In high-throughput proteomics, accuracy, coverage and analysis time are crucial requirements. Sample introduction via automated nanoelectrospray infusion has definite advantages when compared to liquid chromatographic (LC)-electrospray ionization ESI sample delivery, including high-throughput operation, automation, no carry-over or cross-contamination, and a constant sample matrix. We have evaluated and optimized the use of a chip-based nanoelectrospray device (Advion Nanomate) interfaced to a hybrid quadrupole time of flight mass spectrometer (Applied Biosystems / MDS Sciex QStarXL) for the analysis of tryptically digested samples: cytochrome C and a six-protein mixture (containing cytochrome C, lysozyme, alcohol dehydrogenase, bovine serum albumin, apo-transferrin, and b-galactosidase) at concentrations from 5 to 500 fmol/µl. We quantified the effects on the identification and peptide coverage of MS and MS/MS scan times, Zip-Tip C18 sample clean-up, in-gel digestion residues and sample concentrations. Different survey (1-10 seconds) and MS/MS (1-10 seconds) scan times were evaluated, and spectra were searched with Mascot against generic and species specific databases. Each infusion experiment consisted of 22 cycles in the mass spectrometer, and each cycle consisted of 1 survey and 8 MS/MS scans. Infusion results were compared to those from nanoflow LC-ESI-MS/MS, which was run isocratically to maintain a quasi-uniform sample introduction. The results from the isocratic LC-ESI-MS/MS suggest that the chip-based infusion yields greater coverage, with marginally lower MOWSE scores. The optimal MS method, selected to take advantage of the extended infusion without losing the high-throughput requirement, includes a 10 second survey scan and 8 cycles of 3 second MS/MS scans. Each isocratic LC experiment, from injection to injection, took approximately twice as long as each chip-based experiment. We discuss the feasibility of extending this methodology to more complex proteomes, such as those obtained from the model cyanobacterium, Synechocystis sp. PCC6803, as we have extensive experience with this organism [1]. Furthermore, we discuss the role of this configuration in aiding identification of post-translational modifications.
[1] Gan CS, Reardon KF, Wright PC, Comparison of protein and peptide prefractionation methods for the shotgun proteomic analysis of Synechocystis sp. PCC 6803, Proteomics, 5(9), 2005, 2468-2478.