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Proteomics Clin Appl. 2012 Jun;6(5-6):304-8. doi: 10.1002/prca.201100107.

Precursor ion exclusion for enhanced identification of plasma biomarkers.

Author information

  • 1Metabolism Unit, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.

Abstract

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.

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PMID:
22641611
[PubMed - indexed for MEDLINE]
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