Precursor ion exclusion for enhanced identification of plasma biomarkers.

PURPOSE: Our study aims to establish a plasma biomarker analysis workflow, with fewer fractionation steps, for enhanced identification of plasma biomarkers by precursor ion exclusion (PIE). EXPERIMENTAL
DESIGN: Plasma samples were depleted for highly abundant proteins, then further fractionated by molecular weight (MW), before trypsinization for LTQ-Orbitrap mass analysis. Data-dependent acquisition (DDA) was used for baseline analysis. PIE involves the re-injection of samples with exclusion of the previously identified peptides. We compared analyses using multiple PIE iterations, compared to DDA, for plasma interrogation
RESULTS: A higher percentage of unique plasma peptides was identified with PIE, compared to DDA. The first PIE iteration reveals an increase of 75-112% more peptides than the DDA method alone. PIE can interrogate complex plasma samples with the percentage of peptides identified successively increasing with even ≥4 iterations. The total number of peptides identified increases rapidly across the first three PIE iterations and then continues more slowly up to nine iterations. CONCLUSIONS AND CLINICAL RELEVANCE: Iterative injections with PIE resulted in many more peptide identifications in plasma samples of varying degrees of complexity, compared to re-injections using similar DDA parameters. PIE methods may therefore expand our ability to recover plasma peptides for plasma biomarker discovery.