods: Looking for Ligands as title. In order to introduce this method to more researchers who devote to search the bioactive ingredients in natural plant extracts and provide convenience for their works, it is necessary to describe this method in detail and define standard protocol in order to ensure the correct usage of this method. Here we take the screening and identifying ligands from the extract of Phellodendron chinense Schneid cortexes (PE)26 based on G-quadruplex and the extract of Flos Lonicera Japonica (FLJ) based on Avian Influenza Polymerase Protein PAC by using NMR as examples to define well-tested procedures. Experimental design The biomolecules which are reported to have important physiological significance can be used as targets. To be specific, our protocol takes G-quadruplex and Avian Influenza Polymerase Protein PAC (carboxyterminal domain of PA) as examples. G-quadruplexes27,28 can inhibit the activity of telomerase29 and play an important role in suppression of carcinogenesis30-33. The ligands of G-quadruplex have the potential for the arrest of cancer-cell growth and may be potentially valuable as antitumor drugs34-36. The interaction of G-quadruplex and its ligand affects the chemical shifts of imino protons in G-quadruplex, which locate low-field region (10-12 ppm) of NMR spectra. Additionally, the ligand of protein also can be obtained by using spectrally-filtered editing NMR techniques. For PAC, the drugs discovered by it may be effective against most influenza strains less susceptible to drug resistance due to the high conservation of the active sites in PAC37. The medicinal plant extracts which are reported to treat some kind of disease can be used as candidates for the corresponding targets. PE is chosen as a candidate because it is reported to be benefit for the treatment of cancer and contain G-quadruplex ligands38. FLJ is chosen as a candidate because it is reported to be benefit for the treatment of viral infection such as influenza A virus39. The concentration of medicinal plant extracts depends on the concentration of bioactive ingredients. The concentration of medicinal plant extracts should be benefit for observing the obvious changes from the characteristic signals of the targets. There are three steps in this process (Figure 1): First, it is necessary to choose the suitable NMR sequence according to the target. To be specific, if there are not clear and characteristic signals of target in NMR spectroscopy, relaxation-edited NMR is preferred. Otherwise, DOSY is suitable. For example, G-quadruplex has the characteristic peaks around δ 10-12 ppm which are clear and not easy to be interfered by other components. In this case, DOSY can be chose. But for proteins they usually have high molecular weights, thus their peak profiles are broadened and there are uneasy to be identified. In this case, relaxation-edited NMR can be chose. Second, judge the existence of ligand(s) and determine the proton signals of ligand(s) by spectral editing NMR techniques in the extracts. For example, G-quadruplex is taken as target in DOSY. As long as the ligand(s) binds to G-quadruplex, the chemical shifts and intensities of imino protons of G-quadruplex which are clear and characteristic signals of G-quadruplex change and the change is easy to be detected. Then extract the signal of ligand(s) by DOSY. In the relaxation-edited NMR spectra, through the comparison with the spectrum of extract without PAC, the peak of ligand can be picked out. Third, further structural illumination is used to identify the ligand(s) by 1H-13C HSQC and HMBC from the signal obtained in step 2. Limits of applicability and practical considerations If there were bioactive ingredients in medicinal plant extracts and their structures were unknown, this protocol could be employed to find them out. As long as the signals of the ingredients which could interact with the target could be distinguished in NMR spectroscopy, this protocol could be used to identify their structures without isolation. Although it provided a fast approach to structurally determine bioactive ingredients, this method had some limitations. Firstly, the content of bioactive ingredients in the extracts is a key factor that influences the result. For example, the G-quadruplex ligand detected in PE is about 0.06% (mass concentration). If there is more than one ligand in test extracts, the content of each ligand dominates the priority of ligands identified in test extracts in this protocol. For a multiligand system such as extracts of Coptis chinensis Franch rhizomes (CE)26 which contains at least two ligands (berberine and palmatine) with similar G-quadruplex binding ability, the ligand with the higher content tend to be identified and that with lower content may be omitted. The second factor is the requirement of the target. Because our protocol bases on NMR techniques, generally the target must meet the demands in NMR experiments, such as the concentration and molecular weight40. To be specific, it requires the relatively high concentration (mM level) of target when the molecular weight of target is about ten thousand owing to the sensitivity of DOSY. ### Reagents REAGENTS 1. DNA d(TTGGGTT) (Tsingke Biotechnology Co., Ltd) - TSP (3-(trimethylsilyl) propionic acid-d4 sodium salt), berberine (Sigma Co.) - Lentinan extracts (Shanghai kangzhou Fungi extract Co., Ltd) EQUIPMENT 1. Avance 600 NMR spectrometer (Bruker-Biospin). 600 MHz is not the only frequency that can be used in this method, but it is a good compromise for sensitivity and dispersion versus capital cost. - A 5 mm BBI probe capable of delivering z-field gradients. - Eppendorf pipette and pipette tips or similar REAGENT SETUP G-quadruplex In practice, the sample preparation for this experiment is simple. The preparation of G-quadruplex d(TTGGGTT)4 is the same as the procedure described in the literature41. G-quadruplex d(TTGGGTT)4 was formed by dissolving primer d(TTGGGTT) in phosphate buffer (10 mM K2HPO4/KH2PO4, 90% H2O/10% D2O, pH 7.0). The solution was equilibrated at room temperature for 24 h before experiments. The absorbance of DNA d(TTGGGTT) at 260nm is obtained by absorption spectroscopy. It is hypothesized that all DNA d(TTGGGTT) can form d(TTGGGTT)4 and the concentration of d(TTGGGTT)4 is . The minimum concentration of G-quadruplex d(TTGGGTT)4 is 0.25 mM. Under the concentration the characteristic signals of d(TTGGGTT)4 are easily distinguished in NMR spectroscopy. PAC Methods for the preparation of PAC protein were previously described5. Briefly, residues 257–716 of the PA subunit of avian H5N1 influenza A virus (A/goose/Guangdong/1/96) were cloned into a pGEX-6p vector (GE Healthcare) and transformed into Escherichia coli strain BL21. Cells were cultured in LB medium at 37℃ with 100 mg/L of Ampicillin. When the OD600 reached 0.6–0.8, the culture was induced with 0.5 mM isopropyl-thio-Dglactosidase (IPTG) at 16℃. After 20 hours of incubation, the cells expressing PAC were harvested and combined by centrifugation at 5000 rpm for 10 min. Recombinant protein was purified with a glutathione affinity column (GE Healthcare). Glutathione S-transferase (GST) was cleaved with PreScission protease (GE Healthcare), and the protein complex was further purified by Q sepharose FF ion exchange chromatography and Superdex-200 gel filtration chromatography (GE Healthcare). Extracts of Phellodendron chinense Schneid cortexes (PE) A 500 mL three-neck flask is equipped with a magnetic stirrer, thermometer, heating mantle, and a reflux condenser, 60 g powder of Phellodendron chinense Schneid cortexes and 300 ml ethanol-water solution (the volume ratio of water and ethanol was 1:3) are added to the flask. Then the mixture is heated up to 80 degrees Celsius with stirring for 5 h. The crude product was collected by filtration, and dried in a vacuum desiccator at 60 degrees Celsius for 48 h. 31.5 g product is obtained. Extracts of Flos Lonicera Japonica (FLJ) 10 g powder of FLJ was reflux extracted with 300 mL 15% ethanol-water solution for 5h. The residue was then removed by filtration, and the filtrate was desiccated in a vacuum desiccator at 60 ºC for 48 h. 1 g of FLJ extracts was finally obtained, respectively. ### Equipment EQUIPMENT SETUP NMR setup The high-resolution NMR spectroscopy could be used for screening and identifying new bioactive ingredients42. In the following, experimental methods are described using a Bruker NMR spectrometer as an example. Some common pulse techniques can be employed to screen bioactive ingredients with futher structure identification, such as standard Bruker pulse program p3919gp, stebpgp1s19, cpmgpr1d, hsqcetgp and hmbcgplpndqf. Setup on NMR spectrometers of other vendors will require adjustment accordingly. In order to obtain maximum benefit from acquiring NMR spectra, the parameters need to be set accurately. Generally, all NMR experiments should be acquired at 298.2K. Homogenization of the magnetic field (‘shimming’) for samples is important for conventional NMR probes. This can be done with TopShim on Bruker spectrometers (TopSpin 2.1). The homogenization of the magnetic field can be confirmed by the peak profile of TSP which is a single peak at 0ppm. The number of scans per experiment for different medicinal plant extracts can be adjusted according to the signal-to-noise ratio of the components of interest, but this will have an impact on the overall experiment time per sample. Water suppression When observing protons, the dynamic range of the detection can be strongly limited by the size of the water peak and the results in the signal loss for low concentration substances. Hence, the water peak has to be suppressed by using the pulse program p3919gp that applies 3–9–19 pulses with gradients for water suppression43 to improve the signal-to-noise ratio for the detection of ligands. Other spectrometer manufacturers provide the same pulse sequences, but they have different names and syntax; please discuss with the supplier. The parameters are optimized to give optimum suppression of the water resonance without reducing the signal intensity of the compound signals next to it. It is recommended to automatically adjust the receiver gain before acquiring the spectra. All NMR experiments require correct setting of 90° pulse length. It is a key to determine the offset of the water signal for the water suppression. DOSY To get the actual gradient strength in absolute values, it is necessary to obtain a gradient calibration constant, which is used by the AU program dosy to calculate and store the list (difflist) containing absolute gradient strength values. This list is used by the processing tools to calculate the correct diffusion constants. The gradient strength (g), diffusion time big delta (Δ) and diffusion gradient length little delta (δ) are important factors in DOSY experiment. It is necessary to optimize these three parameters to detect the whole decay function properly. Selecting the right values for Δ or δ is important to get good diffusion constants with less error. Using 1D versions of the diffusion pulse program for optimizing the Δ (d20) and δ (p30), experiments are carried out by comparing two spectra with different amplitude (gpz6) 2% and 95%. The parameters are set when the signal decay goes down to 5% residual signal. The comparison is convenient in dual display. Cpmgpr: The relaxation-edited NMR experiments utilized a [D/pre-saturation-90x-(Δ-180y-Δ)n-acquire] pulse sequence, in which the CPMG sequence was used for the spin-lock. Structure identification using 2D NMR spectroscopy Two-dimensional heteronuclear NMR spectroscopy is employed for identifying the connectivity between signals. 1H–13C HSQC and HMBC NMR spectra are used for structural identification44, as they highlight connectivities of protons directly attached to the carbon atoms in a molecule. ### Procedure - **Sample preparation ● TIMING 10–12 min** 1. Dissolving target in appropriate buffer solution and adjust the concentration. For G-quadruplex d(TTGGGTT)4, the concentration is 0.25 mM. For PAC, the concentration is 8.7×10-3mM. - ▲ CRITICAL STEP In the work with G-quadruplex, the formation of G-quadruplex is of paramount importance. Usually triethylamine used in the synthesis of d(TTGGGTT) usually affects the binding between G-quadruplex and ligands. Thus triethylamine needs to be removed by dialysis before the usage of d(TTGGGTT) in this protocol. - For DOSY, make the mixture of d(TTGGGTT)4 and PE and ensure the concentration is 0.25 mM and 3.5 mg mL-1, respectively. For relaxation-edited NMR, prepare two samples. One is the mixture of PAC and FLJ and the concentration is 8.7×10e-3mM and 3.5 mg mL-1, respectively. The other is FLJ and the concentration is 3.5 mg mL-1. - Put the above samples into 5mm NMR tubes and the total volume is 5×10-4L for each sample, respectively. - NMR setup ● TIMING 15–17 min - Insert the NMR tube into the spinner and measure the correct height with the gauge. - Enter the and wait for 5–10 s before entering the button. The sample is now positioned in the probe. - Wait for 5–10 minutes for the temperature equilibrium. - Optimization of NMR spectrometer: (1) tune and match the probe; (2) lock onto the lock solvent; and (3) shim the sample using the lock level. - Determine the sample-specific settings for NMR pulse sequences: (1) Set the offset value to the HDO resonance. (2) Determine the 90° pulse length at a given power level. (3) Re-adjust the frequency offset for water signal suppression, if necessary. - ？TROUBLESHOOTING - ▲ CRITICAL STEP The suppression of water properly is beneficial for the observation of tiny constituent. - **NMR acquisition ● TIMING 24 h** - First, 1D p3919gp sequence (option A) is used to test whether the extracts contain G-quadruplex ligand(s). (TIMING 12-15 min) Second, DOSY (option B) is used to pick out the peak of ligand. (TIMING 2 h) Third, based on the peak of ligand, HSQC and HMBC (option C) are used to identify the structure of ligand. (TIMING 24 h) Adjust the receiver gain per sequence and sample by using automatic receiver gain adjustment. Processing parameters: if not mentioned otherwise, the one-dimensional spectra are generally processed by applying a line broadening of 0.3–1 Hz. - ？TROUBLESHOOTING - (A) 1D p3919gp sequence (Bruker spectrometer: p3919gp) - (i) The parameters are set as follows: spectral width = 20 ppm; number of time domain data points = 32 k; relaxation delay (RD) = 2.0 s; number of scans = 128; and the receiver gain is set to fill the digitizer as closely as possible. This results in a total acquisition time of about 11 min per sample. - (ii) For processing, a target spectral resolution is difficult to define, but typically with a line-broadening of 1 Hz. (TIMING 1 min) - (B) DOSY (Bruker terminology: stebpgp1s19)45 - (i) Acquire diffusion-edited spectra using a pulse sequence with stebpgp1s19. The settings are relaxation delay = 2 s; number of scans = 128 (or higher, depending on requirements); data points in the F2 dimension = 32 k; data points (gradient strengths) in the F1 dimension = 32; spectral width = 20 ppm; diffusion delay Δ = 0.1 s; the gradient length δ = 5.6 ms; line broadening factor in F2 dimension = 0.3 Hz. The data analyses were applied to the raw experimental data using the standard 2D DOSY processing protocol in TOPSPIN (Bruker, Version 2.1) software with logarithmic scaling in the F1 (diffusion coefficient) dimension. - (C) Heteronuclear correlation spectroscopy (Bruker terminology: hsqcetgp and hmbcgplpndqf)46 - (i) Acquire 1H–13C HSQC spectrum using sensitivity improvement with echo-anti echo-TPPI. GARP decoupling of 13C is carried out during the acquisition time. 0.1 μs trim pulses are employed in the INEPT transfer and gradients in back-INEPT. The parameters are as follows: resolution in F2/F1 = 2 k/512 (depending on available experiment time); number of scans = 128 (depending on experiment time); number of dummy scans = 16; sweep width in F2/F1 = 20 ppm/170 ppm in hsqcetgp, and relaxation delay = 2 s. C-H coupling constant is 145 Hz. 1024×1024 points are used in Fourier transformation. - (ii) Collect 1H-13C phase-sensitive (TPPI) HMBC spectra. GARP decoupling of 13C is carried out during the acquisition time. The parameters are as follows: resolution in F2/F1 = 2 k/512 (depending on available experiment time); number of scans = 128 (depending on experiment time); number of dummy scans = 16; sweep width in F2/F1 = 20 ppm/250 ppm in hmbcgplpndqf, and relaxation delay = 2 s. The long range C-H coupling constant for HMBC is 6.25 Hz. 1024×1024 points are used in Fourier transformation. Optional: 9| First, 1D cpmgpr1d sequence (option A) is used in FLJ in the absence (a) and presence (b) of PAC with different spin-lock time.(generally 100-1500ms) (TIMING 24-30 min) Second, the difference spectrum is made with (a) and (b) and find out the changed peaks which belong to ligand. (5min) Third, based on the peaks of ligand, HSQC and HMBC (option C) are used to identify the structure of ligand. (TIMING 24 h) Adjust the receiver gain per sequence and sample by using automatic receiver gain adjustment. Processing parameters: if not mentioned otherwise, the one-dimensional spectra are generally processed by applying a line broadening of 0.3–1 Hz. (A) 1D cpmgpr1d sequence (Bruker spectrometer: cpmgpr1d) (i) The parameters are set as follows: pre-saturation water suppression was applied in pre-acquisition delay (D = 3 s); Δ = 1.5 ms, and 2×n×Δ = total spin-lock time; spectral width = 20 ppm; number of time domain data points = 64 k; relaxation delay (RD) = 2.0 s; number of scans = 32; and the receiver gain is set to fill the digitizer as closely as possible. This results in a total acquisition time of about 1-30 min per sample depending on the spin-lock time. **TIMING** - Steps 1–3 Sample preparation: 10–12 min - Steps 4-8 NMR setup: 15–17 min - Step 9 NMR acquisition: 24 h **？TROUBLESHOOTING** - Step 8 - The main problems can be avoided by ensuring that the water offset and 90° pulse length are adjusted on samples (90° pulse length needs to be adjusted on a sample-by-sample basis). - Step 9 - The receiver gain requires automatic receiver gain adjustment. This will avoid problems with baseline rolling artifacts. ### Anticipated Results Accurate application of the steps in this protocol will lead to consistent NMR spectra. Once these spectra have been obtained, they will require phasing and baseline correction before being subjected to further analysis. The 1H spectra of G-quadruplex d(TTGGGTT)4 with different plant extracts [with or without G-quadruplex ligand(s)], are shown in Figure 2. The evident differences in the imino region (10–12 ppm) in the 1H NMR spectra of free and bound G-quadruplexes47 can be utilized as a spectroscopic means indicating the existence/nonexistence of G-quadruplex ligand(s) in the test extracts. As shown in Figure 2, when lentinan (without G-quadruplex ligand) is added into the d(TTGGGTT)4, the chemical shift and intensity of G3-G5 are unchanged. However, when PE (with G-quadruplex ligand) is added into the d(TTGGGTT)4, a clear upfield shift of the guanine imino proton resonance signals was observed and signal intensity, especially that of G5 dropped greatly. Figure 3 shows that DOSY analysis of a mixture of PE and d-(TTGGGTT)4. Based on the principle of DOSY, if the component binds to d-(TTGGGTT)4, the component has the same diffusion coefficient with d-(TTGGGTT)4. The characteristic peaks of d-(TTGGGTT)4 are around δ 10-12. In Figure 3, the peak around δ 9 ppm has the same ordinate with the peaks around δ 10-12 which means the components from δ 9 ppm have the same diffusion coefficient with d-(TTGGGTT)4. Additionally no such peak appears in the spectrum of the G-quadruplex alone. This result, together with a consideration of the sharp drop in the diffusion coefficient of the proton that resonates around δ 9 ppm in the two samples (4.8×10-10 m2 s-1 in PE and 1.4×10-10 m2 s-1 in a mixture of PE and d-(TTGGGTT)4 respectively), led us to reasonably conclude that this peak resulted from the compound in PE bound to G-quadruplex. According to the peak at δ 9 ppm, the structure of the berberine could be recognized by analyzing the HSQC and HMBC spectra of PE26. (Supplement) The relaxation-edited NMR spectra of FLJ in the absence and presence of PAC and the difference spectrum are shown in Figure 4. In order to identify the PAC ligand from the mixtures, relaxation-edited NMR with Carr-Purcell-Meiboom-Gill (CPMG) spin-lock was applied to FLJ extract in the absence and presence of PAC. As shown in Figure 4b, the application of CPMG spin-lock will reduce the ligand peaks due to its relatively fast R2 upon binding. However, these signals were not influenced in the absence of target (Figure 4a). This difference was highlighted in the difference spectrum (Figure 4c). These attenuated peaks can be ascribed to the PAC ligand in the extract. Thus, the ligand peaks can be “picked out” from the mixture without previous isolation. The next step is to identify which molecule in the mixture is bound to PAC. Based on the characteristic signals picked out by relaxation-edited NMR, structure elucidation was carried out using the subsequent 2DNMR experiments, such as HSQC, HMBC, and total correlation spectroscopy (TOCSY). Followed by proton-carbon connections, proton-carbon long-range correlations, and proton-proton correlations provided by these 2DNMR experiments, the molecular frame of the ligand can be built. The PAC ligand can be then identified to be chlorogenic acid. ### References 1. Schmidt, B. M., Ribnicky, D. M., Lipsky, P. E. & Raskin, I. Revisiting the ancient concept of botanical therapeutics. *Nat Chem Biol* 3, 360-366, (2007). - Koehn, F. E. High impact technologies for natural products screening. Progress in drug research. Fortschritte der Arzneimittelforschung. *Progres des recherches pharmaceutiques* 65, 175, 177-210 (2008). - Balandrin, M. F., Klocke, J. A., Wurtele, E. S. & Bollinger, W. H. Natural plant chemicals: sources of industrial and medicinal materials. *Science* 228, 1154-1160 (1985). - Newman, D. J. & Cragg, G. M. Natural products as sources of new drugs over the last 25 years. *J Nat Prod* 70, 461-477, (2007). - Corson, T. W. & Crews, C. M. Molecular understanding and modern application of traditional medicines: triumphs and trials. *Cell* 130, 769-774, (2007). - Cragg, G. M., Newman, D. J. & Snader, K. M. Natural products in drug discovery and development. *J Nat Prod* 60, 52-60, (1997). - Mahdi, J. G., Mahdi, A. J. & Bowen, I. D. The historical analysis of aspirin discovery, its relation to the willow tree and antiproliferative and anticancer potential. *Cell proliferation* 39, 147-155, (2006). - Cavalla, D. APT drug R&D;: the right active ingredient in the right presentation for the right therapeutic use. *Nat Rev Drug Discov* 8, 849-853, (2009). - Li, L. et al. NMR identification of anti-influenza lead compound targeting at PA(C) subunit of H5N1 polymerase. *Chinese Chemical Letters* 23, 89-92, (2012). - Mercier, K. A., Shortridge, M. D. & Powers, R. A multi-step NMR screen for the identification and evaluation of chemical leads for drug discovery. *Combinatorial chemistry & high throughput screening* 12, 285-295 (2009). - Yagura, T. et al. Anticarcinogenic compounds in the Uzbek medicinal plant, Helichrysum maracandicum. *Nat Med *(Tokyo) 62, 174-178 (2008). - Bai, G. et al. Direct screening of G-quadruplex ligands from Kalopanax septemlobus (Thunb.) Koidz extract by high performance liquid chromatography. *Journal of Chromatography A* 1218, 6433-6438 (2011). - Fedoroff, O. Y. et al. NMR-based model of a telomerase-inhibiting compound bound to G-quadruplex DNA. *Biochemistry* 37, 12367-12374 (1998). - Manzenrieder, F., Frank, A. O. & Kessler, H. Phosphorus NMR spectroscopy as a versatile tool for compound library screening. *Angewandte Chemie-International Edition* 47, 2608-2611, (2008). - McCoy, M. A., Senior, M. M. & Wyss, D. F. Screening of protein kinases by ATP-STD NMR spectroscopy. *J Am Chem Soc* 127, 7978-7979 (2005). - Tengel, T. et al. Use of 19F NMR spectroscopy to screen chemical libraries for ligands that bind to proteins. *Org Biomol Chem* 2, 725-731 (2004). - Li, L., Chang, S. H., Y L Tang & Liu, Y. F. NMR identification of anti-influenza lead compound targeting at PAc subunit of H5N1 polymerase. *Chinese chem lett* 23, 89-92 (2012). - Zhang, X. L., Sanger, A., Hemmig, R. & Jahnke, W. Ranking of High-Affinity Ligands by NMR Spectroscopy. *Angewandte Chemie-International Edition* 48, 6691-6694, (2009). - Zhou, Q., Li, L., Xiang, J., Sun, H. & Tang, Y. Fast screening and structural elucidation of G-quadruplex ligands from a mixture via G-quadruplex recognition and NMR methods. *Biochimie* 91, 304-308 (2009). - Lin, M. F., Shapiro, M. J. & Wareing, J. R. Diffusion-edited NMR-affinity NMR for direct observation of molecular interactions. *Journal of the American Chemical Society* 119, 5249-5250 (1997). - Lin, M. F., Shapiro, M. J. & Wareing, J. R. Screening mixtures by affinity NMR. *Journal of Organic Chemistry* 62, 8930-8931 (1997). - Hodge, P., Monvisade, P., Morris, G. A. & Preece, I. A novel NMR method for screening soluble compound libraries. *Chem Commun*, 239-240 (2001). - Lepre, C. A., Moore, J. M. & Peng, J. W. Theory and applications of NMR-based screening in pharmaceutical research. Chem Rev 104, 3641-3676 (2004). - Pagano, B. et al. State-of-the-Art Methodologies for the Discovery and Characterization of DNA G-Quadruplex Binders. *Current Pharmaceutical Design* 18, 1880-1899 (2012). - Li, Q., Xiang, J. F., Zhang, H. & Tang, Y. L. Searching Drug-Like Anti-cancer Compound(s) Based on G-Quadruplex Ligands. *Current Pharmaceutical Design* 18, 1973-1983 (2012). - Zhou, Q. et al. Screening potential antitumor agents from natural plant extracts by G-quadruplex recognition and NMR methods. *Angew Chem Int Edit* 47, 5590-5592 (2008). - Moyzis, R. K. et al. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. *Proc Natl Acad Sci U S A* 85, 6622-6626 (1988). - Phan, A. T., Kuryavyi, V. & Patel, D. J. DNA architecture: from G to Z. *Curr Opin Struct Biol* 16, 288-298 (2006). - Zahler, A. M., Williamson, J. R., Cech, T. R. & Prescott, D. M. Inhibition of telomerase by G-quartet DNA structures. *Nature* 350, 718-720 (1991). - Davis, J. T. G-quartets 40 years later: from 5’-GMP to molecular biology and supramolecular chemistry. *Angew Chem Int Ed Engl* 43, 668-698, (2004). - Dai, J. et al. An intramolecular G-quadruplex structure with mixed parallel/antiparallel G-strands formed in the human BCL-2 promoter region in solution. *J Am Chem Soc* 128, 1096-1098, (2006). - Hurley, L. H. DNA and its associated processes as targets for cancer therapy. *Nat Rev Cancer* 2, 188-200, (2002). - Siddiqui-Jain, A., Grand, C. L., Bearss, D. J. & Hurley, L. H. Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. *Proc Natl Acad Sci U S A* 99, 11593-11598, (2002). - Mergny, J. L. & Helene, C. G-quadruplex DNA: a target for drug design. *Nat Med* 4, 1366-1367, (1998). - Mergny, J. L. et al. The development of telomerase inhibitors: the G-quartet approach. *Anti-cancer drug design* 14, 327-339 (1999). - Mergny, J. L., Riou, J. F., Mailliet, P., Teulade-Fichou, M. P. & Gilson, E. Natural and pharmacological regulation of telomerase. *Nucleic Acids Res* 30, 839-865 (2002). - Nakazawa, M. et al. PA subunit of RNA polymerase as a promising target for anti-influenza virus agents. *Antiviral Research* 78, 194-201, doi:DOI 10.1016/j.antiviral.2007.12.010 (2008). - Kumar, A. P. et al. Akt/cAMP-responsive element binding protein/cyclin D1 network: a novel target for prostate cancer inhibition in transgenic adenocarcinoma of mouse prostate model mediated by Nexrutine, a Phellodendron amurense bark extract. *Clin Cancer Res* 13, 2784-2794, (2007). - Ko, H. C., Wei, B. L. & Chiou, W. F. The effect of medicinal plants used in Chinese folk medicine on RANTES secretion by virus-infected human epithelial cells. *Journal of Ethnopharmacology* 107, 205-210 (2006). - Meyer, B. & Peters, T. NMR spectroscopy techniques for screening and identifying ligand binding to protein receptors. *Angew Chem Int Ed Engl* 42, 864-890, (2003). - Sun, H., Xiang, J., Tang, Y. & Xu, G. Regulation and recognization of the extended G-quadruplex by rutin. Biochem Biophys Res Commun 352, 942-946, (2007). - Schuetz, A. et al. RDC-enhanced NMR Spectroscopy in structure elucidation of sucro-neolambertellin. *Angewandte Chemie-International Edition* 47, 2032-2034, (2008). - Piotto, M., Saudek, V. & Sklenar, V. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. *J Biomol NMR* 2, 661-665 (1992). - Whittaker, D. V., Parolis, L. A. S. & Parolis, H. Structural Elucidation of the Capsular Polysaccharide Expressed by Escherichia-Coli O20-K83-H26 by High-Resolution Nmr-Spectroscopy. *Carbohyd Res* 253, 247-256 (1994). - Wu, D. H., Chen, A. D. & Johnson, C. S. An Improved Diffusion-Ordered Spectroscopy Experiment Incorporating Bipolar-Gradient Pulses. *J Magn Reson Ser A* 115, 260-264 (1995). - Schleucher, J. et al. A General Enhancement Scheme in Heteronuclear Multidimensional Nmr Employing Pulsed-Field Gradients. *Journal of Biomolecular* Nmr 4, 301-306 (1994). - Gavathiotis, E., Heald, R. A., Stevens, M. F. & Searle, M. S. Recognition and Stabilization of Quadruplex DNA by a Potent New Telomerase Inhibitor: NMR Studies of the 2:1 Complex of a Pentacyclic Methylacridinium Cation with d(TTAGGGT)(4) *Angew Chem Int Edit* 40, 4749-4751 (2001). ### Acknowledgements This work is supported by the Major Research Program of the National Natural Science Foundation of China (Grant No. 91027033), the General Program of the National Natural Science Foundation of China (Grant No. 81072576) and the Key Program of the Chinese Academy of Sciences (Grant No. KJCX2-EW-N06-01). ### Figures **Figure 1: The steps in this protocol** [Download Figure 1](http://www.nature.com/protocolexchange/system/uploads/2366/original/Figure_1.doc?1354781342) **Figure 2: The 1H NMR spectra of a) d(TTGGGTT)4, b) a mixture of d-(TTGGGTT)4 and lentinan extract (without G-quadruplex ligand), and c) a mixture of d(TTGGGTT)4 and PE (with G-quadruplex ligand, berberine).** [Download Figure 2](http://www.nature.com/protocolexchange/system/uploads/2367/original/Figure_2.doc?1354781426) *The concentrations of d(TTGGGTT)4, lentinan extract, and PE are 0.25 mM, 3.50 mg mL-1, and 1.0 mg mL-1, respectively. The region of 10.5–12.0 ppm is broadened and the imino proton resonance signals are labeled as G3–G5. (Reproduced with permission from ref. 25)* **Figure 3: DOSY analysis of a mixture of PE (1.0 mg mL-1) and d- (TTGGGTT)4 (0.25 mM). The peak of the d(TTGGGTT)4 ligand(s) is designated by “↓” in the F2 projection. (Reproduced with permission from ref. 25)** [Download Figure 3](http://www.nature.com/protocolexchange/system/uploads/2369/original/Figure_3.doc?1354796378) **Figure 4: The relaxation-edited NMR spectra of FLJ in the absence (a) and presence (b) of PAC and the difference spectrum (c) of a and b**. [Download Figure 4](http://www.nature.com/protocolexchange/system/uploads/2370/original/Figure_4.doc?1354796475) *The spin-lock time in a and b were both 1500 ms. Contents of FLJ extract were 3.3mg mL-1, and concentration of PAC was 8.7 × 10-3mM. The water peak located at δ 4.8, and 1mM of TSP was added to the sample as a reference (δ 0). The region of δ 6.2-7.8 is broadened, and the ligand peaks attenuated upon the addition of the target were marked with “*” in plot c*. ### Associated Publications 1. **Screening Potential Antitumor Agents from Natural Plant Extracts by G‐Quadruplex Recognition and NMR Methods**. Qiuju Zhou, Lin Li, Junfeng Xiang, Yalin Tang, Hong Zhang, Shu Yang, Qian Li, Qianfan Yang, and Guangzhi Xu. *Angewandte Chemie International Edition* 47 (30) 5590 - 5592 14/07/2008 doi:10.1002/anie.200800913 - **Screening Anti-influenza Agents that Target Avian Influenza Polymerase Protein PAC from Plant Extracts Based on NMR Methods**. Lin Li, Sheng-Hai Chang, Jun-Feng Xiang, Qian Li, Huan-Huan Liang, Jian Li, Hong-Juan Bao, Ya-Lin Tang, and Ying-Fang Liu.* Chemistry Letters* 40 (8) 801 - 803 doi:10.1246/cl.2011.801 - **Screening α-glucosidase inhibitors from mulberry extracts via DOSY and relaxation-edited NNR**. Qian Shang, Jun-Feng Xiang, and Ya-Lin Tang. *Talanta* 97 () 362 - 367 doi:10.1016/j.talanta.2012.04.046 - **Fast screening and structural elucidation of G-quadruplex ligands from a mixture via G-quadruplex recognition and NMR methods**. Qiuju Zhou, Lin Li, Junfeng Xiang, Hongxia Sun, and Yalin Tang. *Biochimie* 91 (2) 304 - 308 doi:10.1016/j.biochi.2008.10.011 - **Fishing potential antitumor agents from natural plant extracts pool by dialysis and G-quadruplex recognition**. Qian Shang, Jun-Feng Xiang, Xiu-Feng Zhang, Hong-Xia Sun, Lin Li, and Ya-Lin Tang. *Talanta* 85 (1) 820 - 823 doi:10.1016/j.talanta.2011.04.011 - **Direct screening of G-quadruplex ligands from Kalopanax septemlobus (Thunb.) Koidz extract by high performance liquid chromatography**. Ge Bai, Xueli Cao, Hong Zhang, Junfeng Xiang, Hong Ren, Li Tan, and Yalin Tang. *Journal of Chromatography A* 1218 (37) 6433 - 6438 doi:10.1016/j.chroma.2011.07.028 - **Screen Anti-influenza Lead Compounds That Target the PAC Subunit of H5N1 Viral RNA Polymerase**. Lin Li, Shenghai Chang, Junfeng Xiang, Qian Li, Huanhuan Liang, Yalin Tang, Yingfang Liu, and Jun Liu. *PLoS ONE* 7 (8) 24/08/2012 doi:10.1371/journal.pone.0035234 - **NMR identification of anti-influenza lead compound targeting at PAC subunit of H5N1 polymerase**. Lin Li, Sheng Hai Chang, Jun Feng Xiang, Qian Li, Huan Huan Liang, Ya Lin Tang, and Ying Fang Liu. *Chinese Chemical Letters* 23 (1) 89 - 92 doi:10.1016/j.cclet.2011.09.006 ### Author information **Yalin Tang**, Tang yalin **Qian Shang, Junfeng Xiang, Qianfan Yang, Qiuju Zhou, Lin Li, Hong Zhang, Qian Li, Hongxia Sun, Aijiao Guan, Wei Jiang & Wei Gai**, Unaffiliated Correspondence to: Yalin Tang (tangyl@iccas.ac.cn) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2534) (2012) doi:10.1038/protex.2012.060. Originally published online 7 December 2012*.