Tandem affinity purification combined with mass spectrometry to identify components of protein complexes

Methods Mol Biol. 2008:439:309-26. doi: 10.1007/978-1-59745-188-8_21.

Abstract

Most biological processes are governed by multiprotein complexes rather than individual proteins. Identification of protein complexes therefore is becoming increasingly important to gain a molecular understanding of cells and organisms. Mass spectrometry-based proteomics combined with affinity-tag-based protein purification is one of the most effective strategies to isolate and identify protein complexes. The development of tandem-affinity purification approaches has revolutionized proteomics experiments. These two-step affinity purification strategies allow rapid, effective purification of protein complexes and, at the same time, minimize background. Identification of even very low-abundant protein complexes with modern sensitive mass spectrometers has become routine. Here, we describe two general strategies for tandem-affinity purification followed by mass spectrometric identification of protein complexes.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Blotting, Western
  • Cells, Cultured
  • Chromatography, Affinity / methods*
  • Chromatography, Ion Exchange
  • Electrophoresis, Polyacrylamide Gel
  • Mass Spectrometry / methods*
  • Proteins / chemistry
  • Proteins / isolation & purification*

Substances

  • Proteins