A mass spectrometry view of stable and transient protein interactions

Abstract

Through an impressive range of dynamic interactions, proteins succeed to carry out the majority of functions in a cell. These temporally and spatially regulated interactions provide the means through which one single protein can perform diverse functions and modulate different cellular pathways. Understanding the identity and nature of these interactions is therefore critical for defining protein functions and their contribution to health and disease processes. Here, we provide an overview of workflows that incorporate immunoaffinity purifications and quantitative mass spectrometry (frequently abbreviated as IP-MS or AP-MS) for characterizing protein-protein interactions. We discuss experimental aspects that should be considered when optimizing the isolation of a protein complex. As the presence of nonspecific associations is a concern in these experiments, we discuss the common sources of nonspecific interactions and present label-free and metabolic labeling mass spectrometry-based methods that can help determine the specificity of interactions. The effective regulation of cellular pathways and the rapid reaction to various environmental stresses rely on the formation of stable, transient, and fast-exchanging protein-protein interactions. While determining the exact nature of an interaction remains challenging, we review cross-linking and metabolic labeling approaches that can help address this important aspect of characterizing protein interactions and macromolecular assemblies.

Figure 1. Common workflow for immunoaffinity purification mass spectrometry experiments

Cells expressing the protein of interest are lysed and protein complexes are isolated by immunoaffinity purification. Eluted proteins are processed for MS analysis. MS spectra are analyzed to identify proteins within isolated complex(es) and bioinformatics tools are implemented to generate protein interaction networks.

Figure 2. Dependence of interaction specificity on IP conditions

Optimized lysis and incubation conditions, such as stringency of lysis buffer and incubation time with beads and antibodies, allow retention of specific stable and transient interactions, while reducing the number of non-specific associations. The latter include proteins that bind to beads, tag, immunoglobulin molecules, or to specific isolated proteins.

Figure 3. Determining specificity and relative stability of interactions using label-free and metabolic labeling approaches

A) In the I-DIRT approach, wild type cells grown in “heavy” medium and cells expressing the tagged protein of interest grown in “light” medium are mixed prior to IP. Isolated complexes are analyzed by MS and isotopic ratios for each protein are indicative of the specificity and stability of the interaction. B) When label-free quantification (e.g., SAINT) is combined with a metabolic labeling approach (e.g., I-DIRT), the relative stability of interactions can be assessed. Specific transient interactions can be observed with high SAINT scores and low I-DIRT ratios.