Ions of the interactome: The role of MS in the study of protein interactions in proteomics and structural biology

Abstract

The role of MS in the study of protein-protein interactions in solution is described from a proteomics perspective, in terms of high-throughput analyses of protein complexes in vivo, through to chemical and biochemical treatments ahead of MS analysis in the context of complementary experimental approaches in structural biology. The use of MS to characterise protein-protein interactions is described following the single and tandem affinity purification of protein complexes and assemblies of expressed proteins in host cells, the isolation and preservation of protein complexes on surfaces and microarrays, and their prior treatment with chemical and biochemical probes by hydrogen exchange, radical probe, chemical cross-linking, and limited proteolysis. The advantages and disadvantages of each of the approaches are presented. These new and emerging applications, which further demonstrate the power of MS, continue to ensure that the mass spectrometer will remain at the heart of discoveries in proteomics in the foreseeable future.

A yEGFP‐based reporter system for high‐throughput yeast two‐hybrid assay by flow cytometry

“…To understand the protein-protein interaction network on a large scale, the genome-wide protein linkage mapping or ''interactome'' analyses have therefore become a major objective in the field of proteomics (1). Although a number of promising technologies have been developed to analyze and monitor protein-protein interactions in vitro and in vivo, and the need for confirmation by independent methods is recognized (2), only the yeast two-hybrid (Y2H) and mass spectrometrybased affinity copurification approaches have been proven robust and versatile enough for systematic large-scale analysis (3)(4)(5)(6).…”

Gene expression changes in normal haematopoietic cells

“…101,102 Another major strength of MS is the ability to detect PTMs on peptide fragments, and the technique is versatile enough to use many sources of materials. 103,104 Thus, MS has been increasingly adapted to provide specific protein identification and high throughput analyses of the proteome. 104 However, maintaining a system to simultaneously investigate the biological context of a range of protein interactions during manipulation will require creative approaches and sophisticated experimental design.…”

“…103,104 Thus, MS has been increasingly adapted to provide specific protein identification and high throughput analyses of the proteome. 104 However, maintaining a system to simultaneously investigate the biological context of a range of protein interactions during manipulation will require creative approaches and sophisticated experimental design. 102,103 Examples of the tools used to evaluate the proteome are listed in Table 2, but experimental approaches to interrogating the proteome are diverse and manifold.…”

Integrating physical and genetic maps: from genomes to interaction networks

“…For instance, genetic interactions mapped by synthetic genetic array (SGA) analysis are influenced by artefacts caused by gene deletion 12,29 , such as mistargeted deletion constructs and deletions that alter the metabolism of the drug that is used for mutant selection. Physical interactions mapped by Y2H or TAP-MS are influenced by artefacts that result from gene tagging, which can influence the functioning of the protein that is produced 30,31 .…”