Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy. Author Todd Greco, Amanda Guise, Ileana Cristea Publication Year 2016 Type Journal Article Abstract In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability. Keywords Animals, Humans, Mass Spectrometry, Proteomics, Proteins, Isotope Labeling, Protein Stability Journal Methods Mol Biol Volume 1410 Pages 39-63 Date Published 2016 ISSN Number 1940-6029 DOI 10.1007/978-1-4939-3524-6_3 Alternate Journal Methods Mol Biol PMCID PMC4916643 PMID 26867737 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML