Gas-phase separations of complex tryptic peptide mixtures

High-resolution ion mobility and time-of-flight mass spectrometry technique s have been used to analyze complex mixtures of peptides generated from try ptic digestion of fourteen common proteins (albumin, bovine, dog, horse, pi g, and sheep; aldolase, rabbit: beta -casein, bovine; cytochrome L, horse: beta -lactoglobulin. bovine; myoglobin, horse: hemoglobin. human, pig, rabb it, and sheep). In this approach, ions are separated based on differences i n mobilities in helium in a drift tube and on differences in their mass-to- charge ratios in a mass spectrometer. From data recorded for fourteen indiv idual proteins: lover a m/z range of 405 to 1000), we observe 428 peaks, of which 205 are assigned to fragments that are expected from tryptic digesti on. In a separate analysis, the fourteen mixtures have been combined and an alyzed as one system. In the single dataset. we resolve 260 features and ar e able to assign 168 peaks to unique peptide sequences. Many other unresolv ed features are observed. Methods for assigning peptides based on the use o f m/z information and existing mobilities or mobilities that are predicted by use of intrinsic size parameters are described.