Abstract
Here we used a data-independent acquisition (DIA) method, Precursor Acquisition Independent from Ion Count (PAcIFIC), to systematically profile the S. cerevisiae proteome. Direct PAcIFIC analysis of a yeast whole cell lysate (WCL) yielded 90% reproducibility between replicates and detected approximately 2000 proteins. When combined with sub-cellular fractionation, reproducibility was equally high and the number of detected yeast proteins approached 5000. As noted previously, this unbiased DIA approach identified so-called "orphan" peptides that could only be detected by tandem mass spectra because there was no detectable precursor ion. Using this unique dataset we examined features associated with peptide detectability and demonstrated that orphans were more likely to arise from low copy number proteins than proteins with median or high copy number. Finally, an investigation into why some orphans also arose from high copy number proteins found that, aside from protein copy number, there was a bias toward physiochemical factors associated with regions flanking the proteolytic cleavage sites of orphan peptides. This suggested that those orphan peptides originating from high abundance proteins were likely the result of inefficient protease release, which has implications for quantitative bottom-up proteomics.