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Concurrent quantification of proteome and phosphoproteome to reveal system-wide association of protein phosphorylation and gene expression.

ABSTRACT: Reversible phosphorylation of proteins is an important process modulating cellular activities from upstream, which mainly involves sequential phosphorylation of signaling molecules, to downstream where phosphorylation of transcription factors regulates gene expression. In this study, we combined quantitative labeling with multidimensional liquid chromatography-mass spectrometry to monitor the proteome and phosphoproteome changes in the initial period of adipocyte differentiation. The phosphorylation level of a specific protein may be regulated by a kinase or phosphatase without involvement of gene expression or as a phenomenon that accompanies the alteration of its gene expression. Concurrent quantification of phosphopeptides and non-phosphorylated peptides makes it possible to differentiate cellular phosphorylation changes at these two levels. Furthermore, on the system level, certain proteins were predicted as the targeted gene products regulated by identified transcription factors. Among them, several proteins showed significant expression changes along with the phosphorylation alteration of their transcription factors. This is to date the first work to concurrently quantify proteome and phosphoproteome changes during the initial period of adipocyte differentiation, providing an approach to reveal the system-wide association of protein phosphorylation and gene expression.

SUBMITTER: Wu YB

PROVIDER: S-EPMC2816019 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Concurrent quantification of proteome and phosphoproteome to reveal system-wide association of protein phosphorylation and gene expression.

Wu Yi-Bo YB Dai Jie J Yang Xing-Lin XL Li Su-Jun SJ Zhao Shi-Lin SL Sheng Quan-Hu QH Tang Jia-Shu JS Zheng Guang-Yong GY Li Yi-Xue YX Wu Jia-Rui JR Zeng Rong R

Molecular & cellular proteomics : MCP 20090812 12

Reversible phosphorylation of proteins is an important process modulating cellular activities from upstream, which mainly involves sequential phosphorylation of signaling molecules, to downstream where phosphorylation of transcription factors regulates gene expression. In this study, we combined quantitative labeling with multidimensional liquid chromatography-mass spectrometry to monitor the proteome and phosphoproteome changes in the initial period of adipocyte differentiation. The phosphoryla ...[more]

PMID: 19674963

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Project description:Diabetic nephropathy is the most common chronic kidney disease in the world and the main cause of end-stage renal disease (ESRD). The structural integrity of podocytes is fundamental to the normal function of the glomerulus, and the role of glycogen synthase kinase 3β (GSK-3β) in podocytes is complicated. A thorough understanding of GSK-3β is crucial to understand the mechanism of diabetic nephropathy. To analyze the roles of GSK-3β in podocytes, GSK-3β knockdown lentivirus by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)9 was applied to establish stable cell lines. Mass spectrometry was utilized to search for differentially expressed proteins. Consequently, we found 34 proteins with higher levels and 115 proteins with lower levels in GSk-3β knockdown cells than in control cells and identified 581 phosphosites with higher phosphorylation levels and 288 phosphosites with lower phosphorylation levels. We performed functional enrichment analysis of these proteins and phosphorylated proteins based on public databases. Enrichment analysis revealed that GSK-3β participates in the spliceosome, Hippo signaling pathway, actin binding, structural molecule activity, and other pathways. Then, we used motif analysis of phosphate sites to determine 89 conserved motifs based on 1,068 phosphoserine (pS) sites and 15 conserved motifs in view of 104 phosphothreonine (pT) sites. Additionally, protein-protein interaction network analysis was carried out using the STRING database. Cytoscape's add-on Molecular Complex Detection (MCODE) was used to analyze key and core protein groups. In quantitative differential protein analysis, four MCODEs were obtained, and 22 MCODEs were obtained in the analysis of the phosphoproteome of differentially expressed proteins. Finally, we analyzed the kinase regulatory network in podocytes after GSK-3β knockdown and identified 299 protein kinases and 3,460 significantly changed phosphorylation modification sites on 1,574 proteins. These results will be valuable for further research on GSK-3β.

Dataset Information

Concurrent quantification of proteome and phosphoproteome to reveal system-wide association of protein phosphorylation and gene expression.

Publications

Concurrent quantification of proteome and phosphoproteome to reveal system-wide association of protein phosphorylation and gene expression.

Similar Datasets

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