Format

Send to

Choose Destination
See comment in PubMed Commons below
Methods Mol Biol. 2009;548:187-207. doi: 10.1007/978-1-59745-540-4_11.

Systematic characterization of the protein interaction network and protein complexes in Saccharomyces cerevisiae using tandem affinity purification and mass spectrometry.

Author information

1
Banting and Best Department of Medical Research, University of Toronto, Toronto, ON, Canada, M5S 3E1.

Abstract

Defining protein complexes is a vital aspect of cell biology because cellular processes are often carried out by stable protein complexes and their characterization often provides insights into their function. Accurate identification of the interacting proteins in macromolecular complexes is easiest after purification to near homogeneity. To this end, the tandem affinity purification (TAP) system with subsequent protein identification by high-throughput mass spectrometry was developed (1, 2) to systematically characterize native protein complexes and transient protein interactions under near-physiological conditions. The TAP tag containing two adjacent affinity purification tags (calmodulin-binding peptide and Staphylococcus aureus protein A) separated by a tobacco etch virus (TEV) protease cleavage site is fused with the open reading frame of interest. Using homologous recombination, a fusion library was constructed for the yeast Saccharomyces cerevisiae (3) in which the carboxy-terminal end of each predicted open reading frame is individually tagged in the chromosome so that the resulting fusion proteins are expressed under the control of their natural promoters (3). In this chapter, an optimized protocol for systematic protein purification and subsequent mass spectrometry-based protein identification is described in detail for the protein complexes of S. cerevisiae (4-6).

PMID:
19521826
DOI:
10.1007/978-1-59745-540-4_11
[Indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments

    Supplemental Content

    Full text links

    Icon for Springer
    Loading ...
    Support Center