Quantitative Proteomic Analysis of Protein Complexes

• DOI: 10.1074/mcp.m700282-mcp200
• Publication Date: 2007-10-24T00:19:40Z
• Authors: Delphine Pflieger; Martin A. Jünger; Markus Müller; Oliver Rinner; Hookeun Lee; Peter M. Gehrig; Matthias Gstaiger; Ruedi Aebersold
• Keywords: Phosphopeptides; Proteomics; 0301 basic medicine; 1303 Biochemistry; [CHIM.ANAL] Chemical Sciences/Analytical chemistry; Saccharomyces cerevisiae Proteins; Molecular Sequence Data; insulin pathway; 610 Medicine & health; 10071 Functional Genomics Center Zurich; Saccharomyces cerevisiae; 03 medical and health sciences; [CHIM.ANAL]Chemical Sciences/Analytical chemistry; genuine partners; 1312 Molecular Biology; insulin receptor substrate; [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology; Animals; Drosophila Proteins; Humans; [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology; Amino Acid Sequence; Phosphorylation; 1602 Analytical Chemistry; dynamic; phosphorylation; protein complex; Angiotensin II; Proteins; Phosphoproteins; stoichiometry; Drosophila melanogaster; 10070 Center of Competence Systems Physiology and Metabolic Diseases; iTRAQ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; 570 Life sciences; biology; U7 Systems Biology / Functional Genomics; Protein Binding

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.