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Keywords:

  • Multi-dimensional fractionation;
  • Plasma biomarkers;
  • Precursor ion exclusion

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.