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. 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

Todd M Greco  1 , Amanda J Guise  1 , Ileana M Cristea  2
Affiliations

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

Todd M Greco et al. Methods Mol Biol. 2016.

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.

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Figures

Fig. 1
Fig. 1
Integrated label-free and metabolic label-based approach for profiling protein interactions. (a) Label-free workflow. Cells expressing the tagged protein of interest (bait protein) are cultured in parallel to cells expressing the tag alone. Following cryogenic lysis, immunoaffinity isolation of the tagged protein (with its interactions) is performed using antibody-conjugated magnetic beads. Captured proteins are subjected to enzymatic digestion, sample clean-up and mass spectrometry analysis. Mass spectrometry signals (e.g., spectrum counts or peak intensities) from the bait protein isolation versus control isolations are analyzed to set cut-offs for high-confidence specific interactions. (b) Metabolic labeling workflow. Cells expressing tagged bait protein are cultured in media containing “light” amino acids, while control wild type cells (not expressing a tag) are cultured in “heavy” amino acid media. Cells are mixed in a 1:1 ratio and subjected to cryogenic lysis, immunoaffinity purification, and proteomic analysis as in (a), except the MS data analysis calculates the relative ion intensities of the “heavy” and “light” peptide signals to determine the specificity of interactions
Fig. 2
Fig. 2
Integration of SAINT and I-DIRT methods allows the simultaneous investigation of specificity and relative stability of protein interactions. Specificity determination using the SAINT algorithm assigns interaction specificity scores to individual proteins (SAINT score), while I-DIRT metabolic labeling distinguishes between stable/specific interactions and nonspecific/background contaminants by calculation of isotope ratios (I-DIRT stability ratio). Integrating these two methods (right panel) provides insight into the relative stability and/or fast-exchanging nature of specific protein interactions of a given bait protein
Fig. 3
Fig. 3
SAINT/I-DIRT analysis of HDAC5 and HDAC1 reflect their subcellular localizations and functions in transcriptional regulation and chromatin remodeling. (a) SAINT/I-DIRT plot for immunoisolated HDAC5 highlights fast-exchanging interactions with 14-3-3 chaperone proteins and components of the nuclear corepressor complex. (b) Transient interactions predicted by SAINT/I-DIRT scoring are consistent with the nucleo-cytoplasmic shuttling of HDAC5. In the nucleus, HDAC5 associates with the nuclear NCoR proteins (purple). During nuclear export, HDAC5 can dissociate from the NCoR complex and increase its interaction with 14-3-3 chaperone proteins. (c) SAINT/I-DIRT plot for immunoisolated HDAC1 allows classification of known and novel protein associations of HDAC1, highlighting transient association with proteins associated with transcription and stable association with numerous chromatin remodeling complexes. (d) HDAC1 associates specifically with chromatin remodeling complexes (e.g. NuRD, Sin3a/b, CoREST), the transcriptional regulatory complex CtBP, and the mitotic deacetylase complex MiDAC. The integrated SAINT/I-DIRT method provides functional insight into the relative stabilities of individual proteins within known complexes reported to associate with HDAC1
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