Data Analysis Strategy for Maximizing High-confidence Protein Identifications in Complex Proteomes Such As Human Tumor Secretomes and Human Serum

Detection of biologically interesting, low-abundance proteins in complex proteomes such as serum typically requires extensive fractionation and high-performance mass spectrometers. Processing of the resulting large data sets involves trade-offs between confidence of identification and depth of protein coverage; that is, higher stringency filters preferentially reduce the number of low-abundance proteins identified. In the current study, an alternative database search and results filtering strategies were evaluated using test samples ranging from purified proteins to ovarian tumor secretomes and human serum to maximize peptide and protein coverage. Full and partial tryptic searches were compared because substantial numbers of partial tryptic peptides were observed in all samples, and the proportion of partial tryptic peptides was particularly high for serum. When data filters that yielded similar false discovery rates (FDR) were used, full tryptic searches detected far fewer peptides than partial tryptic searches. In contrast to the common practice of using full tryptic specificity and a narrow precursor mass tolerance, more proteins and peptides could be confidently identified using a partial tryptic database search with a 100 ppm precursor mass tolerance followed by filtering of results using 10 ppm mass error and full tryptic boundaries.