Proteomics analysis based-on reversed-phase liquid chromatography (RPLC) is widely practiced; however, variations providing cutting-edge RPLC performance have generally not been adopted even though their benefits are well established. Here, we describe an automated format 20 kpsi RPLC system for proteomics and metabolomics that includes on-line coupling of micro-solid phase extraction for sample loading and allows electrospray ionization emitters to be readily replaced. The system uses 50 microm i.d. x 40-200 cm fused-silica capillaries packed with 1.4-3-microm porous C18-bonded silica particles to obtain chromatographic peak capacities of 1000-1500 for complex peptide and metabolite mixtures. This separation quality provided high-confidence identifications of >12 000 different tryptic peptides from >2000 distinct Shewanella oneidensis proteins (approximately 40% of the proteins predicted for the S. oneidensis proteome) in a single 12-h ion trap tandem mass spectrometry (MS/MS) analysis. The protein identification reproducibility approached 90% between replicate experiments. The average protein MS/MS identification rate exceeded 10 proteins/min, and 1207 proteins were identified in 120 min through assignment of 5944 different peptides. The proteomic analysis dynamic range of the 20 kpsi RPLC-ion trap MS/MS was approximately 10(6) based on analyses of a human blood plasma sample, for which 835 distinct proteins were identified with high confidence in a single 12-h run. A single run of the 20 kpsi RPLC-accurate mass MS detected >5000 different compounds from a metabolomics sample.

We describe a four-column, high-pressure capillary liquid chromatography (LC) system for robust, high-throughput liquid chromatography-mass spectrometry (LC-MS(/MS)) analyses. This system performs multiple LC separations in parallel, but staggers each of them such that the data-rich region of each separation is sampled sequentially. By allowing nearly continuous data acquisition, this design maximizes the use of the mass spectrometer. Each analytical column is connected to a corresponding ESI emitter in order to avoid the use of postcolumn switching and associated dead volume issues. Encoding translation stages are employed to sequentially position the emitters at the MS inlet. The high reproducibility of this system is demonstrated using consecutive analyses of global tryptic digest of the microbe Shewanella oneidensis.

A high-throughput approach and platform using 15 minute reversed-phase capillary liquid chromatography (RPLC) separations in conjunction with ion mobility spectrometry-mass spectrometry (IMS-MS) measurements was evaluated for the rapid analysis of complex proteomics samples. To test the separation quality of the short LC gradient, a sample was prepared by spiking twenty reference peptides at varying concentrations from 1 ng/mL to 10 µg/mL into a tryptic digest of mouse blood plasma and analyzed with both a LC-Linear Ion Trap Fourier Transform (FT) MS and LC-IMS-TOF MS. The LC-FT MS detected thirteen out of the twenty spiked peptides that had concentrations ≥100 ng/mL. In contrast, the drift time selected mass spectra from the LC-IMS-TOF MS analyses yielded identifications for nineteen of the twenty peptides with all spiking levels present. The greater dynamic range of the LC-IMS-TOF MS system could be attributed to two factors. First, the LC-IMS-TOF MS system enabled drift time separation of the low concentration spiked peptides from the high concentration mouse peptide matrix components, reducing signal interference and background, and allowing species to be resolved that would otherwise be obscured by other components. Second, the automatic gain control (AGC) in the linear ion trap of the hybrid FT MS instrument limits the number of ions that are accumulated to reduce space charge effects and achieve high measurement accuracy, but in turn limits the achievable dynamic range compared to the IMS-TOF instrument.