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Methods Mol Biol. 2016;1410:39-63. doi: 10.1007/978-1-4939-3524-6_3.

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

Author information

1
Department of Molecular Biology, Princeton University, 210 Lewis Thomas Laboratory, Washington Road, Princeton, NJ, 08544, USA.
2
Department of Molecular Biology, Princeton University, 210 Lewis Thomas Laboratory, Washington Road, Princeton, NJ, 08544, USA. icristea@princeton.edu.

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:

Affinity isolation; I-DIRT; Immunoprecipitation; Label-free quantification; Protein complexes; Protein interactions; SAINT; Stable isotope labeling quantification

PMID:
26867737
PMCID:
PMC4916643
DOI:
10.1007/978-1-4939-3524-6_3
[Indexed for MEDLINE]
Free PMC Article

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