Advances in protein complex analysis using mass spectrometry

Abstract

Proteins often function as components of larger complexes to perform a specific function, and formation of these complexes may be regulated. For example, intracellular signalling events often require transient and/or regulated protein-protein interactions for propagation, and protein binding to a specific DNA sequence, RNA molecule or metabolite is often regulated to modulate a particular cellular function. Thus, characterizing protein complexes can offer important insights into protein function. This review describes recent important advances in mass spectrometry (MS)-based techniques for the analysis of protein complexes. Following brief descriptions of how proteins are identified using MS, and general protein complex purification approaches, we address two of the most important issues in these types of studies: specificity and background protein contaminants. Two basic strategies for increasing specificity and decreasing background are presented: whereas (1) tandem affinity purification (TAP) of tagged proteins of interest can dramatically improve the signal-to-noise ratio via the generation of cleaner samples, (2) stable isotopic labelling of proteins may be used to discriminate between contaminants and bona fide binding partners using quantitative MS techniques. Examples, as well as advantages and disadvantages of each approach, are presented.

A Novel, Evolutionarily Conserved Protein Phosphatase Complex Involved in Cisplatin Sensitivity

“…ging and mass spectrometry, in which a protein complex of interest is purified in a two-step affinity enrichment process and its components are identified by the fragment ion spectra of selected tryptic peptides, have proven to be invaluable tools in the characterization of protein complexes (16,(21)(22)(23). As compared with single tag purification strategies, samples isolated by TAP tagging are significantly cleaner, thereby reducing the likelihood of false-positive identifications.…”

MyoD and E-protein heterodimers switch rhabdomyosarcoma cells from an arrested myoblast phase to a differentiated state

“…We used LC MS/MS (liquid chromatography and tandem mass spectrometry) to separate and identify protein complexes purified through a modified tandem affinity purification (TAP) procedure at a physiologic salt concentration (Supplemental Fig. S1; Rigaut et al 1999;Gingras et al 2005). The E2A gene encodes two alternatively spliced transcripts, E12 and E47, which differ in the exon encoding the bHLH region.…”

Application of proteomics to ecology and population biology

“…The two most commonly used techniques are MALDI and electrospray ionization (ESI). Both methods use socalled 'soft' ionization techniques, but differ in important ways for which the reader can acquire additional information in a variety of recent excellent reviews (Aebersold and Mann, 2003;Yates, 2004;Gingras et al, 2005;Guerrera and Kleiner, 2005;Domon and Aebersold, 2006) Suffice it to say that both techniques produce ionized peptides in the gas phase, which enter a mass analyzer that measures the m/z ratio.…”

“…MS is also a prominent tool used by security agencies, for example in rapid detection of explosive materials at airports (Takats et al, 2004(Takats et al, , 2005a Over the past two decades, a number of major advances in MS technologies applied to the analysis of proteins and peptides has elevated MS to a central role in proteomics, including new methods for the introduction of complex protein and peptide mixtures into the mass spectrometer in such a manner as to allow for the de novo sequencing of small peptides (Lin et al, 2003;Yates, 2004;Gingras et al, 2005;Guerrera and Kleiner, 2005;Domon and Aebersold, 2006) Combined with the ever-expanding availability of whole, or partial, genome sequences and bioinformatic tools for their analysis, MS has assumed a primary role in proteomic analyses. In this regard, MS, genomic sequencing and bioinformatics exist in symbiosis, depending and benefiting from advances in each.…”