Analysis of quantitative proteomic data generated via multidimensional protein identification technology

We describe the analysis of quantitative proteomic samples via multidimensional protein identification technology (MudPIT. Ratio amounts of the soluble portion of the S. cerevisiae proteome from cultures of S. cerevisiae strain S288C grown in either N-14 minimal media or N-15-enriched minimal media were mixed and digested into a complex peptide mixture. A 1x N-14/lx N-15 complex peptide mixture was analyzed by single-dimensional reversed-phase chromatography and electrospray ionization quadrapole time-of-flight mass spectrometry in order to demonstrate the replacement of N-14 by N-15 under the growth conditions used. After conformation of the incorporation of N-15 into the labeled sample, three separate samples consisting of a 1 x N-14/ 1 x N-15 complex peptide mixture, a 5 x N-14/1 x N-15 complex peptide mixture, and a 10 x 14N/ 1 x 15N complex peptide mixture were analyzed via MudPIT. We demonstrate the dynamic range of the system by analyzing a 1: 1, 5: 1, and 10: 1 data set using the soluble portion from S. cerevisiae grown in either N-14 or N-15-enriched minimal media. The method described provides an accurate way to undertake a large-scale quantitative proteomic study.