Ultrahigh-throughput proteomics using fast RPLC separations with ESI-MS/MS.

We describe approaches for proteomics analysis using electrospray ionization-tandem mass spectrometry coupled with fast reversed-phase liquid chromatography (RPLC) separations. The RPLC separations used 50-microm-i.d. fused-silica capillaries packed with submicrometer-sized C18-bonded porous silica particles and achieved peak capacities of 130-420 for analytes from proteome tryptic digests. When these separations were combined with linear ion trap tandem mass spectrometry measurements, approximately 1000 proteins could be identified in 50 min from approximately 4000 identified tryptic peptides; approximately 550 proteins in 20 min from approximately 1800 peptides; and approximately 250 proteins in 8 min from approximately 700 peptides for a S. oneidensis tryptic digest. The dynamic range for protein identification with the fast separations was determined to be approximately 3-4 orders of magnitude of relative protein abundance on the basis of known proteins in human blood plasma analyses. We found that 55% of the MS/MS spectra acquired during the entire analysis (and up to 100% of the MS/MS spectra acquired from the most data-rich zone) provided sufficient quality for identifying peptides. The results confirm that such analyses using very fast (minutes) RPLC separations based on columns packed with microsized porous particles are primarily limited by the MS/MS analysis speed.