Journal articles: '2D-DIGE ( Two Dimensional Differential In Gel Electrophoresis)'

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Ercan, Huriye, Ulrike Resch, Felicia Hsu, Goran Mitulovic, Andrea Bileck, Christopher Gerner, Jae-Won Yang, Margarethe Geiger, Ingrid Miller, and Maria Zellner. "A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection." Cells 12, no. 5 (February 26, 2023): 747. http://dx.doi.org/10.3390/cells12050747.

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Proteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quantitative performance of these two fundamentally different methodologies by the parallel measurement of six technical and three biological replicates of the human prostate carcinoma cell line DU145 using its two most common standard techniques, label-free shotgun and two-dimensional differential gel electrophoresis (2D-DIGE). The analytical strengths and limitations were explored, finally focusing on the unbiased detection of proteoforms, exemplified by discovering a prostate cancer-related cleavage product of pyruvate kinase M2. Label-free shotgun proteomics quickly yields an annotated proteome but with reduced robustness, as determined by three times higher technical variation compared to 2D-DIGE. At a glance, only 2D-DIGE top-down analysis provided valuable, direct stoichiometric qualitative and quantitative information from proteins to their proteoforms, even with unexpected post-translational modifications, such as proteolytic cleavage and phosphorylation. However, the 2D-DIGE technology required almost 20 times as much time per protein/proteoform characterization with more manual work. Ultimately, this work should expose both techniques’ orthogonality with their different contents of data output to elucidate biological questions.

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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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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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Di Carli, Mariasole, Anita Zamboni, Mario Enrico Pè, Mario Pezzotti, Kathryn S. Lilley, Eugenio Benvenuto, and Angiola Desiderio. "Two-Dimensional Differential in Gel Electrophoresis (2D-DIGE) Analysis of Grape Berry Proteome during Postharvest Withering." Journal of Proteome Research 10, no. 2 (February 4, 2011): 429–46. http://dx.doi.org/10.1021/pr1005313.

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Dhingra, V., Q. Li, A. B. Allison, D. E. Stallknecht, and Z. F. Fu. "Proteomic Profiling and Neurodegeneration in West-Nile-Virus-Infected Neurons." Journal of Biomedicine and Biotechnology 2005, no. 3 (2005): 271–79. http://dx.doi.org/10.1155/jbb.2005.271.

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West Nile virus, a mosquito-borne flavivirus, is a human, equine, and avian pathogen. High-resolution two-dimensional differential-gel electrophoresis (2D-DIGE) was used to characterize protein expression in primary rat neurons and to examine the proteomic profiling to understand the pathogenesis of West-Nile-associated meningoencephalitis. Three pH ranges, 3–10, 4–7, and 5–6, were used to analyze the protein spots. The proteins are labeled with fluorescent dyes Cy3 and Cy5 before being separated on the basis of charge and size respectively on a two-dimensional platform. About 55 proteins showed altered expression levels. These were then subsequently digested and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis using peptide mass fingerprinting and database searching. These cellular proteins could represent distinct roles during infection related to apoptosis. Our findings show that two-dimensional differential gel electrophoresis combined with mass spectrometry is a powerful approach that permits the identification of proteins whose expression was altered due to West Nile virus infection.

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Orenes-Piñero, Esteban, Marta Cortón, Pilar González-Peramato, Ferrán Algaba, Ignacio Casal, Alvaro Serrano, and Marta Sánchez-Carbayo. "Searching Urinary Tumor Markers for Bladder Cancer Using a Two-Dimensional Differential Gel Electrophoresis (2D-DIGE) Approach." Journal of Proteome Research 6, no. 11 (November 2007): 4440–48. http://dx.doi.org/10.1021/pr070368w.

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Wang, X., A. Kaya, and E. Memili. "195 SPERMATOZOAL PROTEIN MARKERS FOR ANGUS BULL FERTILITY." Reproduction, Fertility and Development 23, no. 1 (2011): 198. http://dx.doi.org/10.1071/rdv23n1ab195.

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Fertility is one of the most economically important traits controlling animal reproduction. Despite its significant economic impact, there are no reliable markers to predict semen quality. The objective of this study was to identify spermatozoal proteins associated with bull fertility, the ability of the sperm to fertilize the oocyte and support embryonic development. To accomplish our objectives, we isolated total spermatozoal proteins from 4 Angus bulls with different fertility phenotypes. Next, differentially expressed proteins were determined using two-dimensional differential in-gel electrophoresis (2D-DIGE), followed by sequencing of the most differentially expressed proteins. Immunoblotting experiments were conducted to confirm expression of key proteins detected by 2D-DIGE. Our results from 2D-DIGE experiments showed approximately 2000 detectable spermatozoal proteins. Of these comprehensive lists of proteins, we identified 80 of the proteins with the highest differential expression in spermatozoa from high- and low-fertility bulls. Diverse sets of differentially expressed proteins known to play roles in sperm motility, metabolism, and cell morphology were identified. These proteins included outer dense fibre of sperm tails 2 and manganous superoxide, heat shock protein, tubulins, α-enolase, and acrosomal vesicle protein-1. Expression profiles of outer dense fibre of sperm tails 2 and manganous superoxide dismutase were confirmed using immunoblotting. The findings are significant because they help us understand the fundamental biology of the male gamete and early development. In addition, the identified proteins can be used as molecular markers to predict bull fertility, an economically important trait. Funded in part by the Mississippi Agricultural and Forestry Experiment Station, American Angus Association, and Alta Genetics, Inc.

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Martínez-Gomariz, M., M. L. HernÁez, D. GutiÉrrez, P. XimÉnez-EmbÚn, and G. PrÉstamo. "Proteomic Analysis by Two-Dimensional Differential Gel Electrophoresis (2D DIGE) of a High-Pressure Effect in Bacillus cereus." Journal of Agricultural and Food Chemistry 57, no. 9 (May 13, 2009): 3543–49. http://dx.doi.org/10.1021/jf803272a.

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Subong, Bryan John J., Arturo O. Lluisma, Rhodora V. Azanza, and Lilibeth A. Salvador-Reyes. "Differentiating Two Closely Related Alexandrium Species Using Comparative Quantitative Proteomics." Toxins 13, no. 1 (December 23, 2020): 7. http://dx.doi.org/10.3390/toxins13010007.

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Alexandrium minutum and Alexandrium tamutum are two closely related harmful algal bloom (HAB)-causing species with different toxicity. Using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics and two-dimensional differential gel electrophoresis (2D-DIGE), a comprehensive characterization of the proteomes of A. minutum and A. tamutum was performed to identify the cellular and molecular underpinnings for the dissimilarity between these two species. A total of 1436 proteins and 420 protein spots were identified using iTRAQ-based proteomics and 2D-DIGE, respectively. Both methods revealed little difference (10–12%) between the proteomes of A. minutum and A. tamutum, highlighting that these organisms follow similar cellular and biological processes at the exponential stage. Toxin biosynthetic enzymes were present in both organisms. However, the gonyautoxin-producing A. minutum showed higher levels of osmotic growth proteins, Zn-dependent alcohol dehydrogenase and type-I polyketide synthase compared to the non-toxic A. tamutum. Further, A. tamutum had increased S-adenosylmethionine transferase that may potentially have a negative feedback mechanism to toxin biosynthesis. The complementary proteomics approach provided insights into the biochemistry of these two closely related HAB-causing organisms. The identified proteins are potential biomarkers for organismal toxicity and could be explored for environmental monitoring.

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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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Huang, Cheng-Yi, Ko-Chao Lee, Shui-Yi Tung, Wen-Shin Huang, Chih-Chuan Teng, Kam-Fai Lee, Meng-Chiao Hsieh, and Hsing-Chun Kuo. "2D-DIGE-MS Proteomics Approaches for Identification of Gelsolin and Peroxiredoxin 4 with Lymph Node Metastasis in Colorectal Cancer." Cancers 14, no. 13 (June 29, 2022): 3189. http://dx.doi.org/10.3390/cancers14133189.

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Background/Aims: A combination of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry approach was used to search for potential markers for prognosis and intervention of colorectal cancer (CRC) at different stages of lymph node metastasis (LMN). This quantitative proteomic survey aimed to investigate the LNM-associated proteins and evaluate the clinicopathological characteristics of these target proteins in CRC from stage I to stage IV. Methods: Sixteen CRC cases were categorized into paired non-LNM and LNM groups, and two-dimensional difference gel electrophoresis and MS proteome analysis were performed. Differential protein expression between non-LNM and LNM CRC was further validated in a tissue microarray, including 40 paraffin-embedded samples by immunohistochemistry staining. Moreover, a Boyden chamber assay, flow cytometry, and shRNA were used to examine the epithelial–mesenchymal transition and mechanism invasiveness of the differentially expressed proteins in DLD-1 cells and in vivo xenograft mouse model. Results: Eighteen differentially expressed proteins were found between non-LNM and LNM CRC tissues. Among them, protein levels of Gelsolin (GSN) and peroxiredoxin 4 (PRDX4) were abundant in node-positive CRC. Downregulation of GSN and PRDX4 markedly suppressed migration and invasiveness and also induced cell cycle G1/S arrest in DLD-1. Mechanistically, the EGFR/RhoA/PKCα/ERK pathways are critical for transcriptional activation of histone modification of H3 lysine 4 trimethylation (H3K4me3) of GSN and PRDX4 promoters, resulting in upregulation of GSN, PRDX4, Twist-1/2, cyclinD1, proliferating cell-nuclear antigen, β-catenin, N-cadherin, and matrix metalloprotein-9. Conclusions: GSN and PRDX4 are novel regulators in CRC lymph node metastasis to potentially provide new insights into the mechanism of CRC progression and serve as a biomarker for CRC diagnosis at the metastatic stage.

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Ferreira, Tamara Aparecida Reis, Hélida Monteiro de Andrade, Paulo Madureira de Pádua, Maria das Graças Carvalho, Simone da Fonseca Pires, Ivana Helena Rocha Oliveira, Bruna Soares Souza Lima, et al. "Identification of potential biomarkers for systemic lupus erythematosus diagnosis using two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry." Autoimmunity 50, no. 4 (May 19, 2017): 247–56. http://dx.doi.org/10.1080/08916934.2017.1344975.

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Repetto, Ombretta, Valli De Re, Paolo Giuffrida, Marco Vincenzo Lenti, Raffaella Magris, Marino Venerito, Agostino Steffan, Antonio Di Sabatino, and Renato Cannizzaro. "Proteomics signature of autoimmune atrophic gastritis: towards a link with gastric cancer." Gastric Cancer 24, no. 3 (February 23, 2021): 666–79. http://dx.doi.org/10.1007/s10120-020-01148-3.

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Abstract Background Autoimmune atrophic gastritis (AAG) is a chronic disease that can progress to gastric cancer (GC). To better understand AAG pathology, this proteomics study investigated gastric proteins whose expression levels are altered in this disease and also in GC. Methods Using two-dimensional difference gel electrophoresis (2D-DIGE), we compared protein maps of gastric corpus biopsies from AAG patients and controls. Differentially abundant spots (|fold change|≥ 1.5, P < 0.01) were selected and identified by LC–MS/MS. The spots were further assessed in gastric antrum biopsies from AAG patients (without and with Helicobacter pylori infection) and from GC patients and unaffected first-degree relatives of GC patients. Results 2D-DIGE identified 67 differentially abundant spots, with 28 more and 39 less abundant in AAG-corpus than controls. LC–MS/MS identified these as 53 distinct proteins. The most significant (adjusted P < 0.01) biological process associated with the less abundant proteins was “tricarboxylic acid cycle”. Of the 67 spots, 57 were similarly differentially abundant in AAG-antrum biopsies irrespective of H. pylori infection status. The differential abundance was also observed in GC biopsies for 14 of 28 more abundant and 35 of 39 less abundant spots, and in normal gastric biopsies of relatives of GC patients for 6 and 25 spots, respectively. Immunoblotting confirmed the different expression levels of two more abundant proteins (PDIA3, GSTP gene products) and four less abundant proteins (ATP5F1A, PGA3, SDHB, PGC). Conclusion This study identified a proteomics signature of AAG. Many differential proteins were shared by GC and may be involved in the progression of AAG to GC.

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Castillejo, Ma Ángeles, Mónica Fernández-Aparicio, and Diego Rubiales. "Proteomic analysis by two-dimensional differential in gel electrophoresis (2D DIGE) of the early response of Pisum sativum to Orobanche crenata." Journal of Experimental Botany 63, no. 1 (September 14, 2011): 107–19. http://dx.doi.org/10.1093/jxb/err246.

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Li, Fei, Ding-nan Chen, Cheng-wu He, You Zhou, Vesa M. Olkkonen, Nan He, Wei Chen, et al. "Identification of urinary Gc-globulin as a novel biomarker for bladder cancer by two-dimensional fluorescent differential gel electrophoresis (2D-DIGE)." Journal of Proteomics 77 (December 2012): 225–36. http://dx.doi.org/10.1016/j.jprot.2012.09.002.

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Sabha, Bassam H., Afshan Masood, Ibrahim O. Alanazi, Assim A. Alfadda, Hussein A. Almehdar, Hicham Benabdelkamel, and Elrashdy M. Redwan. "Comparative Analysis of Milk Fat Globular Membrane (MFGM) Proteome between Saudi Arabia Camelus dromedary Safra and Wadha Breeds." Molecules 25, no. 9 (May 4, 2020): 2146. http://dx.doi.org/10.3390/molecules25092146.

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Camel milk is traditionally known to have medicinal properties and many potential health benefits. Natural milk contains many soluble proteins and nanoparticles, such as a milk fat globule membrane (MFGM), a three-layered membrane covering of milk fat globule mainly composed of proteins and lipids, which plays an important role in human health. MFGM proteins account for 1%–4% of total milk proteins, and their nutritive value and distribution depends on the different breeds. The differential composition of these membrane proteins among different camel breeds has not been explored. The current study, therefore, aimed to quantitatively analyze and compare the MFGM proteome between the milk produced by the two most common Saudi camel breeds, Camelus dromedarius: Safra and Wadha. Two-dimensional difference in gel electrophoresis (2D-DIGE) and mass spectrometry analysis revealed a total of 44 MFGM proteins that were identified with a significant difference in abundance (p ≤ 0.05; fold change ≥ 1.5) between the two breeds. Thirty-one proteins were up-regulated and 13 proteins were down-regulated in the Safra breed compared to the Wadha breed. The proteins identified with an increased abundance included α-lactalbumin, lactadherin, and annexin a8, whereas the down-regulated proteins included butyrophilin subfamily 1 member a1, lactotransferrin, and vinculin. The differentially abundant proteins were analyzed by the UNIPROT system and gene ontology (GO) to reveal their associations with known biological functions and pathways. Enzyme-linked immunosorbent assay (ELISA) confirmed the 2D-DIGE findings of butyrophilin (BTN) and α-lactalbumin (α-LA) levels obtained from Safra and Wadha breeds.

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Visker, Joseph R., Lawrence J. Dangott, Eric C. Leszczynski, and David P. Ferguson. "Postnatal Growth Restriction in Mice Alters Cardiac Protein Composition and Leads to Functional Impairment in Adulthood." International Journal of Molecular Sciences 21, no. 24 (December 12, 2020): 9459. http://dx.doi.org/10.3390/ijms21249459.

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Postnatal growth restriction (PGR) increases the risk for cardiovascular disease (CVD) in adulthood, yet there is minimal mechanistic rationale for the observed pathology. The purpose of this study was to identify proteomic differences in hearts of growth-restricted and unrestricted mice, and propose mechanisms related to impairment in adulthood. Friend leukemia virus B (FVB) mouse dams were fed a control (CON: 20% protein), or low-protein (LP: 8% protein) isocaloric diet 2 weeks before mating. LP dams produce 20% less milk, inducing growth restriction. At birth (postnatal; PN1), pups born to dams fed the CON diet were switched to LP dams (PGR group) or a different CON dam. At PN21, a sub-cohort of CON (n = 3 males; n = 3 females) and PGR (n = 3 males; n = 3 females) were euthanized and their proteome analyzed by two-dimensional differential in-gel electrophoresis (2D DIGE) and mass spectroscopy. Western blotting and silver nitrate staining confirmed 2D DIGE results. Littermates (CON: n = 4 males and n = 4 females; PGR: n = 4 males and n = 4 females) were weaned to the CON diet. At PN77, echocardiography measured cardiac function. At PN80, hearts were removed for western blotting to determine if differences persisted into adulthood. 2D DIGE and western blot confirmation indicated PGR had reductions in p57kip2, Titin (Ttn), and Collagen (Col). At PN77, PGR had impaired cardiac function as measured by echocardiography. At PN80, western blots of p57kip2 showed protein abundance recovered from PN21. PN80 silver staining of large molecular weight proteins (Ttn and Col) was reduced in PGR. PGR reduces cell cycle activity at PN21, which is recovered in adulthood. However, collagen fiber networks are altered into adulthood.

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Zheng, Dawei, Limin Xu, Lebo Sun, Qiang Feng, Zishan Wang, Guofeng Shao, and Yiming Ni. "Comparison of the Ventricle Muscle Proteome between Patients with Rheumatic Heart Disease and Controls with Mitral Valve Prolapse: HSP 60 May Be a Specific Protein in RHD." BioMed Research International 2014 (2014): 1–9. http://dx.doi.org/10.1155/2014/151726.

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Objective. Rheumatic heart disease (RHD) is a serious autoimmune heart disease. The present study was aimed at identifying the differentially expressed proteins between patients with RHD and controls with mitral valve prolapse.Methods. Nine patients with RHD and nine controls with mitral valve prolapsed were enrolled for this study. Two-dimensional difference in-gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were performed.Results. A total of 39 protein spots with differential expressions were identified between the two groups (P<0.05, Average Ratio > 1.2 or Average Ratio < −1.2) and four upregulated proteins (including heat shock protein 60 (HSP 60), desmin, PDZ and LIM domain protein 1, and proteasome subunit alpha type-1) and three downregulated proteins (including tropomyosin alpha-1 chain, malate dehydrogenase, and chaperone activity of bc1 complex homolog) were determined.Conclusion. These seven proteins, especially HSP 60, may serve as potential biomarkers for the diagnosis of RHD and provide evidence to explain the mechanisms of this complex disease in the future.

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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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Abdelmegid, Shaimaa, David Kelton, Jeff Caswell, and Gordon Kirby. "Proteomic 2D-DIGE Analysis of Milk Whey from Dairy Cows with Staphylococcus aureus Mastitis Reveals Overexpression of Host Defense Proteins." Microorganisms 8, no. 12 (November 28, 2020): 1883. http://dx.doi.org/10.3390/microorganisms8121883.

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Bovine mastitis remains a primary focus of dairy cattle disease research due to its considerable negative economic impact on the dairy industry. Subclinical mastitis (SCM), commonly caused by Staphylococcus aureus, lacks overt clinical signs and the diagnosis is based on bacteriological culture and somatic cell counts of milk, both of which have limitations. The main objective of this study was to identify, characterize and quantify the differential abundance of milk whey proteins from cows with S. aureus SCM compared to whey from healthy cows. Using two-dimensional differential gel electrophoresis (2D-DIGE) coupled with liquid chromatography and tandem mass spectrometry, 28 high-abundant proteins were detected in whey from mastitic milk, 9 of which had host defense functions. These included acute phase proteins involved in innate immunity and antimicrobial functions (e.g., serotransferrin, complement C3, fibrinogen gamma-B chain and cathepsin B), and proteins associated with the immune response to pathogens (e.g., polymeric immunoglobulin receptor-like protein, MHC class I antigen and beta-2-microglobulin). These results provide a unique 2D map of the modulated milk proteome during S. aureus mastitis. The broader importance is that the identified proteins, particularly those with host-defense biological functions, represent potential candidate biomarkers of subclinical mastitis in dairy cows.

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Chen, Yi-Wen, Hsiu-Chuan Chou, Szu-Ting Lin, You-Hsuan Chen, Yu-Jung Chang, Linyi Chen, and Hong-Lin Chan. "Cardioprotective Effects of Quercetin in Cardiomyocyte under Ischemia/Reperfusion Injury." Evidence-Based Complementary and Alternative Medicine 2013 (2013): 1–16. http://dx.doi.org/10.1155/2013/364519.

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Quercetin, a polyphenolic compound existing in many vegetables, fruits, has antiinflammatory, antiproliferation, and antioxidant effect on mammalian cells. Quercetin was evaluated for protecting cardiomyocytes from ischemia/reperfusion injury, but its protective mechanism remains unclear in the current study. The cardioprotective effects of quercetin are achieved by reducing the activity of Src kinase, signal transducer and activator of transcription 3 (STAT3), caspase 9, Bax, intracellular reactive oxygen species production, and inflammatory factor and inducible MnSOD expression. Fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can reveal the differentially expressed proteins of H9C2 cells treated with H2O2or quercetin. Although 17 identified proteins were altered in H2O2-induced cells, these proteins such as alpha-soluble NSF attachment protein (α-SNAP), Ena/VASP-like protein (Evl), and isopentenyl-diphosphate delta-isomerase 1 (Idi-1) were reverted by pretreatment with quercetin, which correlates with kinase activation, DNA repair, lipid, and protein metabolism. Quercetin dephosphorylates Src kinase in H2O2-induced H9C2 cells and likely blocks the H2O2-induced inflammatory response through STAT3 kinase modulation. This probably contributes to prevent ischemia/reperfusion injury in cardiomyocytes.

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Cazenave, Jean-Pierre, Philippe Ohlmann, Hervé Isola, and Christian Gachet. "Photochemical Pathogen Inactivation Treatment of Human Plasma (amotosalen + UVA) Has No Major Impact on the Protein expression pattern Assessed by a 2-DIGE Proteomic Assay." Blood 112, no. 11 (November 16, 2008): 1994. http://dx.doi.org/10.1182/blood.v112.11.1994.1994.

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Abstract Background: Photochemical treatment (PCT) using amotosalen HCl and UVA light (3 J/cm2: 320 – 400 nm) inactivates pathogens and leukocytes in therapeutic single donor apheresis plasma prepared within 8 hr of collection (INTERCEPTTM [I-FFP], Cerus Europe, Amersfoort, Netherlands). Clinical trials demonstrated efficacy and safety supporting a Class III CE mark and AFSSAPS (Medicinal Products Agency of France) registration of I-FFP for primary therapeutic indications including: congenital and acquired coagulopathies and TTP. Although no evidence of immune response to neo-antigens has been detected in clinical trials (Transfusion2005; 45:1610) or in post marketing surveillance studies, it remains unknown to what extent plasma proteins may be altered by PCT. We measured the impact of PCT on plasma protein profiles using a quantitative and qualitative proteomic method (2D-DIGE) to further define the possible occurrence of protein modifications in I-FFP. Methods: Plasma units (650 ml) from 8 donors (4 male and 4 female) were collected by apheresis (Haemonetics MCS+, Braintree, MA) with AB16 anticoagulant. Plasma from each donor was separated into two plasma units, one was treated with the INTERCEPTTM system within 8 hr after collection, and the paired untreated unit served as a Control. The proteomes of I-FFP and Control plasma were evaluated using differential two-dimensional in-gel electrophoresis (2D-DIGE). Before fluorescent cyanine (Cy) labeling and 2D-gel electrophoresis, albumin and IgG, the more abundant proteins, were removed from plasma using a depletion kit (GE Healthcare). Prior to 2D gel electrophoresis, proteins from Control and I-FFP were labeled with fluorescent Cy3 (green) and Cy5 (red), respectively, in four samples and vice versa in the other four. Fifteen μg of protein from Control and or I-FFP plasma from each donor were mixed and resolved on the same minigel (7x7 cm). An internal standard (15 μg), labeled with Cy2 (blue), composed of equal amounts of plasma from each donor, was added in all experiment as a normalizing agent to monitor spot intensity for determination of reproducible quantitative differences of statistical significance (ANOVA, p < 0.05). After electrophoresis, images of 2D gels were generated using a fluorescent scanner and data were analyzed using the Samespots software (NonlinearTM). Results: 281 common protein spots were observed in the proteome pattern of plasma from all 8 independent experiments and included in the analysis. INTERCEPTTM treatment did not change any of these 281 spots (ANOVA, p > 0.05). Using the internal standard to monitor quantitative changes, more than 90% of protein spots demonstrated an expression difference of less than 10% while the highest change observed was 1.8 fold. Conclusion: Within 8 hrs after collection, pathogen inactivation of human plasma by the INTERCEPTTM process did not show evidence of plasma protein modification either in a qualitative or a quantitative manner. Further studies would be required to assess in greater detail the impact on protein modification of the process.

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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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Johnson, D. Thor, Robert A. Harris, Stephanie French, Angel Aponte, and Robert S. Balaban. "Proteomic changes associated with diabetes in the BB-DP rat." American Journal of Physiology-Endocrinology and Metabolism 296, no. 3 (March 2009): E422—E432. http://dx.doi.org/10.1152/ajpendo.90352.2008.

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These studies were structured with the aim of utilizing emerging technologies in two-dimensional (2D) gel electrophoresis and mass spectrometry to evaluate protein expression changes associated with type 1 diabetes. We reasoned that a broad examination of diabetic tissues at the protein level might open up novel avenues of investigation of the metabolic and signaling pathways that are adversely affected in type 1 diabetes. This study compared the protein expression of the liver, heart, and skeletal muscle of diabetes-prone rats and matched control rats by semiquantitative liquid chromatography-mass spectrometry and differential in-gel 2D gel electrophoresis. Differential expression of 341 proteins in liver, 43 in heart, and 9 (2D gel only) in skeletal muscle was detected. These data were assembled into the relevant metabolic pathways affected primarily in liver. Multiple covalent modifications were also apparent in 2D gel analysis. Several new hypotheses were generated by these data, including mechanisms of net cytosolic protein oxidation, formaldehyde generation by the methionine cycle, and inhibition of carbon substrate oxidation via reduction in citrate synthase and short-chain acyl-CoA dehydrogenase.

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Benabdelkamel, Hicham, Afshan Masood, Meshail Okla, Mohammed Y. Al-Naami, and Assim A. Alfadda. "A Proteomics-Based Approach Reveals Differential Regulation of Urine Proteins between Metabolically Healthy and Unhealthy Obese Patients." International Journal of Molecular Sciences 20, no. 19 (October 3, 2019): 4905. http://dx.doi.org/10.3390/ijms20194905.

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Metabolic dysfunction associated with obesity threatens to inundate health care resources by increasing the incidences of obesity-related diseases. The aim of the present study was to investigate the changes in the urinary proteome of 18 individuals classified into metabolically healthy obese (MHO) and metabolically unhealthy obese (MUHO) patients. Proteome analysis was performed using the two-dimensional difference in gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS). Upon analysis, a total of 54 proteins were found to be affected with ≥1.5-fold change (ANOVA, p ≤ 0.05), of which 44 proteins were upregulated and 10 proteins were downregulated. These differentially abundant proteins were related to nuclear factor κB (NF-κB) and p38 mitogen-activated protein (MAP) kinase pathways and were involved in cellular compromise, inflammatory response, and cancer. Proteins involved in inflammation (fibrinogen alpha (FIBA), serotransferrin (TRFE, and kininogen-1 (KNG1)) and insulin resistance (ADP-ribosylation factor (ARF)-like protein 15 (ARL15) and retinol-binding protein 4 (RET4)) were found to be significantly increased in the urine samples of MUHO compared to MHO patients. Investigating the effects of obesity on urinary proteins can help in developing efficient diagnostic procedures for early detection and prevention of obesity-related complications.

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Merkley, Mark A., Paul M. Weinberger, Lana L. Jackson, Robert H. Podolsky, Jeffrey R. Lee, and William S. Dynan. "2D-DIGE proteomic characterization of head and neck squamous cell carcinoma." Otolaryngology–Head and Neck Surgery 141, no. 5 (November 2009): 626–32. http://dx.doi.org/10.1016/j.otohns.2009.08.011.

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Objective: Identify proteins that are differentially expressed between head and neck squamous cell cancer (HNSCC) and patient-matched normal adjacent tissue, and validate findings in a separate patient cohort. Study Design: Cross-sectional study of surgical specimens. Setting: Tertiary care academic medical center. Subjects and Methods: Laser capture microdissection and two-dimensional difference gel electrophoresis were used previously to establish proteomic profiles for tumor and normal adjacent tissue from 14 patients. Here, significance analysis of microarray was used to rank candidate biomarkers. Spots meeting statistical and biological criteria of significance were analyzed by liquid chromatography and tandem mass spectrometry to obtain protein identifications. The expression pattern of the highest-ranked candidate biomarker (cornulin) was validated in a larger, independent patient cohort (n = 68) by immunohistochemical staining of a tissue microarray. Results: Of 732 spots, 117 (15.9%) met criteria for significance. Identities were obtained for 39 spots, representing 17 different proteins. Four proteins were novel in the context of HNSCC: glutathione synthetase, which was upregulated; and cornulin (squamous epithelial heat shock protein 53), guanylate binding protein 6, and heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa), which were downregulated. Cornulin functions in the stress response in normal squamous epithelium, and reduced expression has been proposed as a marker of susceptibility to laryngopharyngeal reflux and other stressors. Loss of cornulin expression was confirmed in an independent HNSCC patient cohort ( P < 0.001). Conclusions: Downregulation of cornulin is a prominent feature of the molecular signature of HNSCC identified by comparative proteomics. Cornulin may represent a link between HNSCC and other pathologies arising in stratified squamous epithelium.

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Buttacavoli, Miriam, Nadia Ninfa Albanese, Elena Roz, Ida Pucci-Minafra, Salvatore Feo, and Patrizia Cancemi. "Proteomic Profiling of Colon Cancer Tissues: Discovery of New Candidate Biomarkers." International Journal of Molecular Sciences 21, no. 9 (April 28, 2020): 3096. http://dx.doi.org/10.3390/ijms21093096.

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Colon cancer is an aggressive tumor form with a poor prognosis. This study reports a comparative proteomic analysis performed by using two-dimensional differential in-gel electrophoresis (2D-DIGE) between 26 pooled colon cancer surgical tissues and adjacent non-tumoral tissues, to identify potential target proteins correlated with carcinogenesis. The DAVID functional classification tool revealed that most of the differentially regulated proteins, acting both intracellularly and extracellularly, concur across multiple cancer steps. The identified protein classes include proteins involved in cell proliferation, apoptosis, metabolic pathways, oxidative stress, cell motility, Ras signal transduction, and cytoskeleton. Interestingly, networks and pathways analysis showed that the identified proteins could be biologically inter-connected to the tumor-host microenvironment, including innate immune response, platelet and neutrophil degranulation, and hemostasis. Finally, transgelin (TAGL), here identified for the first time with four different protein species, collectively down-regulated in colon cancer tissues, emerged as a top-ranked biomarker for colorectal cancer (CRC). In conclusion, our findings revealed a different proteomic profiling in colon cancer tissues characterized by the deregulation of specific pathways involved in hallmarks of cancer. All of these proteins may represent promising novel colon cancer biomarkers and potential therapeutic targets, if validated in larger cohorts of patients.

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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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Capitanio, Daniele, Pietro Barbacini, Beatrice Arosio, Franca Rosa Guerini, Enrica Torretta, Fabio Trecate, Matteo Cesari, Daniela Mari, Mario Clerici, and Cecilia Gelfi. "Can Serum Nitrosoproteome Predict Longevity of Aged Women?" International Journal of Molecular Sciences 21, no. 23 (November 27, 2020): 9009. http://dx.doi.org/10.3390/ijms21239009.

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Aging is characterized by increase in reactive oxygen (ROS) and nitrogen (RNS) species, key factors of cardiac failure and disuse-induced muscle atrophy. This study focused on serum nitroproteome as a trait of longevity by adopting two complementary gel-based techniques: two-dimensional differential in gel electrophoresis (2-D DIGE) and Nitro-DIGE coupled with mass spectrometry of albumin-depleted serum of aged (A, n = 15) and centenarian (C, n = 15) versus young females (Y, n = 15). Results indicate spots differently expressed in A and C compared to Y and spots changed in A vs. C. Nitro-DIGE revealed nitrosated protein spots in A and C compared to Y and spots changed in A vs. C only (p-value < 0.01). Nitro-proteoforms of alpha-1-antitripsin (SERPINA1), alpha-1-antichimotripsin (SERPINA3), ceruloplasmin (CP), 13 proteoforms of haptoglobin (HP), and inactive glycosyltransferase 25 family member 3 (CERCAM) increased in A vs. Y and C. Conversely, nitrosation levels decreased in C vs. Y and A, for immunoglobulin light chain 1 (IGLC1), serotransferrin (TF), transthyretin (TTR), and vitamin D-binding protein (VDBP). Immunoblottings of alcohol dehydrogenase 5/S-nitrosoglutathione reductase (ADH5/GSNOR) and thioredoxin reductase 1 (TRXR1) indicated lower levels of ADH5 in A vs. Y and C, whereas TRXR1 decreased in A and C in comparison to Y. In conclusion, the study identified putative markers in C of healthy aging and high levels of ADH5/GSNOR that can sustain the denitrosylase activity, promoting longevity.

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Repetto, Ombretta, Federica Lovisa, Caterina Elia, Daniel Enderle, Filippo Romanato, Salvatore Buffardi, Alessandra Sala, et al. "Proteomic Exploration of Plasma Exosomes and Other Small Extracellular Vesicles in Pediatric Hodgkin Lymphoma: A Potential Source of Biomarkers for Relapse Occurrence." Diagnostics 11, no. 6 (May 21, 2021): 917. http://dx.doi.org/10.3390/diagnostics11060917.

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Exosomes and other small extracellular vesicles (EVs) are potential sources of cancer biomarkers. Plasma-derived EVs have not yet been studied in pediatric Hodgkin lymphoma (HL), for which predictive biomarkers of relapse are greatly needed. In this two-part proteomic study, we used two-dimensional difference gel electrophoresis (2D-DIGE) followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) to analyze EV proteins of plasma collected at diagnosis from children with nodular sclerosis HL, relapsed or not. EVs isolated using membrane affinity had radii ranging from 20 to 130 nm and contained the programmed cell death 6-interacting (ALIX) and the tumor susceptibility gene 101 (TSG101) proteins, whereas calnexin (CANX) was not detected. 2D-DIGE identified 16 spots as differentially abundant between non-relapsed and relapsed HL (|fold change| ≥ 1.5, p < 0.05). LC–MS/MS identified these spots as 11 unique proteins, including five more abundant in non-relapsed HL (e.g., complement C4b, C4B; fibrinogen γ chain, FGG) and six more abundant in relapsed HL (e.g., transthyretin, TTR). Shotgun LC–MS/MS on pooled EV proteins from non-relapsed HL identified 161 proteins, including 127 already identified in human exosomes (ExoCarta data). This EV cargo included 89 proteins not yet identified in exosomes from healthy plasma. Functional interrogation by the Database for Annotation, Visualization and Integrated Discovery (DAVID) revealed that the EV proteins participate in platelet degranulation and serine-type endopeptidase activity as the most significant Gene Ontology (GO) biological process and molecular function (p < 0.01).

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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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van Zanten, Gabriella C., Nadja Sparding, Avishek Majumder, Sampo J. Lahtinen, Birte Svensson, and Susanne Jacobsen. "The Differential Proteome of the ProbioticLactobacillus acidophilusNCFM Grown on the Potential Prebiotic Cellobiose Shows Upregulation of Twoβ-Glycoside Hydrolases." BioMed Research International 2015 (2015): 1–9. http://dx.doi.org/10.1155/2015/347216.

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Probiotics, prebiotics, and combinations thereof, that is, synbiotics, are known to exert beneficial health effects in humans; however interactions between pro- and prebiotics remain poorly understood at the molecular level. The present study describes changes in abundance of different proteins of the probiotic bacteriumLactobacillus acidophilusNCFM (NCFM) when grown on the potential prebiotic cellobiose as compared to glucose. Cytosolic cell extract proteomes after harvest at late exponential phase of NCFM grown on cellobiose or glucose were analyzed by two dimensional difference gel electrophoresis (2D-DIGE) in the acidic (pH 4–7) and the alkaline (pH 6–11) regions showing a total of 136 spots to change in abundance. Proteins were identified by MS or MS/MS from 81 of these spots representing 49 unique proteins and either increasing 1.5–13.9-fold or decreasing 1.5–7.8-fold in relative abundance. Many of these proteins were associated with energy metabolism, including the cellobiose related glycoside hydrolases phospho-β-glucosidase (LBA0881) and phospho-β-galactosidase II (LBA0726). The data provide insight into the utilization of the candidate prebiotic cellobiose by the probiotic bacterium NCFM. Several of the upregulated or downregulated identified proteins associated with utilization of cellobiose indicate the presence of carbon catabolite repression and regulation of enzymes involved in carbohydrate metabolism.

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Li, Shenjie, Wei Xiang, Junjie Tian, Haorun Wang, Shuiwang Hu, Ke Wang, Ligang Chen, Changren Huang, and Jie Zhou. "Bone Marrow-Derived Mesenchymal Stem Cells Differentially Affect Glioblastoma Cell Proliferation, Migration, and Invasion: A 2D-DIGE Proteomic Analysis." BioMed Research International 2021 (February 11, 2021): 1–13. http://dx.doi.org/10.1155/2021/4952876.

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Bone marrow-derived mesenchymal stem cells (BM-MSCs) display high tumor tropism and cause indirect effects through the cytokines they secrete. However, the effects of BM-MSCs on the biological behaviors of glioblastoma multiforme remain unclear. In this study, the conditioned medium from BM-MSCs significantly inhibited the proliferation of C6 cells ( P < 0.05 ) but promoted their migration and invasion ( P < 0.05 ). Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) proteomic analysis revealed 17 proteins differentially expressed in C6 cells exposed to the BM-MSC-conditioned medium including five upregulated proteins and 12 downregulated proteins. Among these, six differentially expressed proteins (Calr, Set, Oat, Npm1, Ddah1, and Tardbp) were closely related to cell proliferation and differentiation, and nine proteins (Pdia6, Sphk1, Anxa4, Vim, Tuba1c, Actr1b, Actn4, Rap2c, and Tpm2) were associated with motility and the cytoskeleton, which may modulate the invasion and migration of tumor cells. Above all, by identifying the differentially expressed proteins using proteomics and bioinformatics analysis, BM-MSCs could be genetically modified to specifically express tumor-suppressive factors when BM-MSCs are to be used as tumor-selective targeting carriers in the future.

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Takeda, K., M. Tasai, S. Akagi, S. Watanabe, M. Oe, K. Chikuni, M. Ohnishi-Kameyama, et al. "62 COMPARATIVE PROTEOMIC ANALYSIS OF LIVER MITOCHONDRIA DERIVED FROM DECEASED NEWBORN CLONED CALVES AND ADULT CLONES BY TWO-DIMENSIONAL DIFFERENTIAL GEL ELECTROPHORESIS." Reproduction, Fertility and Development 23, no. 1 (2011): 137. http://dx.doi.org/10.1071/rdv23n1ab62.

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Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. The inability to establish functional interactions between donor nucleus and recipient mitochondria is also likely responsible for developmental deficiency. However, an understanding of the expressed proteins in cattle is lacking. In the present study, alterations in mitochondrial protein levels between somatic cell nuclear transferred (SCNT) and control animals (mostly produced by AI) were investigated. Nuclear transfer was performed using donor cells prepared from cumulus cells (B1), ear skin, or skeletal muscle from adult Japanese Black cattle, and enucleated in vitro matured oocytes (Holstein or Japanese Black) as previously reported (Akagi et al. 2003). Liver samples were collected from postmortem SCNT calves (CB1-3; 0, 1, and 9 days postnatally) and adult SCNT cattle (CA1-4; 6, 6, 6, and 5 years of age) produced from the same cell line (B1) and preserved at –80°C. Mitochondrial fractions were prepared from the frozen–thawed liver samples by mechanical homogenization and differential centrifugation, and subjected to two-dimensional difference in gel electrophoresis (2D-DIGE) using CyDye™ dyes (Cy2, Cy3, Cy5; GE Healthcare) for specific labelling. Protein expression changes were confirmed by ImageMaster 2D Platinum software with a volume ratio greater than 2.0 (Student’s t-test; P < 0.05). The expression of 5 proteins were up-regulated in SCNT calves compared to control calves (n = 6; Day 250 fetus, 0, 4, 8, 8, and 8 days after birth; P < 0.05). Expressed protein patterning compared to control groups was different among SCNT calves. The protein spots of CB-1 showed great differences compared with other SCNT calves; 13 spots were up-regulated, and 18 spots were down-regulated. In adult SCNT cattle, the concentrations of 3 proteins were higher when compared to control cattle (n = 4; 2, 2, 6, and 8 years of age; P < 0.05). Protein expression was different among individual SCNT animals even if they were produced from the same donor cell source. For example, 9 spots were up-regulated and 7 spots were down-regulated in CA-1. In contrast, no differences were detected in 2 of the SCNT cattle (CA-3 and 4; P < 0.05). Novel proteins were not identified in any of the SCNT cattle or calves. In conclusion, alteration of mitochondrial protein expression levels were observed in non-viable neonatal SCNT calves and varied among SCNT individuals; suggesting that mitochondrial related gene expression may be implicated in early losses. Comparative proteomic analysis represents an important tool for further studies on SCNT animals. We thank Dr. C. A. Pinkert (Auburn Univ.) and Dr. Somfai (NARO) for their assistance. This work was supported by a grant from the NARO, Japan.

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Kratochwill, Klaus, Thorsten O. Bender, Anton M. Lichtenauer, Rebecca Herzog, Silvia Tarantino, Katarzyna Bialas, Achim Jörres, and Christoph Aufricht. "Cross-Omics Comparison of Stress Responses in Mesothelial Cells Exposed to Heat- versus Filter-Sterilized Peritoneal Dialysis Fluids." BioMed Research International 2015 (2015): 1–12. http://dx.doi.org/10.1155/2015/628158.

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Recent research suggests that cytoprotective responses, such as expression of heat-shock proteins, might be inadequately induced in mesothelial cells by heat-sterilized peritoneal dialysis (PD) fluids. This study compares transcriptome data and multiple protein expression profiles for providing new insight into regulatory mechanisms. Two-dimensional difference gel electrophoresis (2D-DIGE) based proteomics and topic defined gene expression microarray-based transcriptomics techniques were used to evaluate stress responses in human omental peritoneal mesothelial cells in response to heat- or filter-sterilized PD fluids. Data from selected heat-shock proteins were validated by 2D western-blot analysis. Comparison of proteomics and transcriptomics data discriminated differentially regulated protein abundance into groups depending on correlating or noncorrelating transcripts. Inadequate abundance of several heat-shock proteins following exposure to heat-sterilized PD fluids is not reflected on the mRNA level indicating interference beyond transcriptional regulation. For the first time, this study describes evidence for posttranscriptional inadequacy of heat-shock protein expression by heat-sterilized PD fluids as a novel cytotoxic property. Cross-omics technologies introduce a novel way of understanding PDF bioincompatibility and searching for new interventions to reestablish adequate cytoprotective responses.

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Wang, Ruomei, Jisu Wu, Xiong Deng, Dongmiao Liu, and Yueming Yan. "Drought-responsive protein identification in developing grains of a wheat–Haynaldia villosa 6VS/6AL translocation line." Crop and Pasture Science 69, no. 12 (2018): 1182. http://dx.doi.org/10.1071/cp18303.

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Drought is a widespread abiotic stress that has a detrimental effect on both yield and quality of wheat. Discovery and utilisation of drought-resistant gene resources from wheat-related species may help to mitigate effects of drought and decrease yield loss. In this study, we used a comparative proteome approach to identify potential drought-resistance proteins from a wheat (Triticum aestivum L.)–Haynaldia villosa (L.) Schur 6VS/6AL translocation line. Drought experiments showed that introgression of the H. villosa 6VS chromosome short arm into common wheat cultivar Yangmai 5 through 6VS/6AL translocation led to better drought resistance. Two-dimensional difference gel electrophoresis (2D-DIGE) identified 99 differentially accumulated protein (DAP) spots in the wheat–H. villosa 6VS/6AL translocation line, 42 of which were specifically present or showed a significantly upregulated accumulation. Of these, 20 DAPs representing 19 unique proteins in the wheat–H. villosa 6VS/6AL translocation line were upregulated under drought stress. These proteins were mainly involved in defence–stress, energy metabolism, carbon metabolism, nitrogen metabolism, and protein metabolism or folding. Protein–protein interaction analysis of key DAPs displayed a complex interaction network that synergistically regulated drought response. Dynamic transcriptional expression analysis revealed the differential expression of six key DAP genes involved in drought-stress response in the protein–protein interaction network. Our results indicated that H. villosa may have gene resources for wheat drought-resistance improvement.

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Manivannan, Bhagyashree, Pisana Rawson, T. William Jordan, Diana M. S. Karanja, Pauline N. M. Mwinzi, William Evan Secor, and Anne Camille La Flamme. "Identification of Cytokeratin 18 as a Biomarker of Mouse and Human Hepatosplenic Schistosomiasis." Infection and Immunity 79, no. 5 (February 28, 2011): 2051–58. http://dx.doi.org/10.1128/iai.01214-10.

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ABSTRACTPreviously, we demonstrated unique protein expression patterns in 20-week-Schistosoma mansoni-infected CBA/J mice with moderate splenomegaly syndrome (MSS) or hypersplemomegaly syndrome (HSS). To better understand the development of severe pathology, we compared the two-dimensional differential in-gel electrophoresis (2D-DIGE) proteomic signatures of livers from uninfected mice and mice infected for 6, 8, 12, or 20 weeks and found significant changes in collagen isoforms, interleukin-2 (IL-2), cytokeratin 18, hydroxyproline,S. mansoniphosphoenolpyruvate carboxykinase, major urinary protein isoforms, and peroxiredoxin 6. Cytokeratin 18, hydroxyproline, and connective tissue growth factor (CTGF) were chosen for analysis in mouse and human sera using targeted biochemical assays. Consistent with the liver analysis, cytokeratin 18, CTGF, and hydroxyproline were significantly elevated in sera from mice with HSS compared to those from uninfected mice or mice with MSS. Moreover, cytokeratin 18 and CTGF were found to be markers for subjects with hepatosplenic and intestinal schistosomiasis, respectively, while serum hydroxyproline was a strong indicator of fibrosis for severe HS. These findings indicate that schistosome-associated changes to the liver can be detected in the serum and reveal the potential for cytokeratin 18 to be used as a diagnostic marker for early detection of hepatosplenic schistosomiasis.

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Fuhrmann, Dominik C., Michaela Tausendschön, Ilka Wittig, Mirco Steger, Martina G. Ding, Tobias Schmid, Nathalie Dehne, and Bernhard Brüne. "Inactivation of Tristetraprolin in Chronic Hypoxia Provokes the Expression of Cathepsin B." Molecular and Cellular Biology 35, no. 3 (December 1, 2014): 619–30. http://dx.doi.org/10.1128/mcb.01034-14.

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Macrophages play important roles in many diseases and are frequently found in hypoxic areas. A chronic hypoxic microenvironment alters global cellular protein expression, but molecular details remain poorly understood. Although hypoxia-inducible factor (HIF) is an established transcription factor allowing adaption to acute hypoxia, responses to chronic hypoxia are more complex. Based on a two-dimensional differential gel electrophoresis (2D-DIGE) approach, we aimed to identify proteins that are exclusively expressed under chronic but not acute hypoxia (1% O2). One of the identified proteins was cathepsin B (CTSB), and a knockdown of either HIF-1α or -2α in primary human macrophages pointed to an HIF-2α dependency. Although chromatin immunoprecipitation (ChIP) experiments confirmed HIF-2 binding to a CTSB enhancer in acute hypoxia, an increase of CTSB mRNA was evident only under chronic hypoxia. Along those lines, CTSB mRNA stability increased at 48 h but not at 8 h of hypoxia. However, RNA stability at 8 h of hypoxia was enhanced by a knockdown of tristetraprolin (TTP). Inactivation of TTP under prolonged hypoxia was facilitated by c-Jun N-terminal kinase (JNK), and inhibition of this kinase lowered CTSB mRNA levels and stability. We postulate a TTP-dependent mechanism to explain delayed expression of CTSB under chronic hypoxia.

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von Löhneysen, Katharina, Thomas M. Scott, Katrin Soldau, Xiuling Xu, and Jeffrey S. Friedman. "Assessment of the Red Cell Proteome of Young Patients with Unexplained Hemolytic Anemia by Two-Dimensional Differential In-Gel Electrophoresis (DIGE)." PLoS ONE 7, no. 4 (April 3, 2012): e34237. http://dx.doi.org/10.1371/journal.pone.0034237.

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Zhuang, Zhengping, Meng Qi, Jie Li, Hiroaki Okamoto, David S. Xu, Rajiv R. Iyer, Jie Lu, et al. "Proteomic identification of glutamine synthetase as a differential marker for oligodendrogliomas and astrocytomas." Journal of Neurosurgery 115, no. 4 (October 2011): 789–95. http://dx.doi.org/10.3171/2011.5.jns11451.

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Object Astrocytomas and oligodendrogliomas are primary CNS tumors that remain a challenge to differentiate histologically because of their morphological variability and because there is a lack of reliable differential diagnostic markers. To identify proteins that are differentially expressed between astrocytomas and oligodendrogliomas, the authors analyzed the proteomic expression patterns and identified uniquely expressed proteins in these neoplasms. Methods Proteomes of astrocytomas and oligodendrogliomas were analyzed using 2D gel electrophoresis and subsequent computerized gel analysis to detect differentially expressed proteins. The proteins were identified using high-performance liquid chromatography accompanied by tandem mass spectrometry. To determine the role of the differentially expressed proteins in astrocytes, undifferentiated glial cell cultures were treated with dibutyryl–cyclic adenosine monophosphate (cAMP). Results Two-dimensional gel electrophoresis revealed that glutamine synthetase was differentially expressed in astrocytomas and oligodendrogliomas. Western blot and immunohistochemical analyses confirmed the increased expression of glutamine synthetase in astrocytomas compared with oligodendrogliomas. Whereas glutamine synthetase expression was demonstrated across all grades of astrocytomas (Grade II–IV [15 tumors]) and oligoastrocytomas (4 tumors), it was expressed in only 1 oligodendroglioma (6% [16 tumors]). Treatment of undifferentiated glial cell cultures with dibutyryl-cAMP resulted in astrocyte differentiation that was associated with increased levels of glial fibrillary acidic protein and glutamine synthetase. Conclusions These data indicate that glutamine synthetase expression can be used to distinguish astrocytic from oligodendroglial tumors and may play a role in the pathogenesis of astrocytomas.

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Martínez-Aguilar, Mayeli M., Diana I. Aparicio-Bautista, Eric G. Ramírez-Salazar, Juan P. Reyes-Grajeda, Aldo H. De la Cruz-Montoya, Bárbara Antuna-Puente, Alberto Hidalgo-Bravo, et al. "Serum Proteomic Analysis Reveals Vitamin D-Binding Protein (VDBP) as a Potential Biomarker for Low Bone Mineral Density in Mexican Postmenopausal Women." Nutrients 11, no. 12 (November 21, 2019): 2853. http://dx.doi.org/10.3390/nu11122853.

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Osteoporosis is a skeletal disease mainly affecting women over 50 years old and it represents a serious public health problem because of the high socioeconomic burden. This disease is characterized by deterioration of bone microarchitecture, low bone mineral density (BMD), and increased risk of fragility fractures. This study aimed to identify serum useful proteins as biomarkers for the diagnosis and/or prognosis of osteoporosis and fracture risk. We collected 446 serum samples from postmenopausal women aged ≥45 years old. Based on the BMD measurement, we classified the participants into three groups: osteoporotic, osteopenic, and normal. In an initial discovery stage, we conducted a proteomic approach using two-dimensional differential gel electrophoresis (2D-DIGE). The peptides into the spots of interest were identified through matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF/TOF). Enzyme-linked immunosorbent assay (ELISA) was performed to validate the proteins of interest. We identified 27 spots of interest when comparing low BMD versus normal BMD postmenopausal women. Based on their relevance in bone metabolism, we analyzed three proteins: ceruloplasmin (CP), gelsolin (GSN), and vitamin D-binding protein (VDBP). Our results demonstrated that low serum VDBP levels correlate with low BMD (osteopenic and osteoporotic). Therefore, VDBP could be considered as a novel, potential, and non-invasive biomarker for the early detection of osteoporosis.

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Insenser, María, Nuria Vilarrasa, Joan Vendrell, and Héctor F. Escobar-Morreale. "Remission of Diabetes Following Bariatric Surgery: Plasma Proteomic Profiles." Journal of Clinical Medicine 10, no. 17 (August 28, 2021): 3879. http://dx.doi.org/10.3390/jcm10173879.

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Bariatric surgery restores glucose tolerance in many, but not all, severely obese subjects with type 2 diabetes (T2D). We aimed to evaluate the plasma protein profiles associated with the T2D remission after obesity surgery. We recruited seventeen women with severe obesity submitted to bariatric procedures, including six non-diabetic patients and eleven patients with T2D. After surgery, diabetes remitted in 7 of the 11 patients with T2D. Plasma protein profiles at baseline and 6 months after bariatric surgery were analyzed by two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight coupled to mass spectrometry (MALDI-TOF/TOF MS). Remission of T2D following bariatric procedures was associated with changes in alpha-1-antichymotrypsin (SERPINA 3, p < 0.05), alpha-2-macroglobulin (A2M, p < 0.005), ceruloplasmin (CP, p < 0.05), fibrinogen beta chain (FBG, p < 0.05), fibrinogen gamma chain (FGG, p < 0.05), gelsolin (GSN, p < 0.05), prothrombin (F2, p < 0.05), and serum amyloid p-component (APCS, p < 0.05). The resolution of diabetes after bariatric surgery is associated with specific changes in the plasma proteomic profiles of proteins involved in acute-phase response, fibrinolysis, platelet degranulation, and blood coagulation, providing a pathophysiological basis for the study of their potential use as biomarkers of the surgical remission of T2D in a larger series of severely obese patients.

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Thamaga, L., O. Ruzvidzo, and T. B. Dikobe. "Morphological and Proteomic Evaluation of Zea Mays in Response to Osmotic Stress." Open Biotechnology Journal 15, no. 1 (March 18, 2021): 19–26. http://dx.doi.org/10.2174/1874070702115010019.

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Introduction: Drought is the main abiotic stress responsible for crop loss worldwide. Maize (Zea mays L.) is a widely grown drought-sensitive crop used as a staple food by the growing population. Therefore, it is imperative to assess the molecular mechanisms behind drought response and tolerance in maize. Transcriptomic profiling of abiotic stress responsive pathways in various crops appeared to be an unreliable approach due to post-transcriptional modifications, while there is limited published data on molecular mechanisms of osmotic-stress response in maize. Hence our study aimed at profiling osmotic stress responsive proteins augmented by their associated morphological features in Z. mays. Materials and Methods: In this regard, morphological and proteomic investigations were carried out on 16-day maize seedlings exposed to 5% (w/v) and 10% (w/v) polyethylene glycol(PEG) to induce osmotic-stress. Proteomics approach (one-dimensional (1D) and two-dimensional (2D) gel electrophoresis) compared differential protein abundance between controls and the osmotic stressed maize plants. Results: Morphological parameters such as plant growth, height, shoot diameter, leaf area, and colour were highly affected with PEG treatment as compared to the untreated ones. Molecular evaluation by 1D gel electrophoresis revealed that the separated protein patterns were highly expressed in the experiments than the controls. Using 2D gel electrophoresis, a total of seven and eight protein spots were revealed in experimental plants under 5% (w/v) and 10% (w/v) PEG treatment respectively while the control plants only expressed one protein. Increased drought stress resulted in a greater number of proteins with differential abundance. Conclusion: This study has successfully profiled the total osmotic stress responsive proteins and revealed the efficiency of proteomic tools in the qualitative detection of differential proteins from maize.

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Martin, Petra, Sinead Noonan, Blathnaid Nolan, Caitriona Scaife, Giuliano Elia, Diarmuid O'Donoghue, David William Fennelly, and Jacintha O'Sullivan. "Characterization of serum proteome in patients with metastatic colorectal cancer responsive and nonresponsive to bevacizumab using 2 d-DIGE." Journal of Clinical Oncology 30, no. 4_suppl (February 1, 2012): 484. http://dx.doi.org/10.1200/jco.2012.30.4_suppl.484.

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484 Background: Treatment of patients with metastatic colorectal cancer includes chemotherapy and a monoclonal antibody (cetuximab or bevacizumab). Patients who have k-ras mutated tumors are given bevacizumab. However, no biomarker exists to determine those patients who will respond to this targeted treatment. The objective of this study was to investigate the differential protein expression between patients who do and do not respond to bevacizumab and also compare this with normal controls. Methods: Serum from 24 patients diagnosed with metastatic colorectal cancer and 11 normal controls were collected pre-treatment. All patients received bevacizumab along with chemotherapy. Progression free and overall survival data was collected on all patients. Serum was depleted of high abundant proteins and protein expression analysed using fluorescence two-dimensional differential in-gel electrophoresis (2 D-DIGE). Gels were scanned using a Typhoon 9410 Variable Mode Imager (GE Healthcare) and exported into Progenesis SameSpots v3.3 (Nonlinear Dynamics, UK) for quantitative analysis. Selected differentially expressed were excised, digested with trypsin and analysed using the LTQ-Orbitrap XL mass spectrometer. Results: 66 proteins were identified to be statistically expressed between the responders and non-responding group (p<0.05). 30 proteins were differentially expressed between the cancer and normal group (p<0.05). Conclusions: There is a significant difference in protein expression patterns between responders and non responders to bevacizumab. Further screening required to assess for instability proteins that may have functional importance in governing treatment resistance.

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Glancy, Brian, and Robert S. Balaban. "Protein composition and function of red and white skeletal muscle mitochondria." American Journal of Physiology-Cell Physiology 300, no. 6 (June 2011): C1280—C1290. http://dx.doi.org/10.1152/ajpcell.00496.2010.

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Red and white muscles are faced with very different energetic demands. However, it is unclear whether relative mitochondrial protein expression is different between muscle types. Mitochondria from red and white porcine skeletal muscle were isolated with a Percoll gradient. Differences in protein composition were determined using blue native (BN)-PAGE, two-dimensional differential in gel electrophoresis (2D DIGE), optical spectroscopy, and isobaric tag for relative and absolute quantitation (iTRAQ). Complex IV and V activities were compared using BN-PAGE in-gel activity assays, and maximal mitochondrial respiration rates were assessed using pyruvate (P) + malate (M), glutamate (G) + M, and palmitoyl-carnitine (PC) + M. Without the Percoll step, major cytosolic protein contamination was noted for white mitochondria. Upon removal of contamination, very few protein differences were observed between red and white mitochondria. BN-PAGE showed no differences in the subunit composition of Complexes I–V or the activities of Complexes IV and V. iTRAQ analysis detected 358 mitochondrial proteins, 69 statistically different. Physiological significance may be lower: at a 25% difference, 48 proteins were detected; at 50%, 14 proteins were detected; and 3 proteins were detected at a 100%. Thus any changes could be argued to be physiologically modest. One area of difference was fat metabolism where four β-oxidation enzymes were ∼25% higher in red mitochondria. This was correlated with a 40% higher rate of PC+M oxidation in red mitochondria compared with white mitochondria with no differences in P+M and G+M oxidation. These data suggest that metabolic demand differences between red and white muscle fibers are primarily matched by the number of mitochondria and not by significant alterations in the mitochondria themselves.

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Centlow, Magnus, Stefan R. Hansson, and Charlotte Welinder. "Differential Proteome Analysis of the Preeclamptic Placenta Using Optimized Protein Extraction." Journal of Biomedicine and Biotechnology 2010 (2010): 1–9. http://dx.doi.org/10.1155/2010/458748.

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The human placenta is a difficult tissue to work with using proteomic technology since it contains large amounts of lipids and glycogen. Both lipids and glycogen are known to interfere with the first step in the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), the isoelectric focusing. In order to gain the best possible protein separation on 2D-PAGE, an optimized sample preparation protocol for placental proteins was developed. Two different buffers, urea/CHAPS and Hepes, were used for solubilization in combination with six different precipitation methods. The removal of glycogen from the samples by centrifugation was crucial for the final proteome maps. Solubilization with urea/CHAPS in combination with dichloromethane/methanol or acidified acetone proved to be the best precipitation procedures. When applied to clinical placenta samples apolipoprotein A1 was found to be accumulated in the preeclamptic placenta, where it may either have a nutritional effect or act as a modifier of signal transduction.

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Futcher, B., G. I. Latter, P. Monardo, C. S. McLaughlin, and J. I. Garrels. "A Sampling of the Yeast Proteome." Molecular and Cellular Biology 19, no. 11 (November 1, 1999): 7357–68. http://dx.doi.org/10.1128/mcb.19.11.7357.

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ABSTRACT In this study, we examined yeast proteins by two-dimensional (2D) gel electrophoresis and gathered quantitative information from about 1,400 spots. We found that there is an enormous range of protein abundance and, for identified spots, a good correlation between protein abundance, mRNA abundance, and codon bias. For each molecule of well-translated mRNA, there were about 4,000 molecules of protein. The relative abundance of proteins was measured in glucose and ethanol media. Protein turnover was examined and found to be insignificant for abundant proteins. Some phosphoproteins were identified. The behavior of proteins in differential centrifugation experiments was examined. Such experiments with 2D gels can give a global view of the yeast proteome.

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Li, Sung-Chou, Kuo-Chung Lan, Hsuan-Ning Hung, Wan-Ting Huang, Yun-Ju Lai, Hsin-Hsin Cheng, Chih-Chang Tsai, Kun-Long Huang, Huey-Ling You, and Te-Yao Hsu. "HSPA4 Is a Biomarker of Placenta Accreta and Enhances the Angiogenesis Ability of Vessel Endothelial Cells." International Journal of Molecular Sciences 23, no. 10 (May 19, 2022): 5682. http://dx.doi.org/10.3390/ijms23105682.

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Placenta accreta spectrum (PAS) accounts for 7% of maternal mortality and is associated with intraoperative and postoperative morbidity caused by massive blood loss, infection, and adjacent organ damage. The aims of this study were to identify the protein biomarkers of PAS and to further explore their pathogenetic roles in PAS. For this purpose, we collected five placentas from pregnant subjects with PAS complications and another five placentas from normal pregnancy (NP) cases. Then, we enriched protein samples by specifically isolating the trophoblast villous, deeply invading into the uterine muscle layer in the PAS patients. Next, fluorescence-based two-dimensional difference gel electrophoresis (2D-DIGE) and MALDI-TOF/MS were used to identify the proteins differentially abundant between PAS and NP placenta tissues. As a result, nineteen spots were determined as differentially abundant proteins, ten and nine of which were more abundant in PAS and NP placenta tissues, respectively. Then, specific validation with western blot assay and immunohisto/cytochemistry (IHC) assay confirmed that heat shock 70 kDa protein 4 (HSPA4) and chorionic somatomammotropin hormone (CSH) were PAS protein biomarkers. Further tube formation assays demonstrated that HSPA4 promoted the in vitro angiogenesis ability of vessel endothelial cells, which is consistent with the in vivo scenario of PAS complications. In this study, we not only identified PAS protein biomarkers but also connected the promoted angiogenesis with placenta invasion, investigating the pathogenetic mechanism of PAS.

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Reichert, Gerd H. "Two-dimensional Gel Analysis of Proteins from Mouse Fetuses with Trisomy 19 after DEAE-Sepharose Chromatography." Genetical Research 47, no. 3 (June 1986): 193–97. http://dx.doi.org/10.1017/s0016672300023120.

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SummaryIsoelectrofocusing two-dimensional polyacrylamide gel electrophoresis (IEF-2D-PAGE) offers the opportunity to detect typical alterations in the protein pattern of trisomic mouse foetuses at a given time of development. The fractionation of the cell lysate by differential centrifugation into various subcellular components (nuclei, membranes, polyribosomes, cytoplasmic proteins) and fractionation of the proteins through DEAE-Sepharose chromatography allows detection of protein differences.It is possible to detect eight differences in the protein patterns between trisomy 19 (Ts 19) mouse foetuses and euploid mouse fetuses at day 15. Five of these differences are quantitative in nature, three are qualitative. One of these proteins is synthesized in Ts 19 foetuses at a higher level than in euploid mouse fetuses (primary gene dosage effect). The other seven proteins are reduced or not present in trisomic foetuses (consequences of primary gene dosage effects).The molecular mass of the individual proteins ranges from 13 to 41 kDa.

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Journal articles on the topic '2D-DIGE ( Two Dimensional Differential In Gel Electrophoresis)'

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