Characterizing Protein-Protein Interactions Using Mass Spectrometry: Challenges and
2016
During the past decades, mass spectrometry (MS)-based proteomics has become an important technology to identify protein–protein interactions (PPIs). The application of a quantitative filter in protein enrichments from crude lysates to discriminate bona fide interactors from background proteins has proved to be particularly powerful. Recently, many different approaches to identify PPIs have been developed, including proximity-ligation technology and global interactome profiling based on the co-behavior of protein complexes in biochemical purification or perturbation experiments. Furthermore, methodologies have been introduced that provide information regarding the stoichiometry and topology of detected PPIs. We review these novel methodologies and emphasize the need to miniaturize workflows to analyze protein interactions in biological and pathological contexts where sample amounts are limited. Identifying PPIs Using MS Many proteins exert their functions within cells in the context of protein complexes. Therefore, identifying protein–protein interactions (PPIs, see Glossary) is pivotal to gaining insight into the biological function of proteins. During the past decades, MS-based proteomics has become the method of choice to comprehensively identify PPIs. Initial studies used tandem-affinity purification (TAP) to purify the protein of interest and its interactors [1,2]. Sequential affinity purifications typically result in a relatively pure sample. However, modern mass spectrometers detect even the smallest contaminations in these samples. To distinguish contaminants from real interactors, a quantitative filter thus needs to be introduced. In quantitative MS-based proteomics these filters typically rely on the introduction of isotope labels or the use of sophisticated normalization algorithms for label-free quantification [3]. These workflows enable high-confidence PPI identification from single-step affinity purifications (reviewed in [4]).
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