Making Broad Proteome Protein Measurements in 1−5 min Using High-Speed RPLC Separations and High-Accuracy Mass Measurements

The throughput of proteomics measurements that provide broad protein coverage is limited by the quality and speed of both the separations as well as the subsequent mass spectrometric analysis; at present, analysis times can range anywhere from hours (high throughput) to days or longer (low throughput). We have explored the basis for proteomics analyses conducted on the order of minutes using high-speed capillary RPLC combined through on-line electrospray ionization interface with high-accuracy mass spectrometry (MS) measurements. Short 0.8-μm porous C18 particle-packed 50-μm-i.d. capillaries were used to speed the RPLC separations while still providing high-quality separations. Both time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) MS were applied for identifying peptides using the accurate mass and time (AMT) tag approach. Peptide RPLC relative retention (elution) times that were generated by solvent gradients that differed by at least 25-fold were found to provide relative elution times that agreed to within 5%, which provides the basis for using peptide AMT tags for higher throughput proteomics measurements. For fast MS acquisition speeds (e.g., 0.2 s for TOF and either ∼0.3 or ∼0.6 s for FTICR), peptide mass measurement accuracies of better than ±15 ppm were obtained with the high-speed RPLC separations. The ability to identify peptides and the overall proteome coverage was determined by factors that include the separation peak capacity, the sensitivity of the MS (with fast scanning), and the accuracy of both the mass measurements and the relative RPLC peptide elution times. The experimental RPLC relative elution time accuracies of 5% (using high-speed capillary RPLC) and mass measurement accuracies of better than ±15 ppm allowed for the confident identification of >2800 peptides and >760 proteins from >13 000 different putative peptides detected from a <i>Shewanella </i><i>oneidensis</i> tryptic digest. Initial results for both RPLC-ESI-TOF and RPLC-ESI-FTICR MS were similar, with ∼2000 different peptides from ∼600 different proteins identified within 2−3 min. For <120-s proteomic analysis, TOF MS analyses were more effective, while FTICR MS was more effective for the >150-s analysis due to the improved mass accuracies attained using longer spectrum acquisition times.