Genome-specific gas-phase fractionation strategy for improved shotgun proteomic profiling of proteotypic peptides

Gas-phase fractionation (GPF) is an efficient and straightforward method to increase proteome coverage. In this report, optimal m/z ranges were calculated based on genomic complexity and experimental data. Then, theoretical precursor ion densities were calculated in silico from various organisms' genomes and found to corroborate the empirical selection of m/z ranges based on ion density mapping. According to both calculations, the choice of m/z range for most efficient GPF coverage in the lower m/z range should be very narrow and increase as m/z value increases. Next, a systematic LC-MS/MS analysis was performed to confirm this observation. The behavior of data-dependent precursor ion selection and the origin of the observed variability was investigated under three different scan modes of an LTQ-Orbitrap hybrid mass spectrometer. Finally, GPF combined with data-dependent analysis was compared to a targeted, pseudo-multiple reaction monitoring analysis of proteotypic peptides that should be, based on empirical observation of LC-ESI-MS/MS data, detectable. The result of the latter experiment supported our conclusion that data-dependent analysis using rational gas-phase fractionation was sufficient for comprehensive proteomic analysis of the proteotypic peptides in an unfractionated cell lysate.