Improving protein and proteome coverage through data-independent multiplexed peptide fragmentation

Blackburn, K. and Mbeunkui, F. and Mitra, S. K. and Mentzel, T. and Goshe, M. B.. (2010) Improving protein and proteome coverage through data-independent multiplexed peptide fragmentation. Journal of Proteome Research, Vol. 9, H. 7. pp. 3621-3637.

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Official URL: http://edoc.unibas.ch/dok/A5842384

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Performance differences in protein and proteome characterization achieved by data-independent acquisition (DIA) LC/MSE and data-dependent acquisition (DDA) LC/MS/MS approaches were investigated. LC/MSE is a novel mode of generating product ion data for all coeluting precursors in parallel as opposed to LC/MS/MS where coeluting precursors must be serially fragmented one at a time. During LC/MSE analysis, alternating MS scans of "normal" and "elevated" collision energy are collected at regular intervals, providing nearly a 100% duty cycle for precursor detection and fragmentation because all precursors are fragmented across their full chromatographic elution profile. This is in contrast to DDA-based MS/MS where serial selection of precursor ions is biased toward interrogation and detection of the highest abundance sample components by virtue of the intensity-driven interrogation scheme employed. Both modes of acquisition were applied to a simple four-protein standard mixture with a 16-fold dynamic range in concentration, an in-gel digest of the Arabidopsis thaliana protein FLS2 purified by immunoprecipitation, and a solution-digested tomato leaf proteome sample. Dramatic improvement for individual protein sequence coverage was obtained for all three samples analyzed by the DIA approach, particularly for the lowest abundance sample components. In many instances, precursors readily detected and identified during DIA were either interrogated by MS/MS during DDA at inopportune points in their chromatographic elution profiles resulting in poor quality product ion spectra or not interrogated at all. Detailed evaluation of both DDA and DIA raw data and timing of the MS-to-MS/MS switching events clearly revealed the fundamental limitations of serial MS/MS interrogation and the advantages of parallel fragmentation by DIA for more comprehensive protein identification and characterization which holds promise for enhanced isoform and post-translational modification analysi
Faculties and Departments:05 Faculty of Science > Departement Umweltwissenschaften > Ehemalige Einheiten Umweltwissenschaften > Pflanzenphysiologie Pathogenabwehr (Boller)
UniBasel Contributors:Mentzel, Tobias
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:American Chemical Society
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:11 Oct 2012 15:31
Deposited On:11 Oct 2012 15:17

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