Hybrid Proteome Samples for MS Analysis

Commercial tryptic digests of S.cerevisiae (Yeast, Promega, #V746A), human K562 cells (Promega, #V695A) and E.coli (MassPREP standard, Waters, #186003196) were reconstituted in 2% ACN/0.1% FA to final concentration of 0.1 μg/ul. To generate the hybrid proteome samples, purified peptides from each of the three species were combined in different proportions as previously described [42 (link)] and as follows: sample YHE211 consisted of 30% w/w Yeast (3 μg), 65% w/w Human (6.5 μg) and 5% w/w E.coli (0.5 μg); sample YHE114 consisted of 15% w/w Yeast (1.5 μg), 65% w/w Human (6.5 μg) and 20% w/w E.coli (2 μg); sample YHE010 consisted of 0% w/w Yeast (0 μg), 100% w/w Human (6.5 μg), and 0% w/w E.coli (0 μg). Ten replicates of each proteome mixture were subjected to LC-MS/MS analysis on a timsTOF Pro mass spectrometer.