Journal articles: 'Difference gel electrophoresis (DIGE)'

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Lilley, Kathryn S., and David B. Friedman. "Difference gel electrophoresis DIGE." Drug Discovery Today: Technologies 3, no. 3 (September 2006): 347–53. http://dx.doi.org/10.1016/j.ddtec.2006.09.013.

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Kondo, Tadashi. "Cancer biomarker development and two-dimensional difference gel electrophoresis (2D-DIGE)." Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1867, no. 1 (January 2019): 2–8. http://dx.doi.org/10.1016/j.bbapap.2018.07.002.

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Paasch, U., F. Heidenreich, S. Grunewald, K. Kettner, H. J. Glander, and T. M. Kriegel. "Application of difference gel electrophoresis (DIGE) to identify obesity-associated sperm cell proteins." Fertility and Sterility 90 (September 2008): S322. http://dx.doi.org/10.1016/j.fertnstert.2008.07.1672.

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Rukmangadachar, Lokesh A., Jitender Kataria, Gururao Hariprasad, Jyotish C. Samantaray, and Alagiri Srinivasan. "Two-dimensional difference gel electrophoresis (DIGE) analysis of sera from visceral leishmaniasis patients." Clinical Proteomics 8, no. 1 (2011): 4. http://dx.doi.org/10.1186/1559-0275-8-4.

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Albuquerque, Lidiane M., Monique R. O. Trugilho, Alex Chapeaurouge, Patrícia B. Jurgilas, Patrícia T. Bozza, Fernando A. Bozza, Jonas Perales, and Ana G. C. Neves-Ferreira. "Two-Dimensional Difference Gel Electrophoresis (DiGE) Analysis of Plasmas from Dengue Fever Patients." Journal of Proteome Research 8, no. 12 (December 4, 2009): 5431–41. http://dx.doi.org/10.1021/pr900236f.

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Rottensteiner, Hanspeter, Christina Monetti, Herbert Gritsch, Alfred Weber, Hartmut J. Ehrlich, Friedrich Scheiflinger, and Peter L. Turecek. "2D-DIGE As a Tool to Analyze Lot-to-Lot Consistency of Complex Therapeutic Products Such As BAX 855, a PEGylated Recombinant FVIII." Blood 118, no. 21 (November 18, 2011): 4360. http://dx.doi.org/10.1182/blood.v118.21.4360.4360.

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Abstract Abstract 4360 2-Dimensional difference gel electrophoresis (2-D DIGE) is a method that circumvents the gel-to-gel variability inherent in conventional 2-dimensional gel electrophoresis (2-DE). We developed a 2-D DIGE protocol for recombinant factor VIII (rFVIII), a therapeutic protein used for the treatment of hemophilia A. The FVIII heterodimer is composed of heterogenous, strongly glycosylated heavy and light chains that are held together by a divalent cation. 2-DE of rFVIII led to a separation of the various fragments and their identity could be determined by Western blot. A comparison of two rFVIII batches by 2-D DIGE revealed their identical composition, whereas an rFVIII variant lacking its central B domain was congruent with the smallest heavy and light chain fragments of rFVIII only. A simpler pattern was obtained upon removal of the terminal sialic acids of rFVIII’s glycans due to a better focusing in the first dimension. 2-D DIGE was also well suited to structurally evaluate BAX 855, a PEGylated longer-acting variant of recombinant FVIII. 2-D DIGE thus proved an excellent and straightforward method for structural analysis of rFVIII. Our data suggest that the method could serve as a tool to characterize and control quality of very complex pharmaceutically active ingredients such as PEGylated proteins. Disclosures: Rottensteiner: Baxter Innovations GmbH: Employment. Monetti:Baxter Innovations GmbH: Employment. Gritsch:Baxter Innovations GmbH: Employment. Weber:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.

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Hannigan, Adele, Richard Burchmore, and Joanna B. Wilson. "The Optimization of Protocols for Proteome Difference Gel Electrophoresis (DiGE) Analysis of Preneoplastic Skin." Journal of Proteome Research 6, no. 9 (September 2007): 3422–32. http://dx.doi.org/10.1021/pr0606878.

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Hoffert, Jason D., Bas W. M. van Balkom, Chung-Lin Chou, and Mark A. Knepper. "Application of difference gel electrophoresis to the identification of inner medullary collecting duct proteins." American Journal of Physiology-Renal Physiology 286, no. 1 (January 2004): F170—F179. http://dx.doi.org/10.1152/ajprenal.00223.2003.

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In this study, we present a standardized approach to purification of native inner medullary collecting duct (IMCD) cells from rat kidney for proteomic analysis and apply the approach to identification of abundant proteins utilizing two-dimensional difference gel electrophoresis (DIGE) coupled with matrix-assisted laser desorption-ionization-time of flight mass spectrometry. Fractionation of inner medullary cell suspensions by low-speed centrifugation gave a highly purified IMCD cell fraction in which aquaporin-2 was enriched 10-fold. When DIGE was initially applied to rat inner medullas fractionated into IMCD cells (labeled with Cy3) and non-IMCD cells (labeled with Cy5), we identified 50 highly abundant proteins expressed in the IMCD cells. These proteins, identifiable without subcellular fractionation, included chiefly enzymes, structural proteins, and signaling intermediates. An additional 35 proteins were found predominantly in the non-IMCD cell types. Proteins that were highly enriched in the IMCD fraction included cytokeratin 8, cytokeratin 18, transglutaminase II, aminopeptidase B, T-plastin, heat shock protein (HSP) 27, HSP70, and lactate dehydrogenase A. Semiquantitative immunoblotting and immunohistochemistry confirmed relative expression levels and distribution of selected proteins. An additional 40 IMCD proteins were identified in separate experiments aimed at further enrichment of proteins through optimization of sample loading. These studies document the applicability of a standardized approach to purification of IMCD cells for proteomic analysis of IMCD proteins and demonstrate the feasibility of largescale identification of proteins in the native IMCD cell.

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McNamara, Laura E., Fahsai A. Kantawong, Matthew J. Dalby, Mathis O. Riehle, and Richard Burchmore. "Preventing and troubleshooting artefacts in saturation labelled fluorescence 2-D difference gel electrophoresis (saturation DiGE)." PROTEOMICS 11, no. 24 (November 23, 2011): 4610–21. http://dx.doi.org/10.1002/pmic.201100135.

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Nilo, Ricardo, Carlos Saffie, Kathryn Lilley, Ricardo Baeza-Yates, Verónica Cambiazo, Reinaldo Campos-Vargas, Mauricio González, et al. "Proteomic analysis of peach fruit mesocarp softening and chilling injury using difference gel electrophoresis (DIGE)." BMC Genomics 11, no. 1 (2010): 43. http://dx.doi.org/10.1186/1471-2164-11-43.

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&NA;. "A DETERMINATION OF HUMAN ISLET VIABILITY FOR TRANSPLANTATION USING DIFFERENCE-IN-GEL ELECTROPHORESIS (DIGE) PROTEOMICS." Transplantation 82, Suppl 2 (July 2006): 422. http://dx.doi.org/10.1097/00007890-200607152-01053.

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Hobson, D. J., P. Rupa, G. J. Diaz, H. Zhang, M. Yang, Y. Mine, P. V. Turner, and G. M. Kirby. "Proteomic analysis of ovomucoid hypersensitivity in mice by two-dimensional difference gel electrophoresis (2D-DIGE)." Food and Chemical Toxicology 45, no. 12 (December 2007): 2372–80. http://dx.doi.org/10.1016/j.fct.2007.06.039.

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Oliva, Karen, Gillian Barker, Clyde Riley, Mark J. Bailey, Michael Permezel, Gregory E. Rice, and Martha Lappas. "The effect of pre-existing maternal obesity on the placental proteome: two-dimensional difference gel electrophoresis coupled with mass spectrometry." Journal of Molecular Endocrinology 48, no. 2 (February 1, 2012): 139–49. http://dx.doi.org/10.1530/jme-11-0123.

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Our aim was to study the protein expression profiles of placenta obtained from lean and obese pregnant women with normal glucose tolerance at the time of term Caesarean section. We used two-dimensional difference gel electrophoresis (2D-DIGE), utilising narrow-range immobilised pH gradient strips that encompassed the broad pH range of 4–5 and 5–6, followed by MALDI-TOF mass spectrometry of selected protein spots. Western blot and quantitative RT-PCR (qRT-PCR) analyses were performed to validate representative findings from the 2D-DIGE analysis. Eight proteins were altered (six down-regulated and two up-regulated on obese placentas). Annexin A5 (ANXA5), ATP synthase subunit beta, mitochondria (ATPB), brain acid soluble protein 1 (BASP1), ferritin light chain (FTL), heterogeneous nuclear ribonucleoprotein C (HNRPC) and vimentin (VIME) were all lower in obese patients. Alpha-1-antitrypsin (A1AT) and stress-70 protein, mitochondrial (GRP75) were higher in obese patients. Western blot analysis of ANXA5, ATPB, FTL, VIME, A1AT and GRP75 confirmed the findings from the 2D-DIGE analysis. For brain acid soluble protein 1 and HNRPC, qRT-PCR analysis also confirmed the findings from the 2D-DIGE analysis. Immunohistochemical analysis was also used to determine the localisation of the proteins in human placenta. In conclusion, proteomic analysis of placenta reveals differential expression of several proteins in patients with pre-existing obesity. These proteins are implicated in a variety of cellular functions such as regulation of growth, cytoskeletal structure, oxidative stress, inflammation, coagulation and apoptosis. These disturbances may have significant implications for fetal growth and development.

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Chouchani, Edward T., Thomas R. Hurd, Sergiy M. Nadtochiy, Paul S. Brookes, Ian M. Fearnley, Kathryn S. Lilley, Robin A. J. Smith, and Michael P. Murphy. "Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol difference in gel electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation." Biochemical Journal 430, no. 1 (July 28, 2010): 49–59. http://dx.doi.org/10.1042/bj20100633.

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The S-nitrosation of mitochondrial proteins as a consequence of NO metabolism is of physiological and pathological significance. We previously developed a MitoSNO (mitochondria-targeted S-nitrosothiol) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, SNO-DIGE (S-nitrosothiol difference in gel electrophoresis). Protein thiols in control and MitoSNO-treated samples were blocked, then incubated with copper(II) and ascorbate to selectively reduce S-nitrosothiols. The samples were then treated with thiol-reactive Cy3 (indocarbocyanine) or Cy5 (indodicarbocyanine) fluorescent tags, mixed together and individual protein spots were resolved by 2D (two-dimensional) gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by MS. Parallel analysis by Redox-DIGE enabled us to distinguish S-nitrosated thiol proteins from those which became oxidized due to NO metabolism. We identified 13 S-nitrosated mitochondrial proteins, and a further four that were oxidized, probably due to evanescent S-nitrosation relaxing to a reversible thiol modification. We investigated the consequences of S-nitrosation for three of the enzymes identified using SNO-DIGE (aconitase, mitochondrial aldehyde dehydrogenase and α-ketoglutarate dehydrogenase) and found that their activity was selectively and reversibly inhibited by S-nitrosation. We conclude that the reversible regulation of enzyme activity by S-nitrosation modifies enzymes central to mitochondrial metabolism, whereas identification and functional characterization of these novel targets provides mechanistic insight into the potential physiological and pathological roles played by this modification. More generally, the development of SNO-DIGE facilitates robust investigation of protein S-nitrosation across the proteome.

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Gao, L., X. Yan, X. Li, G. Guo, Y. Hu, W. Ma, and Y. Yan. "Proteome analysis of wheat leaf under salt stress by two-dimensional difference gel electrophoresis (2D-DIGE)." Phytochemistry 72, no. 10 (July 2011): 1180–91. http://dx.doi.org/10.1016/j.phytochem.2010.12.008.

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Morra, I., M. Leone, M. Forni, G. Mandili, and C. Zanini. "P04.20 * 2-D FLUORESCENCE DIFFERENCE GEL ELECTROPHORESIS (DIGE) AND MASS SPECTROMETRY ANALYSIS IN EPENDIMOMAS: PRELIMINARY DATA." Neuro-Oncology 16, suppl 2 (September 1, 2014): ii41. http://dx.doi.org/10.1093/neuonc/nou174.152.

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Swatton, J. E., S. Prabakaran, N. A. Karp, K. S. Lilley, and S. Bahn. "Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE)." Molecular Psychiatry 9, no. 2 (January 6, 2004): 128–43. http://dx.doi.org/10.1038/sj.mp.4001475.

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Tajima, Takashi, Fusako Kito, Akihiko Yoshida, Akira Kawai, and Tadashi Kondo. "Calreticulin as A Novel Potential Metastasis-Associated Protein in Myxoid Liposarcoma, as Revealed by Two-Dimensional Difference Gel Electrophoresis." Proteomes 7, no. 2 (April 10, 2019): 13. http://dx.doi.org/10.3390/proteomes7020013.

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Myxoid liposarcoma (MLS) is a mesenchymal malignancy. To identify innovate seeds for clinical applications, we examined the proteomes of primary tumor tissues from 10 patients with MLS with different statuses of postoperative metastasis. The protein expression profiles of tumor tissues were created, and proteins with differential expression associated with postoperative metastasis were identified by two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. The validation was performed using specific antibodies and in vitro analyses. Using 2D-DIGE, we observed 1726 protein species and identified proteins with unique expression levels in metastatic MLS. We focused on the overexpression of calreticulin in metastatic MLS. The higher expression of calreticulin was confirmed by Western blotting, and gene silencing assays demonstrated that reduced expression of calreticulin inhibited cell growth and invasion. Our findings suggested the important roles of calreticulin in MLS metastasis and supported its potential utility as a prognostic biomarker in MLS. Further investigations of the functional properties of calreticulin and other proteins identified in this study will improve our understanding of the biology of MLS and facilitate novel clinical applications.

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Yoshdia, Yutaka, and Tadashi Yamamoto. "Proteomic analysis of human kidney glomerulus with fluoresent 2D difference gel electrophoresis (2D DIGE) using saturation labeling." SEIBUTSU BUTSURI KAGAKU 50, no. 3Special (2006): 211–15. http://dx.doi.org/10.2198/sbk.50.3special_211.

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Henkel, C., M. Roderfeld, R. Weiskirchen, B. Scheibe, S. Matern, and E. Roeb. "Identification of Fibrosis-Relevant Proteins Using DIGE (Difference in Gel Electrophoresis) in Different Models of Hepatic Fibrosis." Zeitschrift für Gastroenterologie 43, no. 01 (January 13, 2005): 23–29. http://dx.doi.org/10.1055/s-2004-813911.

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Swatton, J. E., S. Prabakaran, N. A. Karp, K. S. Lilley, and S. Bahn. "Protein profiling of human post-mortem brain using two-dimensional fluorescence difference gel electrophoresis (2-D DIGE)." Molecular Psychiatry 9, no. 2 (January 27, 2004): 121. http://dx.doi.org/10.1038/sj.mp.4001480.

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FERNÁNDEZ-GARCÍA, AURORA, GEMA ALVAREZ-GARCÍA, VIRGINIA MARUGÁN-HERNÁNDEZ, PAULA GARCÍA-LUNAR, ADRIANA AGUADO-MARTÍNEZ, VERÓNICA RISCO-CASTILLO, and LUIS M. ORTEGA-MORA. "Identification ofBesnoitia besnoitiproteins that showed differences in abundance between tachyzoite and bradyzoite stages by difference gel electrophoresis." Parasitology 140, no. 8 (April 18, 2013): 999–1008. http://dx.doi.org/10.1017/s003118201300036x.

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SUMMARYBovine besnoitiosis is a chronic and debilitating disease, caused by the apicomplexan parasiteBesnoitia besnoiti. Infection of cattle byB. besnoitiis governed by the tachyzoite stage, which is related to acute infection, and the bradyzoite stage gathered into macroscopic cysts located in subcutaneous tissue in the skin, mucosal membranes and sclera conjunctiva and related to persistence and chronic infection. However, the entire life cycle of this parasite and the molecular mechanisms underlying tachyzoite-to-bradyzoite conversion remain unknown. In this context, a different antigenic pattern has been observed between tachyzoite and bradyzoite extracts. Thus, to identify stage-specific proteins, a difference gel electrophoresis (DIGE) approach was used on tachyzoite and bradyzoite extracts followed by mass spectrometry (MS) analysis. A total of 130 and 132 spots were differentially expressed in bradyzoites and tachyzoites, respectively (average ratio±1·5,P<0·05 int-test). Furthermore, 25 differentially expressed spots were selected and analysed by MALDI-TOF/MS. As a result, 5 up-regulated bradyzoite proteins (GAPDH, ENO1, LDH, SOD and RNA polymerase) and 5 up-regulated tachyzoite proteins (ENO2; LDH; ATP synthase; HSP70 and PDI) were identified. The present results set the basis for the identification of new proteins as drug targets. Moreover, the role of these proteins in tachyzoite-to-bradyzoite conversion and the role of the host cell environment should be a subject of further research.

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Jin, Mi, Nicolas Szapiel, Jennifer Zhang, John Hickey, and Sanchayita Ghose. "Profiling of host cell proteins by two-dimensional difference gel electrophoresis (2D-DIGE): Implications for downstream process development." Biotechnology and Bioengineering 105, no. 2 (February 1, 2010): 306–16. http://dx.doi.org/10.1002/bit.22532.

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Della Corte, Anna, Norma Maugeri, Agnieszka Pampuch, Chiara Cerletti, Giovanni de Gaetano, and Domenico Rotilio. "Application of 2-dimensional difference gel electrophoresis (2D-DIGE) to the study of thrombin-activated human platelet secretome." Platelets 19, no. 1 (January 2008): 43–50. http://dx.doi.org/10.1080/09537100701609035.

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Szulc, Małgorzata, and Alain Maquet. "Extraction Protocol for Winter Wheat Whole Grain Proteins Compatible with Two-Dimensional Fluorescence Difference Gel Electrophoresis (2D DIGE)." Cereal Chemistry Journal 86, no. 6 (November 2009): 692–94. http://dx.doi.org/10.1094/cchem-86-6-0692.

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Kondo, Tadashi, and Setsuo Hirohashi. "Application of highly sensitive fluorescent dyes (CyDye DIGE Fluor saturation dyes) to laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE) for cancer proteomics." Nature Protocols 1, no. 6 (December 2006): 2940–56. http://dx.doi.org/10.1038/nprot.2006.421.

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Schnaars, Vanessa, Marvin Dörries, Michael Hutchins, Lars Wöhlbrand, and Ralf Rabus. "What’s the Difference? 2D DIGE Image Analysis by DeCyder^TM versus SameSpots^TM." Journal of Molecular Microbiology and Biotechnology 28, no. 3 (2018): 128–36. http://dx.doi.org/10.1159/000494083.

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The efficiency and reproducibility of two-dimensional difference gel electrophoresis (2D DIGE) depends on several crucial steps: (i) adequate number of replicate gels, (ii) accurate image acquisition, and (iii) statistically confident protein abundance analysis. The latter is inherently determined by the image analysis system. Available software solutions apply different strategies for consecutive image alignment and protein spot detection. While DeCyderTM performs spot detection on single gels prior to the alignment of spot maps, SameSpotsTM completes image alignment in advance of spot detection. In this study, the performances of DeCyderTM and SameSpotsTM were compared considering all protein spots detected in 2D DIGE resolved proteomes of three different environmental bacteria with minimal user interference. Proteome map-based analysis by SameSpotsTM allows for fast and reproducible abundance change determination, avoiding time-consuming, manual spot matching. The different raw spot volumes, determined by the two software solutions, did not affect calculated abundance changes. Due to a slight factorial difference, minor abundance changes were very similar, while larger differences in the case of major abundance changes did not impact biological interpretation in the studied cases. Overall, affordable fluorescent dyes in combination with fast CCD camera-based image acquisition and user-friendly image analysis still qualify 2D DIGE as a valuable tool for quantitative proteomics.

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Ura, Blendi, Stefania Biffi, Lorenzo Monasta, Giorgio Arrigoni, Ilaria Battisti, Giovanni Di Lorenzo, Federico Romano, et al. "Two Dimensional-Difference in Gel Electrophoresis (2D-DIGE) Proteomic Approach for the Identification of Biomarkers in Endometrial Cancer Serum." Cancers 13, no. 14 (July 20, 2021): 3639. http://dx.doi.org/10.3390/cancers13143639.

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Endometrial cancer is the most common gynecologic malignancy arising from the endometrium. Identification of serum biomarkers could be beneficial for its early diagnosis. We have used 2D-Difference In Gel Electrophoresis (2D-DIGE) coupled with Mass Spectrometry (MS) procedures to investigate the serum proteome of 15 patients with endometrial cancer and 15 non-cancer subjects. We have identified 16 proteins with diagnostic potential, considering only spots with a fold change in %V ≥ 1.5 or ≤0.6 in intensity, which were statistically significant (p < 0.05). Western blotting data analysis confirmed the upregulation of CLU, ITIH4, SERPINC1, and C1RL in endometrial and exosome cancer sera compared to those of control subjects. The application of the logistic regression constructed based on the abundance of these four proteins separated the controls from the cancers with excellent levels of sensitivity and specificity. After a validation phase, our findings support the potential of using the proposed algorithm as a diagnostic tool in the clinical stage.

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Doliwa, Christelle, Dong Xia, Sandie Escotte-Binet, Emma L. Newsham, Sanderson Sanya J., Dominique Aubert, Nadine Randle, Jonathan M. Wastling, and Isabelle Villena. "Identification of differentially expressed proteins in sulfadiazine resistant and sensitive strains of Toxoplasma gondii using difference-gel electrophoresis (DIGE)." International Journal for Parasitology: Drugs and Drug Resistance 3 (December 2013): 35–44. http://dx.doi.org/10.1016/j.ijpddr.2012.12.002.

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Gao, Liyan, Aili Wang, Xiaohui Li, Kun Dong, Ke Wang, Rudi Appels, Wujun Ma, and Yueming Yan. "Wheat quality related differential expressions of albumins and globulins revealed by two-dimensional difference gel electrophoresis (2-D DIGE)." Journal of Proteomics 73, no. 2 (December 2009): 279–96. http://dx.doi.org/10.1016/j.jprot.2009.09.014.

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Brechlin, Peter, Olaf Jahn, Petra Steinacker, Lukas Cepek, Hartmut Kratzin, Stefan Lehnert, Sarah Jesse, et al. "Cerebrospinal fluid-optimized two-dimensional difference gel electrophoresis (2-D DIGE) facilitates the differential diagnosis of Creutzfeldt-Jakob disease." PROTEOMICS 8, no. 20 (September 22, 2008): 4357–66. http://dx.doi.org/10.1002/pmic.200800375.

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Jedmowski, Christoph, Ahmed Ashoub, Tobias Beckhaus, Thomas Berberich, Michael Karas, and Wolfgang Brüggemann. "Comparative Analysis of Sorghum bicolor Proteome in Response to Drought Stress and following Recovery." International Journal of Proteomics 2014 (October 1, 2014): 1–10. http://dx.doi.org/10.1155/2014/395905.

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The adaptive response of Sorghum bicolor landraces from Egypt to drought stress and following recovery was analyzed using two-dimensional difference gel electrophoresis, 2D-DIGE. Physiological measurements and proteome alterations of accession number 11434, drought tolerant, and accession number 11431, drought sensitive, were compared to their relative control values after drought stress and following recovery. Differentially expressed proteins were analysed by Matrix assisted laser desorption ionisation time-of-flight mass spectrometry, MALDI-TOF-MS. Alterations in protein contents related to the energy balance, metabolism (sensu Mewes et al. 1997), and chaperons were the most apparent features to elucidate the differences between the drought tolerant and sensitive accessions. Further alterations in the levels of proteins related to transcription and protein synthesis are discussed.

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Feugang, J. M., C. Rozanas, A. Kaya, E. Topper, and E. Memili. "100 PROTEOME OF BULL SPERMATOZOA." Reproduction, Fertility and Development 20, no. 1 (2008): 130. http://dx.doi.org/10.1071/rdv20n1ab100.

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The reproductive performance of a herd is the biggest factor affecting production and product quality of livestock. Thus, a decline of male fertility represents a dramatic economic loss in beef and dairy industries. Caused by molecular defects in the spermatozoa, uncompensatory infertility is a current challenge for the cattle industry, because even with normal sperm morphology, motility, and number, fertility of bulls is still sub-par. Thus, the objective of this study was to determine the global proteome of spermatozoa collected from bulls with different fertility and study the proteins playing a role in uncompensatory infertility. We performed difference gel electrophoresis (2D-DIGE) using cryopreserved sperm from a total of 6 bulls. Spermatozoa were thawed, purified by percoll gradient, and washed in PBS solution. For each bull, a pellet of 100 million sperm cells was resuspended in the 2D-DIGE labeling buffer, and the total protein was quantified. Cell lysates were separately labeled with CyDye DIGE Fluor dyes (and reciprocally with different dyes) and multiplexed in pairs on 3 gels. The samples were focused according to their isoelectric point through an Immobiline DryStrip, pH4-7, using a IPGphor 2, followed by separation on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (21.5–200 kDa) using a DALTsix. The resulting gels were scanned, and their topographic digital maps were used for algorithmic spot matching, background normalization, spot differences quantification, and elimination of artifacts (DeCyder 2-D Differential Analysis Software, GE Healthcare Life Sciences, Piscataway, NJ, USA). The results showed between 2600 and 2800 proteins with high confidence. Compared to the high-fertile bulls, 30 proteins were increased, and 27 were decreased in the low-fertility bulls within a 2-fold range. The largest significant increase and decrease were 3.97- and 2.4-fold, respectively. The identification of these differentially represented proteins is in progress. However, our results provided a panoramic view of sperm proteome from bulls of different fertility and thus paved the way for research on mechanisms of uncompensatory bull infertility.

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Arentz, Georgia, Tim Chataway, Mark R. Condina, Timothy J. Price, Peter Hoffmann, and Jennifer E. Hardingham. "Increased Phospho-Keratin 8 Isoforms in Colorectal Tumors Associated with EGFR Pathway Activation and Reduced Apoptosis." ISRN Molecular Biology 2012 (January 31, 2012): 1–8. http://dx.doi.org/10.5402/2012/706545.

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Hyperphosphorylated keratin (K) 8 acts as a phosphate “sponge” for stress-activated protein kinases thereby inhibiting pro-apoptotic molecules and thus apoptosis. MAP kinase/ERK1 has increased activity in colorectal cancer (CRC) and is known to phosphorylate K8. The aims were to identify the K8 isoforms abundantly present in colon tumors, using 2D difference gel electrophoresis (DIGE), to identify the modifications using mass spectrometry, and to validate the differential abundance of these isoforms in tumors relative to matched normal mucosae. 2D DIGE showed 3 isoforms of K8 significantly increased in tumor ≥2-fold in 6/8 pairs. Metal oxide affinity chromatography mass spectrometry and bioinformatics were used to identify phosphorylated serine residues. Levels of PS24, PS432, and PS74 by western blotting were found to be significantly increased in tumor versus matched normal. Blocking of EGFR signaling in Caco2 cells showed a significant decrease (P<0.0001) in K8 PS74 and PS432 levels by 59% and 66%, respectively, resulting in increased apoptosis.

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Nelson, M. M., A. R. Jones, J. C. Carmen, A. P. Sinai, R. Burchmore, and J. M. Wastling. "Modulation of the Host Cell Proteome by the Intracellular Apicomplexan Parasite Toxoplasma gondii." Infection and Immunity 76, no. 2 (October 29, 2007): 828–44. http://dx.doi.org/10.1128/iai.01115-07.

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ABSTRACT To investigate how intracellular parasites manipulate their host cell environment at the molecular level, we undertook a quantitative proteomic study of cells following infection with the apicomplexan parasite Toxoplasma gondii. Using conventional two-dimensional electrophoresis, difference gel electrophoresis (DIGE), and mass spectrometry, we identified host proteins that were consistently modulated in expression following infection. We detected modification of protein expression in key metabolic pathways, including glycolysis, lipid and sterol metabolism, mitosis, apoptosis, and structural-protein expression, suggestive of global reprogramming of cell metabolism by the parasite. Many of the differentially expressed proteins had not been previously implicated in the response to the parasite, while others provide important corroborative protein evidence for previously proposed hypotheses of pathogen-cell interactions. Significantly, over one-third of all modulated proteins were mitochondrial, and this was further investigated by DIGE analysis of a mitochondrion-enriched preparation from infected cells. Comparison of our proteomic data with previous transcriptional studies suggested that a complex relationship exits between transcription and protein expression that may be partly explained by posttranslational modifications of proteins and revealed the importance of investigating protein changes when interpreting transcriptional data. To investigate this further, we used phosphatase treatment and DIGE to demonstrate changes in the phosphorylation states of several key proteins following infection. Overall, our findings indicate that the host cell proteome responds in a dramatic way to T. gondii invasion, in terms of both protein expression changes and protein modifications, and reveal a complex and intimate molecular relationship between host and parasite.

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Wang, Da-Zhi, Hong-Po Dong, Cheng Li, Zhang-Xian Xie, Lin Lin, and Hua-Sheng Hong. "Identification and Characterization of Cell Wall Proteins of a Toxic DinoflagellateAlexandrium catenellaUsing 2-D DIGE and MALDI TOF-TOF Mass Spectrometry." Evidence-Based Complementary and Alternative Medicine 2011 (2011): 1–11. http://dx.doi.org/10.1155/2011/984080.

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The cell wall is an important subcellular component of dinoflagellate cells with regard to various aspects of cell surface-associated ecophysiology, but the full range of cell wall proteins (CWPs) and their functions remain to be elucidated. This study identified and characterized CWPs of a toxic dinoflagellate,Alexandrium catenella, using a combination of 2D fluorescence difference gel electrophoresis (DIGE) and MALDI TOF-TOF mass spectrometry approaches. Using sequential extraction and temperature shock methods, sequentially extracted CWPs and protoplast proteins, respectively, were separated fromA. catenella. From the comparison between sequentially extracted CWPs labeled with Cy3 and protoplast proteins labeled with Cy5, 120 CWPs were confidently identified in the 2D DIGE gel. These proteins gave positive identification of protein orthologues in the protein database usingde novosequence analysis and homology-based search. The majority of the prominent CWPs identified were hypothetical or putative proteins with unknown function or no annotation, while cell wall modification enzymes, cell wall structural proteins, transporter/binding proteins, and signaling and defense proteins were tentatively identified in agreement with the expected role of the extracellular matrix in cell physiology. This work represents the first attempt to investigate dinoflagellate CWPs and provides a potential tool for future comprehensive characterization of dinoflagellate CWPs and elucidation of their physiological functions.

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Corzett, Todd H., Imola K. Fodor, Megan W. Choi, Vicki L. Walsworth, Kenneth W. Turteltaub, Sandra L. McCutchen-Maloney, and Brett A. Chromy. "Statistical Analysis of Variation in the Human Plasma Proteome." Journal of Biomedicine and Biotechnology 2010 (2010): 1–12. http://dx.doi.org/10.1155/2010/258494.

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Quantifying the variation in the human plasma proteome is an essential prerequisite for disease-specific biomarker detection. We report here on the longitudinal and individual variation in human plasma characterized by two-dimensional difference gel electrophoresis (2-D DIGE) using plasma samples from eleven healthy subjects collected three times over a two week period. Fixed-effects modeling was used to remove dye and gel variability. Mixed-effects modeling was then used to quantitate the sources of proteomic variation. The subject-to-subject variation represented the largest variance component, while the time-within-subject variation was comparable to the experimental variation found in a previous technical variability study where one human plasma sample was processed eight times in parallel and each was then analyzed by 2-D DIGE in triplicate. Here, 21 protein spots had larger than 50% CV, suggesting that these proteins may not be appropriate as biomarkers and should be carefully scrutinized in future studies. Seventy-eight protein spots showing differential protein levels between different individuals or individual collections were identified by mass spectrometry and further characterized using hierarchical clustering. The results present a first step toward understanding the complexity of longitudinal and individual variation in the human plasma proteome, and provide a baseline for improved biomarker discovery.

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Baker, M. A., R. Witherdin, L. Hetherington, K. Cunningham-Smith, and J. Aitken. "209.A proteomic analysis of rat caput and cauda sperm using difference in 2D-gel electrophoresis." Reproduction, Fertility and Development 16, no. 9 (2004): 209. http://dx.doi.org/10.1071/srb04abs209.

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When testicular spermatozoa migrate to the epididymis they are still functionally incompetent. Having lost the cellular machinery to support gene transcription and protein translation, these cells acquire the gamut of biological functions needed to achieve fertilization under the influence of factors provided by the epididymal microenvironment. Although the biological changes exhibited by spermatozoa during epididymal transit have been well established, the molecular basis for these changes is still poorly understood. Difference in 2D Gel electrophoresis (DIGE) is a powerful new technology for comparing up to three different protein samples in the same 2D gel, thus eliminating the variation that occurs with more traditional proteomic approaches. Using a combination of this procedure and matrix assisted laser desorption ionisation time of flight (MALDI-TOF) mass spectrometry, we have unambiguously identified 8 proteins that change significantly during epididymal maturation including a-enolase, hsp60, endoplasmin, phosphatidyl-ethanolamine binding protein, testis lipid binding protein and the b-subunit of the F1 ATPase. The nature of these changes (80 kDa mass shift and increase electronegative charge) suggested a series of phosphorylation events. In order to further characterize these changes, Western blot studies were conducted using anti-phosphoserine antibodies. This analysis revealed a dramatic increase in serine phosphorylation for two major proteins (54 and 73 kDa) during epididymal transit, one of which (54 kDa) and confirmed by MALDI-TOF analysis to be the b-subunit of the mitochondrial ATPase. The phosphorylation of this protein was associated with a 3-fold increase in the ATP content of epididymal spermatozoa as they pass from the caput to the cauda epididymis.� This change clearly identifies mitochondrial ATP production as a key component of the epididymal changes that lead to the generation of vigorously motile functional spermatozoa. The kinase responsible for the F1-ATPase phosphorylation appears to be PKA regulated and a clear potential target for contraceptive intervention.

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Corbett, Mark, Sam Virtue, Kenneth Bell, Paul Birch, Tom Burr, Lysbeth Hyman, Kathryn Lilley, Susannah Poock, Ian Toth, and George Salmond. "Identification of a New Quorum-Sensing-Controlled Virulence Factor in Erwinia carotovora subsp. atroseptica Secreted via the Type II Targeting Pathway." Molecular Plant-Microbe Interactions® 18, no. 4 (April 2005): 334–42. http://dx.doi.org/10.1094/mpmi-18-0334.

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Two-dimensional polyacrylamide gel electrophoresis of the secreted proteins of Erwinia carotovora subsp. atroseptica revealed alow-abundance protein that was identified by mass spectrometry as a homologue of a Xanthomonas campestris avirulence protein with unknown function. The predicted Svx protein has an N-terminal signal sequence and zinc binding-region signature, and the mature protein is post-translationally modified. A 2D difference gel electrophoresis (DIGE) showed that the protein is secreted by the type II (out) secretion apparatus, which is also responsible for the secretion of the major known virulence factors, PelC and CelV. Transcription of the svx gene is under Nacyl- homoserine lactone-mediated quorum-sensing control. The svx gene was inactivated by transposon insertion. The mutant showed a decrease in virulence in potato plant assays, demonstrating a role for Svx in the pathogenicity of E. carotovora subsp. atroseptica. These results show that Svx is a previously unidentified virulence determinant which is secreted by the out machinery and is regulated by quorum sensing, two systems employed by several other virulence factors. Thus, the type II secretory machine is a conduit for virulence factors other than the main pectinnases and cellulase in E. carotovora subsp. atroseptica.

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Li, Xiaomin, Zhao Jin, Fei Gao, Jian Lu, Guolin Cai, Jianjun Dong, Junhong Yu, and Mei Yang. "Comparative Proteomic Analysis of Dan’er Malts Produced from Distinct Malting Processes by Two-Dimensional Fluorescence Difference in Gel Electrophoresis (2D-DIGE)." Journal of Agricultural and Food Chemistry 62, no. 38 (September 15, 2014): 9310–16. http://dx.doi.org/10.1021/jf5030483.

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Carvajal-Serna, Melissa, Meriem Fatnassi, Felipe Torres-Ruda, Jaime Antonio Cardozo, Henry Grajales-Lombana, Mohamed Hammadi, Jose Alfonso Abecia, et al. "Vasectomy and Photoperiodic Regimen Modify the Protein Profile, Hormonal Content and Antioxidant Enzymes Activity of Ram Seminal Plasma." International Journal of Molecular Sciences 21, no. 21 (October 29, 2020): 8063. http://dx.doi.org/10.3390/ijms21218063.

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This work aimed to determine the contribution of the testis and epididymis and the effect of the photoperiodic regimen on ram seminal plasma (SP). Semen was collected from 15 mature rams located in an equatorial (Colombian Creole and Romney Marsh, eight intact and two vasectomized) or a temperate climate (Rasa Aragonesa, three intact and two vasectomized). SP proteins were analyzed by Bradford, SDS-PAGE and difference gel electrophoresis (DIGE). Melatonin and testosterone concentrations were quantified by ELISA, and activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase by enzymatic assays. Vasectomy increased protein concentration and the intensity of high molecular weight bands (p < 0.001), with no differences between breeds. DIGE revealed the absence of six proteins in vasectomized rams: angiotensin-converting enzyme, lactotransferrin, phosphoglycerate kinase, sorbitol dehydrogenase, epididymal secretory glutathione peroxidase and epididymal secretory protein E1. Vasectomy also decreased melatonin concentrations in seasonal rams, and testosterone in all of them (p < 0.001), but did not affect antioxidant enzyme activity. Equatorial rams showed lower melatonin and testosterone concentration (p < 0.01) and catalase, but higher GPx activity (p < 0.05). In conclusion, vasectomy modifies the protein profile and hormonal content of ram seminal plasma, whereas the exposure to a constant photoperiod affects hormonal concentration and antioxidant enzymes activity.

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Martin, Sandra L., L. Elaine Epperson, James C. Rose, Courtney C. Kurtz, Cécile Ané, and Hannah V. Carey. "Proteomic analysis of the winter-protected phenotype of hibernating ground squirrel intestine." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 295, no. 1 (July 2008): R316—R328. http://dx.doi.org/10.1152/ajpregu.00418.2007.

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The intestine of hibernating ground squirrels is protected against damage by ischemia-reperfusion (I/R) injury. This resistance does not depend on the low body temperature of torpor; rather, it is exhibited during natural interbout arousals that periodically return hibernating animals to euthermia. Here we use fluorescence two-dimensional difference gel electrophoresis (DIGE) to identify protein spot differences in intestines of 13-lined ground squirrels in the sensitive and protected phases of the circannual hibernation cycle, comparing sham-treated control animals with those exposed to I/R. Protein spot differences distinguished the sham-treated summer and hibernating samples, as well as the response to I/R between summer and hibernating intestines. The majority of protein changes among these groups were attributed to a seasonal difference between summer and winter hibernators. Many of the protein spots that differed were unambiguously identified by high-pressure liquid chromatography followed by tandem mass spectrometry of their constituent peptides. Western blot analysis confirmed significant upregulation for three of the proteins, albumin, apolipoprotein A-I, and ubiquitin hydrolase L1, that were identified in the DIGE analysis as increased in sham-treated hibernating squirrels compared with sham-treated summer squirrels. This study identifies several candidate proteins that may contribute to hibernation-induced protection of the gut during natural torpor-arousal cycles and experimental I/R injury. It also reveals the importance of enterocyte maturation in defining the hibernating gut proteome and the role of changing cell populations for the differences between sham and I/R-treated summer animals.

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Gemoll, Timo, Svitlana Rozanova, Christian Röder, Sonja Hartwig, Holger Kalthoff, Stefan Lehr, Abdou ElSharawy, and Jens Habermann. "Protein Profiling of Serum Extracellular Vesicles Reveals Qualitative and Quantitative Differences after Differential Ultracentrifugation and ExoQuick™ Isolation." Journal of Clinical Medicine 9, no. 5 (May 12, 2020): 1429. http://dx.doi.org/10.3390/jcm9051429.

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Solid tumor biopsies are the current standard for precision medicine. However, the procedure is invasive and not always feasible. In contrast, liquid biopsies, such as serum enriched for extracellular vesicles (EVs) represent a non-invasive source of cancer biomarkers. In this study, we compared two EV isolation methods in the context of the protein biomarker detection in inflammatory bowel disease (IBD) and colorectal cancer (CRC). Using serum samples of a healthy cohort as well as CRC and IBD patients, EVs were isolated by ultracentrifugation and ExoQuick™ in parallel. EV associated protein profiles were compared by multiplex-fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and subsequent identification by mass spectrometry. Validation of gelsolin (GSN) was performed using fluorescence-quantitative western blot. 2D-DIGE resolved 936 protein spots in all serum-enriched EVs isolated by ultracentrifugation or ExoQuick™. Hereof, 93 spots were differently expressed between isolation approaches. Higher levels of GSN in EVs obtained with ExoQuick™ compared to ultracentrifugation were confirmed by western blot (p = 0.0006). Although patient groups were distinguishable after both EV isolation approaches, sample preparation strongly influences EVs’ protein profile and thus impacts on inter-study reproducibility, biomarker identification and validation. The results stress the need for strict SOPs in EV research before clinical implementation can be reached.

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Tian, Zhongmin, Yu Wang, Chenyang Zhao, Tianshu Wang, Entai Hou, and Na Sun. "Qualitative and Quantitative Analysis of Phosphoproteomic Experimental Workflow Based on Phosphoprotein Enrichment Strategy and Two- Dimensional Difference Gel Electrophoresis (2D-DIGE) Techniques." Current Proteomics 11, no. 4 (January 21, 2015): 252–63. http://dx.doi.org/10.2174/157016461104150121114414.

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Oliva, Karen, Gillian Barker, Gregory E. Rice, Mark J. Bailey, and Martha Lappas. "2D-DIGE to identify proteins associated with gestational diabetes in omental adipose tissue." Journal of Endocrinology 218, no. 2 (May 24, 2013): 165–78. http://dx.doi.org/10.1530/joe-13-0010.

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Gestational diabetes mellitus (GDM) is a significant risk factor for the type 2 diabetes epidemic in many populations. Maternal adipose tissue plays a central role in the pathophysiology of GDM. Thus, the aim of this study was to determine the effect of GDM on the proteome of adipose tissue. Omental adipose tissue was obtained at the time of term Caesarean section from women with normal glucose tolerance (NGT) or GDM. 2D-difference gel electrophoresis (DIGE), followed by mass spectrometry, was used to identify protein spots (n=6 patients per group). Western blotting was used for confirmation of six of the spot differences (n=6 patients per group). We found 14 proteins that were differentially expressed between NGT and GDM adipose tissue (≥1.4-fold,P<0.05). GDM was associated with an up-regulation of four proteins: collagen alpha-2(VI) chain (CO6A2 (COL6A2)), fibrinogen beta chain (FIBB (FGB)), lumican (LUM) and S100A9. On the other hand, a total of ten proteins were found to be down-regulated in adipose tissue from GDM women. These were alpha-1-antitrypsin (AIAT (SERPINA1)), annexin A5 (ANXA5), fatty acid-binding protein, adipocyte (FABP4), glutathione S-transferase P (GSTP (GSTP1)), heat-shock protein beta-1 (HSP27 (HSPB1)), lactate dehydrogenase B chain (LDHB), perilipin-1 (PLIN1), peroxiredoxin-6 (PRX6 (PRDX6)), selenium-binding protein 1 (SBP1) and vinculin (VINC (VCL)). In conclusion, proteomic analysis of omental fat reveals differential expression of several proteins in GDM patients and NGT pregnant women. This study revealed differences in expression of proteins that are involved in inflammation, lipid and glucose metabolism and oxidative stress and added further evidence to support the role of visceral adiposity in the pathogenesis of GDM.

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Fröhlich, T., R. Kashirin, P. Bolbrinker, H. D. Reichenbach, E. Wolf, and G. J. Arnold. "104 ESTROUS CYCLE-DEPENDENT CHANGES IN THE BOVINE ENDOMETRIUM PROTEOME." Reproduction, Fertility and Development 21, no. 1 (2009): 152. http://dx.doi.org/10.1071/rdv21n1ab104.

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Among the reproductive tissues, the endometrium plays a central role in the context of embryo-maternal communication and pregnancy recognition. During the estrous cycle, characteristic morphological and functional changes occur in the bovine endometrium, being crucial for uterine receptivity. These changes are mainly regulated by the hormones progesterone, estradiol, and oxytocin. The bovine estrous cycle, with a length of 21 days, can be divided into 4 stages: i) estrus (considered as Day 0, low progesterone level and time of ovulation); ii) metestrus (Days 1 to 5, corpus luteum formation, rising progesterone level); iii) diestrus (Days 6 to 17, high progesterone); and iv) proestrus (Days 18 to 20, corpus luteum degeneration; declining progesterone). The principles of hormonal regulation during the estrous cycle are well understood; however, in-depth knowledge of the detailed molecular mechanisms is still incomplete. To elucidate the underlying biochemical processes, the proteomes of bovine endometrial samples of all 4 stages (cycle Day 0, Day 3.5, Day 12, and Day 18) were compared in a quantitative manner. To maximize the accuracy of protein quantification, sophisticated 2-dimensional (2D) fluorescence difference gel electrophoresis (2D-DIGE) experiments were performed, using internal pooled standards for inter-gel normalization. To enhance the resolution of 2D-polyacrylamide gel electrophoresis separation, the proteins were analyzed by 2 overlapping pH gradients. In total, 28 individual DIGE experiments (14 2D gels × 2 pH gradients) were performed, corresponding to 84 gel images. With a refined statistical analysis of spot intensities, we were able to identify a total of 91 spots altered by at least a factor of ±2 (P < 0.05) in intensity between at least 2 of the 4 stages. Matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) identification of these spots showed that they originated from 66 different proteins. Moreover, for 14 of these proteins, several polymorphic variants could be identified. Gene ontology analysis of the protein IDs revealed a broad diversity of biological and biochemical functions as well as cellular localizations of these proteins. Several proteins detected (e.g. FK506 and 20 alpha-HSD) are crucial components for uterine receptivity and represent interesting targets for further functional studies. This study was supported by the Deutsche Forschungsgemeinschaft (FOR 478).

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Hu, Jian Da, Min-Hui Lin, Xin-Ji Chen, Ting-Bo Liu, and Lian-Huang Lv. "Differential Protein Expressions Between Leukemia HL-60 and Adriamycin-Resistant HL-60 Cell Lines by Comparative Preteomic Analysis." Blood 112, no. 11 (November 16, 2008): 5052. http://dx.doi.org/10.1182/blood.v112.11.5052.5052.

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Abstract Resistance is a major problem of chemotherapy failure in acute leukemia. Although multi-drug resistance is an important factor, the exact mechanisms of resistance remain to be clarified. With the aim of better understanding the protein involvement in development of resistance mechanisms, a comparative proteomic analysis——two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) was used to compare differential protein expression profiles in adriamycin-resistant acute myeloid leukemia (AML) HL-60/ADR and sensitive HL-60 cell line. Total cellular proteins extracted from HL-60 and adriamycin- resistant HL-60 cells were separated by 2-D gel electrophoresis.Differential expressed proteins were analyzed by mass spectrometry (MALDI-TOF/TOF) and database searching. 16 significantly differentially expressed protein spots were identified, among which 13 protein spots were identified as up-regulated and 3 as down-regulated. The identified proteins were categorized into: (±)metabolic enzymes; (II)proteins related with signal transduction;(III)cell cycle regulators; and(‡W)cellular proliferation and apoptosis proteins. Some of these differential protein expression were confirmed by western blot. In addition, we investigated the expression of distinct proteins in the primary leukemia cells. The results revealed that nucleolar phosphoprotein (B23) over-expressed in the relapsed patients with acute monocytic leukemia (M5). It suggests that B23 might be a useful indicator of prognosis of M5, but the exact role in resistant mechanism is still to be investigated. 2-D DIGE is a useful approach to investigate differential protein expression related treatment resistance.

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Passmore, Ian J., Kahoko Nishikawa, Kathryn S. Lilley, Steven D. Bowden, Jade C. S. Chung, and Martin Welch. "Mep72, a Metzincin Protease That Is Preferentially Secreted by Biofilms of Pseudomonas aeruginosa." Journal of Bacteriology 197, no. 4 (December 8, 2014): 762–73. http://dx.doi.org/10.1128/jb.02404-14.

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In this work, we compared the profile of proteins secreted by planktonic and biofilm cultures ofPseudomonas aeruginosausing two-dimensional difference gel electrophoresis (2D-DiGE). This revealed that a novel metzincin protease, Mep72, was secreted during biofilm growth. Subsequent Western blotting and reverse transcription-PCR (RT-PCR) analyses demonstrated that Mep72 was expressed only during biofilm growth. Mep72 has a tridomain structure comprised of a metzincin protease-like domain and two tandem carbohydrate-binding domains. Unlike the only other metzincin (alkaline protease; AprA) inP. aeruginosa, Mep72 is secreted through the type II pathway and undergoes processing during export. During this processing, the metzincin domain is liberated from the carbohydrate-binding domains. This processing may be self-catalyzed, since purified Mep72 autodegradedin vitro. This autodegradation was retarded in the presence of alginate (an extracellular matrix component of manyP. aeruginosabiofilms). The expression of full-lengthmep72inEscherichia coliwas toxic. However, this toxicity could be alleviated by coexpression ofmep72with the adjacent gene,bamI. Mep72 and BamI were found to form a protein-protein complexin vitro. 2D-DiGE revealed that the electrophoretic mobility of several discrete protein spots was altered in the biofilm secretome of anmep72mutant, including type III secretion proteins (PopD, PcrV, and ExoS) and a flagellum-associated protein (FliD). Mep72 was found to bind directly to ExoS and PcrV and to affect the processing of these proteins in the biofilm secretome. We conclude that Mep72 is a secreted biofilm-specific regulator that affects the processing of a very specific subset of virulence factors.

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Plumel, Marine, Stéphanie Dumont, Pauline Maes, Cristina Sandu, Marie-Paule Felder-Schmittbuhl, Etienne Challet, and Fabrice Bertile. "Circadian Analysis of the Mouse Cerebellum Proteome." International Journal of Molecular Sciences 20, no. 8 (April 15, 2019): 1852. http://dx.doi.org/10.3390/ijms20081852.

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The cerebellum contains a circadian clock, generating internal temporal signals. The daily oscillations of cerebellar proteins were investigated in mice using a large-scale two-dimensional difference in gel electrophoresis (2D-DIGE). Analysis of 2D-DIGE gels highlighted the rhythmic variation in the intensity of 27/588 protein spots (5%) over 24 h based on cosinor regression. Notably, the rhythmic expression of most abundant cerebellar proteins was clustered in two main phases (i.e., midday and midnight), leading to bimodal distribution. Only six proteins identified here to be rhythmic in the cerebellum are also known to oscillate in the suprachiasmatic nuclei, including two proteins involved in the synapse activity (Synapsin 2 [SYN2] and vesicle-fusing ATPase [NSF]), two others participating in carbohydrate metabolism (triosephosphate isomerase (TPI1] and alpha-enolase [ENO1]), Glutamine synthetase (GLUL), as well as Tubulin alpha (TUBA4A). Most oscillating cerebellar proteins were not previously identified in circadian proteomic analyses of any tissue. Strikingly, the daily accumulation of mitochondrial proteins was clustered to the mid-resting phase, as previously observed for distinct mitochondrial proteins in the liver. Moreover, a number of rhythmic proteins, such as SYN2, NSF and TPI1, were associated with non-rhythmic mRNAs, indicating widespread post-transcriptional control in cerebellar oscillations. Thus, this study highlights extensive rhythmic aspects of the cerebellar proteome.

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Al-Khalili, Lubna, Thais de Castro Barbosa, Jörgen Östling, Julie Massart, Pablo Garrido Cuesta, Megan E. Osler, Mutsumi Katayama, Ann-Christin Nyström, Jan Oscarsson, and Juleen R. Zierath. "Proteasome inhibition in skeletal muscle cells unmasks metabolic derangements in type 2 diabetes." American Journal of Physiology-Cell Physiology 307, no. 9 (November 1, 2014): C774—C787. http://dx.doi.org/10.1152/ajpcell.00110.2014.

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Two-dimensional difference gel electrophoresis (2-D DIGE)-based proteome analysis has revealed intrinsic insulin resistance in myotubes derived from type 2 diabetic patients. Using 2-D DIGE-based proteome analysis, we identified a subset of insulin-resistant proteins involved in protein turnover in skeletal muscle of type 2 diabetic patients, suggesting aberrant regulation of the protein homeostasis maintenance system underlying metabolic disease. We then validated the role of the ubiquitin-proteasome system (UPS) in myotubes to investigate whether impaired proteasome function may lead to metabolic arrest or insulin resistance. Myotubes derived from muscle biopsies obtained from people with normal glucose tolerance (NGT) or type 2 diabetes were exposed to the proteasome inhibitor bortezomib (BZ; Velcade) without or with insulin. BZ exposure increased protein carbonylation and lactate production yet impaired protein synthesis and UPS function in myotubes from type 2 diabetic patients, marking the existence of an insulin-resistant signature that was retained in cultured myotubes. In conclusion, BZ treatment further exacerbates insulin resistance and unmasks intrinsic features of metabolic disease in myotubes derived from type 2 diabetic patients. Our results highlight the existence of a confounding inherent abnormality in cellular protein dynamics in metabolic disease, which is uncovered through concurrent inhibition of the proteasome system.

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Journal articles on the topic 'Difference gel electrophoresis (DIGE)'

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