The study of macromolecular complexes by quantitative proteomics.
We describe a generic strategy for determining the specific composition, changes in the composition, and changes in the abundance of protein complexes. It is based on the use of isotope-coded affinity tag (ICAT) reagents and mass spectrometry to compare the relative abundances of tryptic peptides derived from suitable pairs of purified or partially purified protein complexes. In a first application, the genuine protein components of a large RNA polymerase II (Pol II) preinitiation complex (PIC) were distinguished from a background of co-purifying proteins by comparing the relative abundances of peptides derived from a control sample and the specific complex that was purified from nuclear extracts by a single-step promoter DNA affinity procedure. In a second application, peptides derived from immunopurified STE12 protein complexes isolated from yeast cells in different states were used to detect quantitative changes in the abundance of the complexes, and to detect dynamic changes in the composition of the samples. The use of quantitative mass spectrometry to guide identification of specific complex components in partially purified samples, and to detect quantitative changes in the abundance and composition of protein complexes, provides the researcher with powerful new tools for the comprehensive analysis of macromolecular complexes.
Ranish
Jeffrey A
JA
Institute for Systems Biology, 1441 North 34th Street, Seattle, Washington 98103-8904, USA.
Yi
Eugene C
EC
Leslie
Deena M
DM
Purvine
Samuel O
SO
Goodlett
David R
DR
Eng
Jimmy
J
Aebersold
Ruedi
R
D000345:Affinity Labels; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D040901:Proteomics / Q000379:methods*; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics
D000345:Affinity Labels; D005656:Fungal Proteins; D046911:Macromolecular Substances; C489241:STE12 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012319:RNA Polymerase II