Most cellular processes in a cell are mediated by large protein assemblies or multiprotein complexes, which dynamically assemble, store and transduce biological information. A key step toward gaining a full understanding of how these "protein machines" function is to characterize subunit composition and stoichiometry of the protein complexes. Affinity purification/tandem mass spectrometry has been successfully applied to purify and identify subunit composition of protein complexes, whereas native mass spectrometry has been the major tool for determination of subunit stoichiometry. Although it is effective, native mass spectrometry of intact protein complexes remains technically challenging. With its high throughput and sensitivity for protein identification, bottom-up based absolute quantitation strategy has become an attractive and alternative approach for determination of subunit stoichiometry. Absolute quantification of proteins in a complex mixture has been accomplished by isotope dilution using stable isotope-labeled synthetic peptides (AQUA peptide) that are chemically identical to the proteotypic peptides generated by proteolysis of proteins of interest. More recently, absolute multiplexed quantitative analysis of protein mixtures has been achieved by creating and expressing an artificial gene (QconCAT) to code for a protein composed of a concatenated proteotypic peptides of selected proteins in a single experiment. Here, we have developed an integrated quantitative bottom up approach by adopting the QconCAT strategy to quantify absolute abundance and determine subunit stoichiometry of affinity purified multi-protein complexes. Affinity purified human proteasome complexes were used as our target protein complexes for analysis.
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