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Large-scale Identification and Time-course Quantification of Ubiquitylation Events During Maize Seedling De-etiolation. GENOMICS PROTEOMICS & BIOINFORMATICS 2020; 17:603-622. [PMID: 32179194 PMCID: PMC7212306 DOI: 10.1016/j.gpb.2018.05.005] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/27/2017] [Revised: 04/11/2018] [Accepted: 05/04/2018] [Indexed: 01/02/2023]
Abstract
The ubiquitin system is crucial for the development and fitness of higher plants. De-etiolation, during which green plants initiate photomorphogenesis and establish autotrophy, is a dramatic and complicated process that is tightly regulated by a massive number of ubiquitylation/de-ubiquitylation events. Here we present site-specific quantitative proteomic data for the ubiquitylomes of de-etiolating seedling leaves of Zea mays L. (exposed to light for 1, 6, or 12 h) achieved through immunoprecipitation-based high-resolution mass spectrometry (MS). Through the integrated analysis of multiple ubiquitylomes, we identified and quantified 1926 unique ubiquitylation sites corresponding to 1053 proteins. We analyzed these sites and found five potential ubiquitylation motifs, KA, AXK, KXG, AK, and TK. Time-course studies revealed that the ubiquitylation levels of 214 sites corresponding to 173 proteins were highly correlated across two replicate MS experiments, and significant alterations in the ubiquitylation levels of 78 sites (fold change >1.5) were detected after de-etiolation for 12 h. The majority of the ubiquitylated sites we identified corresponded to substrates involved in protein and DNA metabolism, such as ribosomes and histones. Meanwhile, multiple ubiquitylation sites were detected in proteins whose functions reflect the major physiological changes that occur during plant de-etiolation, such as hormone synthesis/signaling proteins, key C4 photosynthetic enzymes, and light signaling proteins. This study on the ubiquitylome of the maize seedling leaf is the first attempt ever to study the ubiquitylome of a C4 plant and provides the proteomic basis for elucidating the role of ubiquitylation during plant de-etiolation.
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Scharfmann R, Staels W, Albagli O. The supply chain of human pancreatic β cell lines. J Clin Invest 2019; 129:3511-3520. [PMID: 31478912 PMCID: PMC6715382 DOI: 10.1172/jci129484] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Patients with type 1 or type 2 diabetes have an insufficiency in their functional β cell mass. To advance diabetes treatment and to work toward a cure, a better understanding of how to protect the pancreatic β cells against autoimmune or metabolic assaults (e.g., obesity, gestation) will be required. Over the past decades, β cell protection has been extensively investigated in rodents both in vivo and in vitro using isolated islets or rodent β cell lines. Transferring these rodent data to humans has long been challenging, at least partly for technical reasons: primary human islet preparations were scarce and functional human β cell lines were lacking. In 2011, we described a robust protocol of targeted oncogenesis in human fetal pancreas and produced the first functional human β cell line, and in subsequent years additional lines with specific traits. These cell lines are currently used by more than 150 academic and industrial laboratories worldwide. In this Review, we first explain how we developed the human β cell lines and why we think we succeeded where others, despite major efforts, did not. Next, we discuss the use of such functional human β cell lines and share some perspectives on their use to advance diabetes research.
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Affiliation(s)
- Raphael Scharfmann
- INSERM U1016, Institut Cochin, Université Paris Descartes, Paris, France
| | - Willem Staels
- INSERM U1016, Institut Cochin, Université Paris Descartes, Paris, France
- Beta Cell Neogenesis (BENE), Vrije Universiteit Brussel, Brussels, Belgium
| | - Olivier Albagli
- INSERM U1016, Institut Cochin, Université Paris Descartes, Paris, France
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3
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Peng F, Huang Y, Li MY, Li GQ, Huang HC, Guan R, Chen ZC, Liang SP, Chen YH. Dissecting characteristics and dynamics of differentially expressed proteins during multistage carcinogenesis of human colorectal cancer. World J Gastroenterol 2016; 22:4515-4528. [PMID: 27182161 PMCID: PMC4858633 DOI: 10.3748/wjg.v22.i18.4515] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/07/2015] [Revised: 02/13/2016] [Accepted: 03/18/2016] [Indexed: 02/06/2023] Open
Abstract
AIM: To discover novel biomarkers for early diagnosis, prognosis or treatment of human colorectal cancer.
METHODS: iTRAQ 2D LC-MS/MS analysis was used to identify differentially expressed proteins (DEPs) in the human colonic epithelial carcinogenic process using laser capture microdissection-purified colonic epithelial cells from normal colon, adenoma, carcinoma in situ and invasive carcinoma tissues.
RESULTS: A total of 326 DEPs were identified, and four DEPs (DMBT1, S100A9, Galectin-10, and S100A8) with progressive alteration in the carcinogenic process were further validated by immunohistochemistry. The DEPs were involved in multiple biological processes including cell cycle, cell adhesion, translation, mRNA processing, and protein synthesis. Some of the DEPs involved in cellular process such as “translation” and “mRNA splicing” were progressively up-regulated, while some DEPs involved in other processes such as “metabolism” and “cell response to stress” was progressively down-regulated. Other proteins with up- or down-regulation at certain stages of carcinogenesis may play various roles at different stages of the colorectal carcinogenic process.
CONCLUSION: These findings give insights into our understanding of the mechanisms of colorectal carcinogenesis and provide clues for further investigation of carcinogenesis and identification of biomarkers.
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Liu CW, Atkinson MA, Zhang Q. Type 1 diabetes cadaveric human pancreata exhibit a unique exocrine tissue proteomic profile. Proteomics 2016; 16:1432-46. [PMID: 26935967 DOI: 10.1002/pmic.201500333] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2015] [Revised: 01/26/2016] [Accepted: 02/17/2016] [Indexed: 12/28/2022]
Abstract
Type 1 diabetes (T1D) is an autoimmune disorder resulting from a self-destruction of pancreatic islet beta cells. The complete proteome of the human pancreas, where both the dysfunctional beta cells and their proximal environment co-exist, remains unknown. Here, we used TMT10-based isobaric labeling and multidimensional LC-MS/MS to quantitatively profile the differences between pancreatic head region tissues from T1D (N = 5) and healthy subjects (N = 5). Among the 5357 (1% false discovery rate) confidently identified proteins, 145 showed statistically significant dysregulation between T1D and healthy subjects. The differentially expressed pancreatic proteome supports the growing notion of a potential role for exocrine pancreas involvement in T1D. This study also demonstrates the utility for this approach to analyze dysregulated proteins as a means to investigate islet biology, pancreatic pathology and T1D pathogenesis.
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Affiliation(s)
- Chih-Wei Liu
- Center for Translational Biomedical Research, University of North Carolina at Greensboro, North Carolina Research Campus, Kannapolis, NC, USA
| | - Mark A Atkinson
- Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL, USA
| | - Qibin Zhang
- Center for Translational Biomedical Research, University of North Carolina at Greensboro, North Carolina Research Campus, Kannapolis, NC, USA.,Department of Chemistry & Biochemistry, University of North Carolina at Greensboro, Greensboro, NC, USA
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Huang W, Liang Q, Chen J, Zhu H, Xie W, Wang Y, Yang B, Peng W, Xiong X. Quantitative proteomic analysis of synovial tissue from rats with collagen-induced arthritis. RSC Adv 2015. [DOI: 10.1039/c5ra18743e] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
The pathway networks involved in RA pathological process were analyzed by Ingenuity pathway analysis (IPA).
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Affiliation(s)
- Wei Huang
- Institute of Integrated Medicine
- Xiangya Hospital
- Central South University
- Changsha
- PR China
| | - Qinghua Liang
- Institute of Integrated Medicine
- Xiangya Hospital
- Central South University
- Changsha
- PR China
| | - Jiang Chen
- Central of Telemedicine
- Xiangya Hospital
- Central South University
- Changsha
- PR China
| | - Hao Zhu
- Institute of Integrated Medicine
- The First Affiliated Hospital of Soochow University
- Soochow
- PR China
| | - Wei Xie
- Department of Pathology & Immunology
- Baylor College of Medicine
- Houston
- USA
| | - Yang Wang
- Institute of Integrated Medicine
- Xiangya Hospital
- Central South University
- Changsha
- PR China
| | - Bo Yang
- Institute of Integrated Medicine
- Xiangya Hospital
- Central South University
- Changsha
- PR China
| | - Weijun Peng
- Department of Integrated Chinese and Western Medicine
- The Second Xiangya Hospital
- Central South University
- Changsha 410011
- PR China
| | - Xingui Xiong
- Institute of Integrated Medicine
- Xiangya Hospital
- Central South University
- Changsha
- PR China
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Liu J, Qin J, Mei W, Zhang H, Yuan Q, Peng Z, Luo R, Yuan X, Huang L, Tao L. Expression of Niban in renal interstitial fibrosis. Nephrology (Carlton) 2014; 19:479-89. [PMID: 24750539 DOI: 10.1111/nep.12266] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/14/2014] [Indexed: 12/30/2022]
Affiliation(s)
- Jishi Liu
- Division of Nephrology; Xiangya Hospital; Central South University; Changsha China
| | - Jiao Qin
- Division of Nephrology; Xiangya Hospital; Central South University; Changsha China
| | - Wenjuan Mei
- Division of Nephrology; Xiangya Hospital; Central South University; Changsha China
| | - Hao Zhang
- Division of Nephrology; The Third Xiangya Hospital; Central South University; Changsha China
| | - Qiongjing Yuan
- Division of Nephrology; Xiangya Hospital; Central South University; Changsha China
| | - Zhangzhe Peng
- Division of Nephrology; Xiangya Hospital; Central South University; Changsha China
| | - Renna Luo
- Division of Nephrology; Xiangya Hospital; Central South University; Changsha China
| | - Xiangning Yuan
- Division of Nephrology; Xiangya Hospital; Central South University; Changsha China
| | - Ling Huang
- Division of Nephrology; Xiangya Hospital; Central South University; Changsha China
| | - Lijian Tao
- Division of Nephrology; Xiangya Hospital; Central South University; Changsha China
- State Key Laboratory of Medical Genetics of China; Central South University; Changsha China
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Tolin S, Arrigoni G, Moscatiello R, Masi A, Navazio L, Sablok G, Squartini A. Quantitative analysis of the naringenin-inducible proteome in Rhizobium leguminosarum by isobaric tagging and mass spectrometry. Proteomics 2013; 13:1961-72. [PMID: 23580418 DOI: 10.1002/pmic.201200472] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2012] [Revised: 02/17/2013] [Accepted: 03/12/2013] [Indexed: 11/05/2022]
Abstract
The rhizobium-legume interaction is a critical cornerstone of crop productivity and environmental sustainability. Its potential improvement relies on elucidation of the complex molecular dialogue between its two partners. In the present study, the proteomic patterns of gnotobiotic cultures of Rhizobium leguminosarum bv. viciae 3841 grown for 6 h in presence or absence of the nod gene-inducing plant flavonoid naringenin (10 μM) were analyzed using the iTRAQ approach. A total of 1334 proteins were identified corresponding to 18.67% of the protein-coding genes annotated in the sequenced genome of bv. viciae 3841. The abundance levels of 47 proteins were increased upon naringenin treatment showing fold change ratios ranging from 1.5 to 25 in two biological replicates. Besides the nod units, naringenin enhanced the expression of a number of other genes, many of which organized in operons, including β(1-2) glucan production and secretion, succinoglycan export, the RopA outer membrane protein with homology to an oligogalacturonide-specific porin motif, other enzymes for carbohydrate and amino acid metabolism, and proteins involved in the translation machinery. Data were validated at the transcriptional and phenotypic levels by RT-PCR and an assay of secreted sugars in culture supernatants, respectively. The current approach provides not only a high-resolution analysis of the prokaryotic proteome but also unravels the rhizobium molecular dialogue with legumes by detecting the enhanced expression of several symbiosis-associated proteins, whose flavonoid-dependency had not yet been reported.
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Affiliation(s)
- Serena Tolin
- Department of Agronomy, Food, Natural Resources, Animals and Environment, DAFNAE, Legnaro, Padova, Italy
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Scharfmann R, Rachdi L, Ravassard P. Concise review: in search of unlimited sources of functional human pancreatic beta cells. Stem Cells Transl Med 2012; 2:61-7. [PMID: 23283495 DOI: 10.5966/sctm.2012-0120] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
It is well-established that insulin-producing pancreatic beta cells are central in diabetes. In type 1 diabetes, beta cells are destroyed by an autoimmune mechanism, whereas in type 2 diabetes, there is a decrease in functional beta-cell mass. In this context, studying beta cells is of major importance. Beta cells represent only 1% of total pancreatic cells and are found dispersed in the pancreatic gland. During the past decades, many tools and approaches have been developed to study rodent beta cells that efficiently pushed the field forward. However, rodent and human beta cells are not identical, and our knowledge of human beta cells has not progressed as quickly as our understanding of rodent beta cells. We believe that one of the reasons for this inefficient progress is the difficulty of accessing unlimited sources of functional human pancreatic beta cells. The main focus of this review concerns recent strategies to generate new sources of human pancreatic beta cells.
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Kim SJ, Jin J, Kim YJ, Kim Y, Yu HG. Retinal proteome analysis in a mouse model of oxygen-induced retinopathy. J Proteome Res 2012; 11:5186-203. [PMID: 23039900 DOI: 10.1021/pr300389r] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
To identify proteins that are involved in the molecular mechanisms of oxygen-induced retinopathy (OIR), a well-established model of blinding ischemic retinopathy, we quantitatively analyzed the retinal proteome in a mouse model of OIR. OIR was induced by exposing C57BL/6 mice on postnatal day 7 (P7) to 75% hyperoxia for 5 days, followed by 5 days in room air. Retinas from mice on P12 and P17, the hyperoxic and hypoxic phases, respectively, and control groups were examined using isobaric tags for relative and absolute quantitation (iTRAQ) and nano-LC-ESI-MS/MS. In total, 1422 retinal proteins were identified: 699 from the iTRAQ experiment and 1074 by nano-LC-ESI-MS/MS. Compared with control retinas in the iTRAQ study, OIR retinas upregulated and downregulated 21 and 17 proteins, respectively, in P17 retinas and 25 and 14 proteins, respectively, in P12 retinas. Of the differentially expressed proteins, the retinal expression of crystallin proteins, Müller cell-associated proteins, neurodegeneration-associated proteins, and angiogenesis-associated proteins, such as 150-kDa oxygen-regulated protein (ORP150), were analyzed. ORP150 colocalized to the neovascular tufts, and knockdown of ORP150 by siRNA decreased the levels of secreted VEGF in cultured retinal pigment epithelial cells. Moreover, intravitreal administration of siRNA targeting ORP150 significantly reduced the retinal neovascularization in OIR. In conclusion, our proteomic discovery method, coupled with targeted approaches, revealed many proteins that were differentially regulated in the mouse model of OIR. These proteins, including ORP150, are potential novel therapeutic targets for the treatment of proliferative ischemic retinopathy.
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Affiliation(s)
- Sang Jin Kim
- Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Republic of Korea
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Zhou JY, Dann GP, Liew CW, Smith RD, Kulkarni RN, Qian WJ. Unraveling pancreatic islet biology by quantitative proteomics. Expert Rev Proteomics 2012; 8:495-504. [PMID: 21819304 DOI: 10.1586/epr.11.39] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
The pancreatic islets of Langerhans play a critical role in maintaining blood glucose homeostasis by secreting insulin and several other important peptide hormones. Impaired insulin secretion due to islet dysfunction is linked to the pathogenesis underlying both Type 1 and Type 2 diabetes. Over the past 5 years, emerging proteomic technologies have been applied to dissect the signaling pathways that regulate islet functions and gain an understanding of the mechanisms of islet dysfunction relevant to diabetes. Herein, we briefly review some of the recent quantitative proteomic studies involving pancreatic islets geared towards gaining a better understanding of islet biology relevant to metabolic diseases.
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Affiliation(s)
- Jian-Ying Zhou
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA
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11
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Fan NJ, Gao CF, Wang CS, Zhao G, Lv JJ, Wang XL, Chu GH, Yin J, Li DH, Chen X, Yuan XT, Meng NL. Identification of the up-regulation of TP-alpha, collagen alpha-1(VI) chain, and S100A9 in esophageal squamous cell carcinoma by a proteomic method. J Proteomics 2012; 75:3977-86. [PMID: 22583932 DOI: 10.1016/j.jprot.2012.05.008] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2012] [Revised: 04/09/2012] [Accepted: 05/04/2012] [Indexed: 12/20/2022]
Abstract
Esophageal squamous cell carcinoma (ESCC) is one of the most common primary malignant tumor of digestive tract. However, the early diagnosis and molecular mechanisms that underlie tumor formation and progression have been progressed less. To identify new biomarkers for ESCC, we performed a comparative proteomic research. Isobaric tags for relative and absolute quantitation-based proteomic method was used to screen biomarkers between ESCC and normal. 802 non-redundant proteins were identified, 39 of which were differentially expressed with 1.5-fold difference (29 up-regulated and 10 down-regulated). Through Swiss-Prot and GO database, the location and function of differential proteins were analyzed, which are related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc. Among the differentially expressed proteins, TP-alpha, collagen alpha-1(VI) chain and S100A9 were verified to be upregulated in 77.19%, 75.44% and 59.65% of ESCC by immunohistochemistry and western-blot. Diagnostic value of these three proteins was validated. These results provide new insights into ESCC biology and potential diagnostic and therapeutic biomarkers, which suggest that TP-alpha, collagen alpha-1(VI) chain and S100A9 are potential biomarkers of ESCC, and may play an important role in tumorigenesis and development of ESCC.
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Affiliation(s)
- Nai-Jun Fan
- Institute of Anal-Colorectal Surgery, No. 150 Central Hospital of PLA, Luoyang, China
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Differential proteome profiling using iTRAQ in microalbuminuric and normoalbuminuric type 2 diabetic patients. EXPERIMENTAL DIABETES RESEARCH 2012; 2012:168602. [PMID: 22536212 PMCID: PMC3318901 DOI: 10.1155/2012/168602] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/02/2011] [Revised: 10/27/2011] [Accepted: 11/22/2011] [Indexed: 11/17/2022]
Abstract
Diabetic nephropathy (DN) is a long-term complication of diabetes mellitus that leads to end-stage renal disease. Microalbuminuria is used for the early detection of diabetic renal damage, but such levels do not reflect the state of incipient DN precisely in type 2 diabetic patients because microalbuminuria develops in other diseases, necessitating more accurate biomarkers that detect incipient DN. Isobaric tags for relative and absolute quantification (iTRAQ) were used to identify urinary proteins that were differentially excreted in normoalbuminuric and microalbuminuric patients with type 2 diabetes where 710 and 196 proteins were identified and quantified, respectively. Some candidates were confirmed by 2-DE analysis, or validated by Western blot and multiple reaction monitoring (MRM). Specifically, some differentially expressed proteins were verified by MRM in urine from normoalbuminuric and microalbuminuric patients with type 2 diabetes, wherein alpha-1-antitrypsin, alpha-1-acid glycoprotein 1, and prostate stem cell antigen had excellent AUC values (0.849, 0.873, and 0.825, resp.). Moreover, we performed a multiplex assay using these biomarker candidates, resulting in a merged AUC value of 0.921. Although the differentially expressed proteins in this iTRAQ study require further validation in larger and categorized sample groups, they constitute baseline data on preliminary biomarker candidates that can be used to discover novel biomarkers for incipient DN.
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Bauer C, Kleinjung F, Rutishauser D, Panse C, Chadt A, Dreja T, Al-Hasani H, Reinert K, Schlapbach R, Schuchhardt J. PPINGUIN: Peptide Profiling Guided Identification of Proteins improves quantitation of iTRAQ ratios. BMC Bioinformatics 2012; 13:34. [PMID: 22340093 PMCID: PMC3368728 DOI: 10.1186/1471-2105-13-34] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2011] [Accepted: 02/16/2012] [Indexed: 01/07/2023] Open
Abstract
Background Recent development of novel technologies paved the way for quantitative proteomics. One of the most important among them is iTRAQ, employing isobaric tags for relative or absolute quantitation. Despite large progress in technology development, still many challenges remain for derivation and interpretation of quantitative results. One of these challenges is the consistent assignment of peptides to proteins. Results We have developed Peptide Profiling Guided Identification of Proteins (PPINGUIN), a statistical analysis workflow for iTRAQ data addressing the problem of ambiguous peptide quantitations. Motivated by the assumption that peptides uniquely derived from the same protein are correlated, our method employs clustering as a very early step in data processing prior to protein inference. Our method increases experimental reproducibility and decreases variability of quantitations of peptides assigned to the same protein. Giving further support to our method, application to a type 2 diabetes dataset identifies a list of protein candidates that is in very good agreement with previously performed transcriptomics meta analysis. Making use of quantitative properties of signal patterns identified, PPINGUIN can reveal new isoform candidates. Conclusions Regarding the increasing importance of quantitative proteomics we think that this method will be useful in practical applications like model fitting or functional enrichment analysis. We recommend to use this method if quantitation is a major objective of research.
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Affiliation(s)
- Chris Bauer
- MicroDiscovery GmbH, Marienburger Str, 1, 10405 Berlin, Germany.
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Ghosh D, Yu H, Tan XF, Lim TK, Zubaidah RM, Tan HT, Chung MCM, Lin Q. Identification of key players for colorectal cancer metastasis by iTRAQ quantitative proteomics profiling of isogenic SW480 and SW620 cell lines. J Proteome Res 2011; 10:4373-87. [PMID: 21854069 DOI: 10.1021/pr2005617] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
This study compared the whole cell proteome profiles of two isogenic colorectal cancer (CRC) cell lines (primary SW480 cell line and its lymph node metastatic variant SW620), as an in vitro metastatic model, to gain an insight into the molecular events of CRC metastasis. Using iTRAQ (isobaric tags for relative and absolute quantitation) based shotgun proteomics approach, we identified 1140 unique proteins, out of which 147 were found to be significantly altered in the metastatic cell. Ingenuity pathway analysis with those significantly altered proteins, revealed cellular organization and assembly as the top-ranked altered biological function. Differential expression pattern of 6 candidate proteins were validated by Western blot. Among these, the low expression level of β-catenin combined with the up-regulation of CacyBP (Calcyclin binding Protein), a β-catenin degrading protein, in the metastatic cell provided a rational guide for the downstream functional assays. The relative expression pattern of these two proteins was further validated in three other CRC cells by Western blot and quantitative immunofluorescence studies. Overexpression of CacyBP in three different primary CRC cell lines showed significant reduction in adhesion characteristics as well as cellular β-catenin level as confirmed by our experiments, indicating the possible involvement of CacyBP in CRC metastasis. In short, this study demonstrates successful application of a quantitative proteomics approach to identify novel key players for CRC metastasis, which may serve as biomarkers and/or drug targets to improve CRC therapy.
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Affiliation(s)
- Dipanjana Ghosh
- Department of Biological Sciences, National University of Singapore , 14 Science Drive 4, Singapore 117543
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Jin J, Kwon YW, Paek JS, Cho HJ, Yu J, Lee JY, Chu IS, Park IH, Park YB, Kim HS, Kim Y. Analysis of differential proteomes of induced pluripotent stem cells by protein-based reprogramming of fibroblasts. J Proteome Res 2011; 10:977-89. [PMID: 21175196 DOI: 10.1021/pr100624f] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
The recent generation of induced pluripotent stem (iPS) cells represents a novel opportunity to complement embryonic stem (ES) cell-based approaches. iPS cells can be generated by viral transduction of specific transcription factors, but there is a potential risk of tumorigenicity by random retroviral integration. We have generated novel iPS (sFB-protein-iPS) cells from murine dermal fibroblasts (FVB-sFB) that have ES cell characteristics, using ES cell-derived cell extracts instead of performing viral transduction. Notably, only cell extracts from an ES cell line (C57-mES) on the C57/BL6 background generated iPS cells in our protocol-not an ES cell line (E14-mES) on the 129 background. Hypothesizing that determining the differences in these 2 mES cell lines will provide vital insight into the reprogramming machinery, we performed proteomic and global gene expression analysis by iTRAQ and mRNA microarray, respectively. We observed that pluripotent ES cells and ES cell extract-derived iPS cells had differential proteomes and global gene expression patterns. Notably, reprogramming-competent C57-mES cells highly expressed proteins that regulate protein synthesis and metabolism, compared with reprogramming-incompetent 129-mES cells, suggesting that there is a threshold that protein synthetic machinery must exceed to initiate reprogramming.
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Affiliation(s)
- Jonghwa Jin
- Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea
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Zhang L, Jia X, Feng Y, Peng X, Zhang Z, Zhou W, Zhang Z, Ma F, Liu X, Zheng Y, Yang P, Yuan Z. Plasma membrane proteome analysis of the early effect of alcohol on liver: implications for alcoholic liver disease. Acta Biochim Biophys Sin (Shanghai) 2011; 43:19-29. [PMID: 21134885 DOI: 10.1093/abbs/gmq108] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
In humans, the over-consumption of alcohol can lead to serious liver disease. To examine the early effects of alcohol on liver disease, rats were given sufficient ethanol to develop liver cirrhosis. Rats before the onset of fibrosis were studied in this work. Plasma membranes (PM) of liver were extracted by twice sucrose density gradient centrifugation. The proteome profiles of PM from ethanol-treated rats and the controls were analyzed using two-dimensional gel electrophoresis (2-DE) and isobaric tag for relative and absolute quantitation (iTRAQ) technology. Ethanol treatment altered the amount of 15 different liver proteins: 10 of them were detected by 2-DE and 5 by iTRAQ. Keratin 8 was detected by both methods. Gene ontology analysis of these differentially detected proteins indicated that most of them were involved in important cell functions such as binding activity (including ion, DNA, ATP binding, etc.), cell structure, or enzyme activity. Among these, annexin A2, keratin 8, and keratin 18 were further verified using western blot analysis and annexin A2 was verified by immunohistochemistry. Our results suggested that alcohol has the potential to affect cell structure, adhesion and enzyme activity by altering expression levels of several relevant proteins in the PM. To the best of our knowledge, this is the first time to study the effect of alcohol on the liver PM proteome and it might be helpful for understanding the possible mechanisms of alcohol-induced liver disease.
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Affiliation(s)
- Lijun Zhang
- Shanghai Public Health Clinical Center, Fudan University, Shanghai, China.
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Hansson SF, Henriksson Å, Johansson L, Korsgren O, Eriksson JW, Tornqvist H, Davidsson P. Membrane Protein Profiling of Human Islets of Langerhans Using Several Extraction Methods. Clin Proteomics 2010. [DOI: 10.1007/s12014-010-9060-1] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Abstract
Introduction
Proteomic characterization of the human pancreatic islets, containing the insulin producing beta-cells, is likely to be of great importance for improved treatment and understanding of the pathophysiology of diabetes mellitus.
Objective
The focus of this study was to characterize the human islet membrane proteome.
Methods
In order to identify as many membrane proteins as possible, five different extraction procedures were used, i.e., phase separation using Triton X-114, a plasma membrane protein kit, cell surface protein biotinylation, total protein extraction, and lipid-based protein immobilization flow cell. Digested protein extracts were analyzed by nanoflow liquid chromatography tandem mass spectrometry. Then the identified proteins were categorized according to cellular location using their gene ontology annotation and by prediction of transmembrane helices in the sequence. This information was used to estimate the amount of membrane proteins identified.
Results
By combining the results from all extraction procedures, the total number of membrane proteins identified from the human islets was increased, accentuating that a combination of methods usually gives a higher coverage of the proteome. A total of 1,700 proteins were identified (≥2 unique peptides), and 735 of these proteins were annotated as membrane proteins while 360 proteins had at least one predicted transmembrane helix. The extraction method using phase separation with Triton X-114 yielded both the highest number and the highest proportion of membrane proteins.
Conclusion
This study gave an enhanced characterization of the human islet membrane proteome which may contribute to a better understanding of islet biology.
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Carey C, Purohit S, She JX. Advances and challenges in biomarker development for type 1 diabetes prediction and prevention using omic technologies. EXPERT OPINION ON MEDICAL DIAGNOSTICS 2010; 4:397-410. [PMID: 20885991 PMCID: PMC2946241 DOI: 10.1517/17530059.2010.508492] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
IMPORTANCE OF THE FIELD: Biomarkers are essential for the identification of high risk children as well as monitoring of prevention outcomes for type 1 diabetes (T1D). AREAS COVERED IN THIS REVIEW: This review discusses progress, opportunities and challenges in biomarker discovery and validation using high throughput genomic, transcriptomic and proteomic technologies. The authors also suggest potential solutions to deal with the current challenges. WHAT THE READER WILL GAIN: Readers will gain an overview of the current status on T1D biomarkers, an integrated review of three omic technologies, their applications and limitations for biomarker discovery and validation, and a critical discussion of the major issues encountered in biomarker development. TAKE HOME MESSAGE: Better biomarkers are still urgently needed for T1D prediction and prevention. The high throughput omic technologies offer great opportunities but also face significant challenges that have to be solved before their potential for biomarker development is fully realized.
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Affiliation(s)
- Colleen Carey
- Medical College of Georgia, Center for Biotechnology and Genomic Medicine, 1120 15th St., Augusta, 30912, USA
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Carranza P, Grunau A, Schneider T, Hartmann I, Lehner A, Stephan R, Gehrig P, Grossmann J, Groebel K, Hoelzle LE, Eberl L, Riedel K. A gel-free quantitative proteomics approach to investigate temperature adaptation of the food-borne pathogen Cronobacter turicensis
3032. Proteomics 2010; 10:3248-61. [DOI: 10.1002/pmic.200900460] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
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Abstract
The complementary disciplines of genomics and proteomics offer better insights into the molecular mechanisms of diseases. While genomics hunts for defining our static genetic substrate, proteomics explores the structure and function of proteins expressed by a cell or tissue type under specified conditions. In the past decade, proteomics has been revolutionized by the application of techniques such as two-dimensional gel electrophoresis (2DGE), mass spectrometry (MS), and protein arrays. These techniques have tremendous potential for biomarker development, target validation, diagnosis, prognosis, and optimization of treatment in medical care, especially in the field of islet and diabetes research. This chapter will highlight the contributions of proteomic technologies toward the dissection of complex network of signaling molecules regulating islet function, the identification of potential biomarkers, and the understanding of mechanisms involved in the pathogenesis of diabetes.
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Zhang Z, Zhang L, Hua Y, Jia X, Li J, Hu S, Peng X, Yang P, Sun M, Ma F, Cai Z. Comparative proteomic analysis of plasma membrane proteins between human osteosarcoma and normal osteoblastic cell lines. BMC Cancer 2010; 10:206. [PMID: 20470422 PMCID: PMC2880991 DOI: 10.1186/1471-2407-10-206] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2009] [Accepted: 05/14/2010] [Indexed: 01/17/2023] Open
Abstract
BACKGROUND Osteosarcoma (OS) is the most common primary malignant tumor of bone in children and adolescents. However, the knowledge in diagnostic modalities has progressed less. To identify new biomarkers for the early diagnosis of OS as well as for potential novel therapeutic candidates, we performed a sub-cellular comparative proteomic research. METHODS An osteosarcoma cell line (MG-63) and human osteoblastic cells (hFOB1.19) were used as our comparative model. Plasma membrane (PM) was obtained by aqueous two-phase partition. Proteins were analyzed through iTRAQ-based quantitative differential LC/MS/MS. The location and function of differential proteins were analyzed through GO database. Protein-protein interaction was examined through String software. One of differentially expressed proteins was verified by immunohistochemistry. RESULTS 342 non-redundant proteins were identified, 68 of which were differentially expressed with 1.5-fold difference, with 25 up-regulated and 43 down-regulated. Among those differential proteins, 69% ware plasma membrane, which are related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc., and interaction with each other. One protein--CD151 located in net nodes was verified to be over-expressed in osteosarcoma tissue by immunohistochemistry. CONCLUSION It is the first time to use plasma membrane proteomics for studying the OS membrane proteins according to our knowledge. We generated preliminary but comprehensive data about membrane protein of osteosarcoma. Among these, CD151 was further validated in patient samples, and this small molecule membrane might be a new target for OS research. The plasma membrane proteins identified in this study may provide new insight into osteosarcoma biology and potential diagnostic and therapeutic biomarkers.
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Affiliation(s)
- Zhiyu Zhang
- Department of Orthopaedics, The 4th Affiliated Hospital, China MedicalUniversity, Shenyang, 110032, China.
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Datta A, Park JE, Li X, Zhang H, Ho ZS, Heese K, Lim SK, Tam JP, Sze SK. Phenotyping of an in vitro model of ischemic penumbra by iTRAQ-based shotgun quantitative proteomics. J Proteome Res 2010; 9:472-84. [PMID: 19916522 DOI: 10.1021/pr900829h] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Cerebral ischemia is a major cause of death and long-term disability worldwide. Ischemic penumbra, the electrically silent but metabolically viable perifocal brain tissue, is the target for the much elusive stroke therapy. To characterize the molecular events of the dynamic penumbra, we applied an iTRAQ-based shotgun proteomic approach in an in vitro neuronal model, using the rat B104 neuroblastoma cell line. Various functional and cytometric assays were performed to establish the relevant time-point and conditions for ischemia to recapitulate the pathology of the penumbra. Two replicate iTRAQ experiments identified 1796 and 1566 proteins, respectively (<or=1.0% false discovery rate). Mining of proteomic data indicated the up-regulation of proteins involved in ammoniagenesis, antiapoptotic, anti-inflammatory and mitochondrial heat shock response and down-regulation of proteins pertaining to antioxidative defense and protein metabolism. Additionally, many proteins (for instance, park7 and VAP-A) involved in the chronic neurological disorders (such as Alzheimer's disease, Parkinson's disease or Bipolar disorder) were also regulated in this model of acute neuronal injury. Our results also provide preliminary evidence about the presence of a relative glucose paradox under in vitro conditions indicating possible application of this cell system to study the mechanisms of transient protection induced by concomitant glucose deprivation under hypoxia. In conclusion, our study shows the potential application of iTRAQ-based quantitative proteomics for the elucidation of pathophysiology and the discovery of novel therapeutic targets in the field of neuroproteomics.
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Affiliation(s)
- Arnab Datta
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore
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Abstract
Islet protein profiling is defined as generation of extended protein expression data sets from islets or islet cells. Islets from rodent control and animal models of type 1 and type 2 diabetes mellitus and healthy humans and insulin- and glucagon-producing cell lines have been used. Protein profiling entails separation, differential expression determination, identification and expression analysis. Protein/peptide separation is either gel-based or by chromatography. Differential expression is based on comparison of visualized spots/proteins between gels or by sample labelling in gel-free systems. Identification of proteins is made by tryptic fragmentation of proteins, fragment mass determination and mass comparison with protein databases. Analysis of expression data sets interprets the complex protein changes into cellular mechanisms to generate hypotheses. The importance of such protein expression sets to elucidate islet cellular events is evidenced by the observation that only about 50% of the differentially expressed proteins and transcripts showed concordance when measured in parallel. Using protein profiling, different areas related to islet dysfunction in type 1 and type 2 diabetes mellitus have been addressed, including dysfunction induced by elevated levels of glucose and fatty acids and cytokines. Because islets from individuals with type 1 or type 2 diabetes mellitus have not yet been protein profiled, islets from rat (BB-DP) and mouse (NOD, ob/ob, MKR) models of the disease have been used, and mechanisms responsible for islet dysfunction delineated offering avenues of intervention.
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Affiliation(s)
- P Bergsten
- Department of Medical Cell Biology, Uppsala University, Sweden.
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