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J Proteome Res DOI:10.1021/pr801017a

Directed sample interrogation utilizing an accurate mass exclusion-based data-dependent acquisition strategy (AMEx).

Publication TypeJournal Article
Year of Publication2009
AuthorsRudomin, EL, Carr, SA, Jaffe, JD
JournalJ Proteome Res
Date Published2009 Jun
KeywordsAlgorithms, Chromatography, Liquid, HeLa Cells, Humans, Mass Spectrometry, Peptide Fragments, Proteins, Proteomics, Trypsin

The ability to perform thorough sampling is of critical importance when using mass spectrometry to characterize complex proteomic mixtures. A common approach is to reinterrogate a sample multiple times by LC-MS/MS. However, the conventional data-dependent acquisition methods that are typically used in proteomics studies will often redundantly sample high-intensity precursor ions while failing to sample low-intensity precursors entirely. We describe a method wherein the masses of successfully identified peptides are used to generate an accurate mass exclusion list such that those precursors are not selected for sequencing during subsequent analyses. We performed multiple concatenated analytical runs to sample a complex cell lysate, using either accurate mass exclusion-based data-dependent acquisition (AMEx) or standard data-dependent acquisition, and found that utilization of AMEx on an ESI-Orbitrap instrument significantly increases the total number of validated peptide identifications relative to a standard DDA approach. The additional identified peptides represent precursor ions that exhibit low signal intensity in the sample. Increasing the total number of peptide identifications augmented the number of proteins identified, as well as improved the sequence coverage of those proteins. Together, these data indicate that using AMEx is an effective strategy to improve the characterization of complex proteomic mixtures.


Alternate JournalJ. Proteome Res.
PubMed ID19344186
PubMed Central IDPMC2753172
Grant List1U24 CA126476 / CA / NCI NIH HHS / United States
U01 HL081341-01 / HL / NHLBI NIH HHS / United States
U01-HL081341 / HL / NHLBI NIH HHS / United States
U01 HL081341 / HL / NHLBI NIH HHS / United States
U24 CA126476 / CA / NCI NIH HHS / United States
U24 CA126476-01 / CA / NCI NIH HHS / United States