Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy.

TitleDetermining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy.
Publication TypeJournal Article
Year of Publication2016
AuthorsGreco, TM, Guise, AJ, Cristea, IM
JournalMethods Mol Biol
Volume1410
Pagination39-63
Date Published2016
ISSN1940-6029
Abstract

<p>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.</p>

DOI10.1007/978-1-4939-3524-6_3
Alternate JournalMethods Mol. Biol.
PubMed ID26867737
PubMed Central IDPMC4916643
Grant ListR01 GM114141 / GM / NIGMS NIH HHS / United States
R01 HL127640 / HL / NHLBI NIH HHS / United States
R01GM114141 / GM / NIGMS NIH HHS / United States
R21 AI102187 / AI / NIAID NIH HHS / United States
R21 HD073044 / HD / NICHD NIH HHS / United States
R21AI102187 / AI / NIAID NIH HHS / United States