Analysis of the Trypanosome Flagellar Proteome Using a Combined Electron Transfer/Collisionally Activated Dissociation Strategy

Sarah Hart, Sarah R. Hart, King Wai Lau, Zhiqi Hao, Richard Broadhead, Neil Portman, Andreas Hühmer, Keith Gull, Paul G. McKean, Simon J. Hubbard, Simon J. Gaskell

    Research output: Contribution to journalArticlepeer-review

    Abstract

    The use of electron-transfer dissociation as an alternative peptide ion activation method for generation of protein sequence information is examined here in comparison with the conventional method of choice, collisionally activated dissociation, using a linear ion trapping instrument. Direct comparability between collisionally and electron-transfer-activated product ion data were ensured by employing an activation-switching method during acquisition, sequentially activating precisely the same precursor ion species with each fragmentation method in turn. Sequest (Thermo Fisher Scientific, San Jose, CA) searching of product ion data generated an overlapping yet distinct pool of polypeptide identifications from the products of collisional and electron-transfer-mediated activation products. To provide a highly confident set of protein recognitions, identification data were filtered using parameters that achieved a peptide false discovery rate of 1%, with two or more independent peptide assignments required for each protein. The use of electron transfer dissociation (ETD) has allowed us to identify additional peptides where the quality of product ion data generated by collisionally activated dissociation (CAD) was insufficient to infer peptide sequence. Thus, a combined ETD/CAD approach leads to the recognition of more peptides and proteins than are achieved using peptide analysis by CAD- or ETD-based tandem mass spectrometry alone. © 2009 American Society for Mass Spectrometry.
    Original languageEnglish
    Pages (from-to)167-175
    Number of pages8
    JournalJournal of the American Society for Mass Spectrometry
    Volume20
    Issue number2
    DOIs
    Publication statusPublished - Feb 2009

    Fingerprint

    Dive into the research topics of 'Analysis of the Trypanosome Flagellar Proteome Using a Combined Electron Transfer/Collisionally Activated Dissociation Strategy'. Together they form a unique fingerprint.

    Cite this