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Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-13775

Pflieger, D; Jünger, M A; Müller, M; Rinner, O; Lee, H; Gehrig, P M; Gstaiger, M; Aebersold, R (2008). Quantitative proteomic analysis of protein complexes: concurrent identification of interactors and their state of phosphorylation. Molecular and Cellular Proteomics, 7(2):326-346.

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Abstract

Protein complexes have largely been studied by immunoaffinity purification and (mass spectrometric) analysis. Although this approach has been widely and successfully used it is limited because it has difficulties reliably discriminating true from false protein complex components, identifying post-translational modifications, and detecting quantitative changes in complex composition or state of modification of complex components. We have developed a protocol that enables us to determine, in a single LC-MALDI-TOF/TOF analysis, the true protein constituents of a complex, to detect changes in the complex composition, and to localize phosphorylation sites and estimate their respective stoichiometry. The method is based on the combination of fourplex iTRAQ (isobaric tags for relative and absolute quantification) isobaric labeling and protein phosphatase treatment of substrates. It was evaluated on model peptides and proteins and on the complex Ccl1-Kin28-Tfb3 isolated by tandem affinity purification from yeast cells. The two known phosphosites in Kin28 and Tfb3 could be reproducibly shown to be fully modified. The protocol was then applied to the analysis of samples immunopurified from Drosophila melanogaster cells expressing an epitope-tagged form of the insulin receptor substrate homologue Chico. These experiments allowed us to identify 14-3-3epsilon, 14-3-3zeta, and the insulin receptor as specific Chico interactors. In a further experiment, we compared the immunopurified materials obtained from tagged Chico-expressing cells that were either treated with insulin or left unstimulated. This analysis showed that hormone stimulation increases the association of 14-3-3 proteins with Chico and modulates several phosphorylation sites of the bait, some of which are located within predicted recognition motives of 14-3-3 proteins.

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Functional Genomics Center Zurich
04 Faculty of Medicine > Center of Competence Systems Physiology and Metabolic Diseases
08 University Research Priority Programs > Systems Biology / Functional Genomics
DDC:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2008
Deposited On:19 Feb 2009 09:12
Last Modified:28 Nov 2013 01:50
Publisher:American Society for Biochemistry and Molecular Biology
ISSN:1535-9476
Additional Information:This research was originally published in Pflieger, D; Jünger, M A; Müller, M; Rinner, O; Lee, H; Gehrig, P M; Gstaiger, M; Aebersold, R (2008). Quantitative proteomic analysis of protein complexes: concurrent identification of interactors and their state of phosphorylation. Molecular & Cellular Proteomics, 7(2):326-346. © the American Society for Biochemistry and Molecular Biology.
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:10.1074/mcp.M700282-MCP200
PubMed ID:17956857
Citations:Web of Science®. Times Cited: 73
Google Scholar™
Scopus®. Citation Count: 74

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