Tandem Affinity Purification Combined with Mass Spectrometry to Identify Components of Protein Complexes
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.
Keywordstandem-affinity purification (TAP) His-Bio tag mass spectrometry protein complex identification in-vivo cross-linking MudPIT
We thank Christian Tagwerker and Cortnie Guerrero for their contributions in developing the protocols described here and Karin Flick for the figure design. Work in the laboratory of P. Kaiser is supported by grants from NIH (RO1GM-66164, R21CA113823) and the California Breast Cancer Research Program (11NB-0177). L. Huang acknowledges support from NIH (GM074830) and the DOD (PC-041126). D. Meierhofer is supported by an Erwin Schroedinger Fellowship (FWF J2665).
- 3.Denison C, Rudner AD, Gerber SA, Bakalarski CE, Moazed D, Gygi SP (2004) A proteomic strategy for gaining insights into protein sumoylation in yeast. Mol Cell ProteomicsGoogle Scholar
- 6.6. Tagwerker C, Flick K, Cu M, Guerrero C, Dou Y, Auer B, Baldi P, Huang L, Kaiser P (2006) A tandem affinity tag for two-step purification under fully denaturing conditions: Application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking. Mol Cell Proteomics 5:737–748PubMedGoogle Scholar
- 8.8. Guerrero C, Tagwerker C, Kaiser P, Huang L (2006) An integrated mass spectrometry-based proteomic approach: quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network. Mol Cell Proteomics 5:366–378PubMedGoogle Scholar
- 13.13. Savage D, Mattson G, Desa, S, Niedlander G, Morgensen S, Conklin E (1994) Avidin-biotin chemistry: A handbook. Pierce Chemical, Rockford, IL.Google Scholar