Littérature scientifique sur le sujet « NanoLC FT MS »

MS, with or without pre-analysis peptide fractionation, can be used to decipher the residues on proteins where oxidative modifications caused by peroxynitrite, singlet oxygen or electrophilic lipids have occurred. Peroxynitrite nitrates tyrosine and tryptophan residues on the surface of actin. Singlet oxygen, formed by the interaction of UVA light with tryptophan, can oxidize neighbouring cysteine, histidine, methionine, tyrosine and tryptophan residues. Dose–response inactivation by 4HNE (4-hydroxynonenal) of hBAT (human bile acid CoA:amino acid N-acyltransferase) and CKBB (cytosolic brain isoform of creatine kinase) is associated with site-specific modifications. FT-ICR (Fourier-transform ion cyclotron resonance)–MS using nanoLC (nano-liquid chromatography)–ESI (electrospray ionization)–MS or direct-infusion ESI–MS with gas-phase fractionation identified 14 4HNE adducts on hBAT and 17 on CKBB respectively. At 4HNE concentrations in the physiological range, one member of the catalytic triad of hBAT (His362) was modified; for CKBB, although all four residues in the active site that were modifiable by 4HNE were ultimately modified, only one, Cys283, occurred at physiological concentrations of 4HNE. These results suggest that future in vivo studies should carefully assess the critical sites that are modified rather than using antibodies that do not distinguish between different modified sites.

11504 Background: There is a need for a reliable breast cancer biomarker that can predict a patient’s response to therapy. Serum glycans, or oligosaccharides, are of particular interest as over half of all proteins are glycosylated and alterations in glycosylation influence growth, adhesion, metastasis and immune surveillance of tumor, among other important functions. Serum glycans can be analyzed by high resolution mass spectrometry. Methods: Sera from patients with known metastatic breast cancer and age-matched healthy controls without medical problems were prospectively analyzed by mass spectroscopy. Women over the age of 18, who were not pregnant or breastfeeding, and who were without other active cancers were eligible. Samples were de-identified for laboratory personnel who analyzed sera by matrix-assisted laser desoprtion/ionization (MALDI) and Fourier transform ion-cyclotron resonance mass sepctrometry (FT ICR MS). Glycans were also profiled by chromatographic separation using a microchip nanoLC (Agilent) with a time-of-flight (TOF) mass analyzers. Results: Sera from 25 patients with metastatic breast cancer and 25 controls were evaluated. The mass profiles were obtained corresponding to both N-linked oligosaccharides (N-glycans) and O-linked oligosaccharides (O-glycans). Distinct variations in glycosylation were observed among sera analyzed from patients with metastatic breast cancer compared to controls. Specific glycan masses were analyzed and found to correspond to N-glycans. The chromatographic glycan profile showed individual glycans that were distinct for the cancer patients. Conclusions: Analysis of serum gylcans by mass spectrometry represents a new paradigm of cancer biomarker studies, focusing on post-translational modifications of proteins, rather than protein expression. Further refinement of this technology may be clinically useful in monitoring response to therapy in metastatic breast cancer. No significant financial relationships to disclose.

Abstract Background Urinary hepcidin is a potential biomarker of renal inflammation and acute kidney injury (AKI) which is elevated in sickle cell disease (SCD). Hepcidin in circulation is filtered through glomeruli filtration barrier and reabsorbed by the renal tubules. Hepcidin can also be synthesized by the kidney tubular cells. Thus, increased urinary levels of hepcidin may reflect either a reduction in tubular uptake or an increase in renal production. Recent studies suggested that urinary hepcidin may protect against AKI by attenuating heme-mediated injury. Thus decreased hepcidin levels in SCD patients may contribute to AKI and serve as potentially informative marker of SCD-associated kidney injury. Previously, hepcidin was measured by ELISA and mass spectrometry. Immunoassays are limited due to the cross-reactivity of antibodies to prohepcidin and truncated hepcidin-20, -22, and -24 isoforms of active hepcidin-25. Mass spectrometric assays are specific for hepcidin-25 but sample preparation remains a challenge. Objective To develop a sensitive, reliable and reproducible nanoLC/FT-MS method with simplified sample preparation for measuring of hepcidin in urine samples. Also to correlate urinary hepcidin with urinary albumin and urinary protein to access the degree of kidney dysfunction. Methods Samples were enriched and purified semi-automaticaly on 10-uL ZipTip and online trap column. Stable isotope-labeled hepcidin was used as internal standard. The standard concentration range was 1.56-800 nM and quality control samples were 5 nM, 20 nM, 80 nM and 400 nM. Samples were subjected to an LC-20AD nano HPLC system coupled to an LTQ XL™ Orbitrap mass spectrometer with an in-house made nano-HPLC column. High resolution/selected reaction monitoring (HR/SRM) scan was carried out and the narrow mass range ([M+H]+ ±0.01 Da) was used to extract ion chromatograms (EICs) for quantification. Urinary samples were collected from 20 SCD patients and 13 controls. Urinary albumin, protein and creatinine were detected by ELISA. The urine hepcidin concentrations were normalized to urine creatinine (Cr) values. Results Semi-automatic approach simplified sample preparation and accelerated the analysis. At least 24 samples could be prepared and processed at the same time. Online column trapping further purified and enriched hepcidin and improved the sensitivity and specificity of this method by eliminating interferences from urine. Hepcidin showed a good linearity within the concentration range of 1.56-800 nM with an r2 value of 0.9994. The precision intraday (n = 5) and interday (n = 5) and the repeatability (n=5) of the method were good with relative standard deviations (RSDs) lower than 5%. The analyzed samples were stable for 3 days at +4°C (RSDs<5%). The percent mean recoveries of hepcidin was within the acceptable range of 89.65-104.79%. We found that SCD patients had significantly lower (about 2-fold) urinary hepcidin levels compared to controls, and urinary hepcidin levels in 2 SCD patients were below the lower limit of detection (<0.5 nM). We found that there was no difference in urine albumin between SCD and control subjects, total urine protein was significantly increased in SCD patients. There was no positive correlation between urine hepcidin and urine albumin or total protein. Conclusion We developed an LC-MS based method for measuring levels of urinary hepcidin. This method is promising in terms of recovery, sensitivity, selectivity, repeatability and simplicity of sample preparation. SCD patients showed significantly decreased hepcidin levels in urine suggesting a potentially novel mechanism of AKI in SCD. Acknowledgments This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005 and 5G12MD007597). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosures No relevant conflicts of interest to declare.

La glycosylation est la forme la plus courante de modification post-traductionnelle (PTM) des protéines humaines, puisque plus de 70% d’entre elles sont glycosylées. Celle-ci régule de nombreuses propriétés biologiques comme leur stabilité, leur demi-vie et leur activité. Néanmoins, les protéines peuvent également présenter d'autres types de PTM, ce qui peut conduire pour une protéine donnée à un très grand nombre d'isoformes variant par leur masse, leurs propriétés biologiques et physico-chimiques et leur concentration dans les échantillons biologiques. Ainsi, caractériser une glycoprotéine comporte de nombreux défis et nécessite la mise en œuvre de méthodes séparatives très performantes et de détection très sensibles et informatives.La gonadotrophine chorionique humaine (hCG) est l’hormone spécifique de la grossesse humaine. Elle est essentielle au développement du placenta et du fœtus. Elle est composée de deux sous-unités hCGα et hCGβ qui sont fortement glycosylées (4 sites de N-glycosylation et 4 sites d’O-glycosylation). Récemment, des travaux ont montré une corrélation entre sa glycosylation et une bonne implantation du fœtus. Une caractérisation des ces glycoformes s’avère donc nécessaire.Par conséquent, de nouvelles méthodes en LC/CE-MS ont été développées pour la caractérisation de la hCG à l’échelle intacte en utilisant deux médicaments à base de hCG ayant des glycosylations différentes. Alors que la méthode en CZE-MS (TQ) a permis de différencier les profils des glycoformes de la sous-unité hCGα des deux médicaments, la complémentarité des méthodes RP- et HILIC-MS (qTOF) a conduit à leur identification.Pour limiter les erreurs potentielles d’identification dues au chevauchement des profils isotopiques, le profil de chaque isoforme a été résolu par FT-ICR MS. Dans ce but, une séparation au format nanoLC en mode RP a été développée, améliorant ainsi la sensibilité de la méthode d’un facteur 500 par rapport au format conventionnel. Cette méthode a permis de confirmer l’identification des glycoformes de la sous-unité hCGα. D’autre part, il a été possible d’obtenir des profils différents de glycosylation de la sous-unité hCGβ en favorisant leur ionisation par réduction de la hCG. Enfin, un traitement à la PNGase a conduit à l’élimination des N-glycanes pour l’obtention des profils d’O-glycosylation de la sous-unité hCGβ
Glycosylation is the most common form of post-translational modifications (PTMs) of human proteins, since more than 70% are glycosylated. It regulates numerous biological properties including their stability, half-life, and activity. Nevertheless, proteins can also exhibit other types of PTMs that lead to a very large number of isoforms, varying in mass, properties and concentration in the biological samples. Therefore, the characterization of a glycoprotein is highly challenging and requires the use of powerful separation techniques and sensitive and informative detection modes.The human chorionic gonadotropin (hCG) is the hormone specific to human pregnancy. It is essential for the development of placenta and fetus. It is based on two heavily glycosylated subunits, hCGα and hCGβ, having 8 glycosylation sites (4 N- and 4 O-glycosylation sites). Some recent studies demonstrated that here is a correlation between the hCG glycosylation state and the fetus implantation. This is why the characterization of the hCG glycoformes is needed.Therefore, new LC/CE-MS methods were developed for the characterisation of hCG at the intact level using two hCG-based drugs having different glycosylation profiles. While the CZE-MS (TQ) method showed its potential for glycosylation fingerprinting, the complementarity of LC-(qTOF) MS methods in RP and HILIC modes allowed the identification of the glycoforms of the hCGα subunit.To limit the identification errors due to the overlapping of isotopic distribution patterns, the profile of each isoform was resolved by FT-ICR MS. For this purpose, a nanoLC separation in RP mode was developed, thus improving the sensitivity of the method by a factor 500 compared to the conventional format. This method allowed the confirmation of the identification of hCGα glycoforms. Then, it was possible to obtain different glycosylation patterns of the hCGβ by promoting its ionization after hCG reduction. Then, a PNGase treatment was carried out to remove the N-glycans in order to obtain the O-glycoprofiles of hCGβ isoforms

Au cours des deux dernières décennies, la spectrométrie de masse (MS) est devenue un outil incontournable dans la caractérisation des protéines comme les IgG. Ces anticorps assurent la protection de l'organisme contre les antigènes, qu'ils reconnaissent grâce aux domaines variables de leurs chaînes lourdes et légères. Des séquences polymorphes existent également au niveau des domaines constants CH2 et CH3 du fragment Fc, impliquant quelques acides aminés, qui définissent ainsi 34 allèles protéiques IGHG des 4 sous-classes d'IgG. Des relations sont établies entre certains de ces polymorphismes et la fonctionnalité de la réponse anticorps. La caractérisation du polymorphisme allélique des IgG par la MS est fréquemment effectuée par la stratégie bottom-up. Cette approche analytique repose sur l'analyse des protéines digérées par des endoprotéases classiques (comme la trypsine) générant des peptides protéolytiques (~30 acides aminés). Nous avons donc utilisé cette méthode combinée à des approches de biologie moléculaire pour étudier ce polymorphisme. Cependant la présence, pour un même individu, de plusieurs allèles d'homologie très forte limite la caractérisation spécifique des séquences de Fc. Dans ce contexte, nous avons développé une approche middle-down permettant d'analyser de grands fragments peptidiques (~ 211 acides aminés). IdeS est une endoprotéase spécifique des IgG qui génère spécifiquement deux fragments : les fragments Fc/2 et F(ab')2. Ces fragments sont alors étudiés par nanoLC-MS/MS à très haute résolution (140000 à m/z 200). La fragmentation des ions précurseurs a été réalisée en mode HCD (higher-energy collisional dissociation). Les données obtenues ont été traitées pour recherche dans les banques de données et les résultats ont été validés manuellement. Malgré la faible performance des logiciels disponibles à ce jour pour automatiser ce traitement, notre approche montre qu'il est possible de discriminer plusieurs allèles IGHG des différentes IgG. Au vu de la forte homologie de séquence il est important d'optimiser la couverture de séquence car certains allèles ne diffèrent que d'un résidu sur les 211 acides aminés séquencés. Cette preuve de concept des capacités d'une analyse middle-down pour identifier les différents Fc/2 pourrait à l'avenir être utilisée comme outil diagnostic pour la caractérisation des polymorphismes des Fc/2 dans un contexte de suspicion d'infections parasitaires congénitales
Over the past two decades, mass spectrometry (MS) has become an essential tool in the characterization of proteins such as IgG. These antibodies protect the organism against antigens, which they recognize through the variable domains of their heavy and light chains. Polymorphic sequences also exist at the constant domains CH2 and CH3 of the Fc fragment, involving a few amino acids, which thus define 34 IGHG protein alleles of the 4 IgG subclasses. Relationships are established between some of these polymorphisms and the functionality of the antibody response. The characterization of allelic polymorphism of IgG by MS is frequently carried out by the bottom-up strategy. This analytical approach relies on the analysis of proteins digested by conventional endoproteases (such as trypsin) generating proteolytic peptides (~ 30 amino acids). We have therefore used this method combined with molecular biology approaches to study this polymorphism. However, the presence, for the same individual, of several alleles of very strong homology limits the specific characterization of the sequences of Fc. In this context, we developed a middle-down approach to analyze large peptide fragments (~ 211 amino acids). IdeS is an IgG-specific endoprotease that specifically generates two fragments: the Fc/2 and F (ab')2 fragments. These fragments are then studied by nanoLC-MS/MS at very high resolution (140000 at m/z 200). The fragmentation of the precursor ions was carried out in HCD (higher-energy collisional dissociation) mode. The data obtained were processed and searched in the databases and the results were validated manually. Despite the poor performance of the software available to date due to limited automation, our approach shows that it is possible to discriminate several IGHG alleles from different IgGs. In view of the high sequence homology it is important to optimize the sequence coverage because some alleles differ only by one residue over the 211 amino acids sequenced. This proof of concept of the capabilities of a middle-down analysis to identify different Fc/2 could be used in the future as a diagnostic tool for the characterization of Fc /2 polymorphisms in a context of suspicion of congenital parasitic infections

Abstract Jurassic's kerogen shale-carbonate reservoir in North Kuwait is categorized as a source rock exhibiting micro- to Nano Darcy permeability and is Kuwait Oil Company's focus in recent years. Although the challenges are significant (formation creep, fracturing initiation, etc.), the efforts toward producing from unconventional reservoirs and applying experience from both USA and Canada in this field are ongoing. As a step toward development, the gas field development group selected a vertical pilot well to measure the inflow of hydrocarbon from a single fracture while minimizing formation creep (flowing of particulate material and formation into the wellbore that blocks the production). This step was required prior to drilling a long horizontal lateral wells and completing it with multiple hydraulic fractures to confirm commercial production. A comprehensive design process was executed with the full integration of operator and service company competencies to achieve the three main objectives: First, characterize the kerogen rock mechanics which allows selection of the most competent kerogen beds to prevent collapse of the hole during fracturing (creep effect) by conducting scratch, unconfined stress, proppant embedment, and fluid compatibility tests. Then, prepare a suit of strength measurements on full core samples to help in fracturing design and minimize creep effect. The second objective was to design and implement a robust proppant fracturing program that avoids the kerogen concerns after selecting the most competent reservoir unit and suitable proppant type. Third, perform controlled flowback to unload the well and attempt to establish clean inflow unlike previous attempts that failed to either suitably stimulate or prevent solids production (deliver clean inflow). After analyzing the lab test results, choosing the optimal fracturing design, and preparing the vertical well for proppant hydraulic fracturing, the treatment was performed. In December 2019, the hydraulic fracturing treatment with resin-coated bauxite proppant was successfully pumped through 6 ft of perforation interval and followed by a controlled flowback. Resin-coated bauxite proppant was specifically selected to overcome the creep and embedment effects during the fracture closure and flowback. Moreover, a properly designed choke schedule was implemented to balance unloading with a delicate enough drawdown to avoid formation failure. This paper discusses in detail the lab testing, evolution of fracturing design, treatment analysis, and the robust workflow that led to successfully achieving all main objectives, paving the way for long horizontal lateral wells. This unconventional undertaking in Kuwait presents a real challenge. It is a departure from traditional methods, yet it points toward a high upside potential should the appraisal campaign be completed effectively.

Abstract Jurassic Kerogen shale/carbonate reservoir in North Kuwait provides the same challenges as North American shales in addition to ones not yet comparable to any other analogue reservoir globally. It is the Kerogen's resource density; however, that makes this play so attractive. Like ‘conventional’ unconventional in the US and Canada this kerogen is believed to be a source rock and is on the order of micro-to nano-Darcy permeability. As such, industry learnings show that likely long horizontal laterals with multiple hydraulic fractures will be necessary to make commercial wells. Following this premise, the immediate objective is to establish clean inflow into wellbore as the previous attempts to appraise failed due to "creep" of particulate material and formation flowing into the wellbore. Achieving this milestone will confirm that this formation is capable of solids free inflow and will open a new era in unconventional in Kuwait. Planning for success, the secondary objective is to then upscale to full field development. The main uncertainties lie in both producibility and ‘frac-ability’, and certainly, these challenges are not trivial. A fully integrated testing program was applied to both better understand the rock mechanical properties and to land on an effective frac design. Scratch, unconfined stress, proppant embedment and fluid compatibility tests were conducted on full core samples for geo-mechanics to prepare a suite of strength measurements ahead of frac design and to custom-design the fracture treatment and "controlled" flowback programs to establish inflow from Kerogen without "creep". Unlike developed shale reservoirs, the Jurassic Kerogen tends to become unconsolidated when treated. The pre-frac geomechanics tests will be outlined in this paper with the primary objective of finding the most competent reservoir unit to select the limited perforation interval to frac through so that formation competency can be maintained. Previous attempts failed to maintain a competent rock matrix even only after pumping data-fracs. Acidizing treatments also turn the treated rock volume into sludgy material with no in-situ stability nor ability to deliver "clean inflow". A propped fracturing treatment with resin-coated bauxite was successfully placed in December 2019 in a vertical appraisal well perforated over 6 ft at 12 spf shot density. "Controlled" flowback carried out in January 2020 achieved the strategically critical "clean inflow" with reservoir fluids established to surface. Special proppant technologies provided by an industry leading manufacturer overcame the embedment effects and to control solids flowback. A properly designed choke schedule to balance unloading with a delicate enough drawdown to avoid formation failure was executed. Local oilfields relied on the vast reserves and produced easily from carbonate reservoirs that required only perforating or acid squeezes to easily meet or exceed high production expectations. This unconventional undertaking in Kuwait presents a real challenge as it is a complete departure from the ways of working yet it points towards a very high upside potential should the appraisal campaign can be completed effectively.