Analysis of protein complexes using mass spectrometry

Abstract

In this article, we review the current status of affinity purification and mass spectrometry (AP–MS) and its promise for better understanding protein complexes, complex structure and the dynamics of complex formation. We describe the general AP–MS strategy, with an emphasis on generic approaches (flag-tag, tandem AP) and how AP–MS of multiple components (that is, high-density AP–MS) can help to reveal the true composition of protein complexes. Recent high-throughput studies with flag-tagging or tandem AP significantly improved our understanding of protein–protein interactions in yeast. AP–MS can be combined with classical biochemical purification approaches to reveal complex composition and to resolve the problem of mutually exclusive complexes co-precipitating with the same tagged protein. Crosslinkers can contribute to AP–MS strategies by stabilizing weak or transient protein interactions and by revealing details concerning complex organization and interacting surfaces. Stoichiometry of protein complexes can be obtained using intact-complex mass measurement and absolute quantitative proteomics tools. Quantitative proteomics approaches can help to decipher the dynamics of protein-complex formation.