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An integrated, directed mass spectrometric approach for in-depth characterization of complex peptide mixtures


Schmidt, Alexander; Gehlenborg, Nils; Bodenmiller, Bernd; Mueller, Lukas N; Campbell, Dave; Mueller, Markus; Aebersold, Ruedi; Domon, Bruno (2008). An integrated, directed mass spectrometric approach for in-depth characterization of complex peptide mixtures. Molecular & Cellular Proteomics, 7(11):2138-2150.

Abstract

LC-MS/MS has emerged as the method of choice for the identification and quantification of protein sample mixtures. For very complex samples such as complete proteomes, the most commonly used LC-MS/MS method, data-dependent acquisition (DDA) precursor selection, is of limited utility. The limited scan speed of current mass spectrometers along with the highly redundant selection of the most intense precursor ions generates a bias in the pool of identified proteins toward those of higher abundance. A directed LC-MS/MS approach that alleviates the limitations of DDA precursor ion selection by decoupling peak detection and sequencing of selected precursor ions is presented. In the first stage of the strategy, all detectable peptide ion signals are extracted from high resolution LC-MS feature maps or aligned sets of feature maps. The selected features or a subset thereof are subsequently sequenced in sequential, non-redundant directed LC-MS/MS experiments, and the MS/MS data are mapped back to the original LC-MS feature map in a fully automated manner. The strategy, implemented on an LTQ-FT MS platform, allowed the specific sequencing of 2,000 features per analysis and enabled the identification of more than 1,600 phosphorylation sites using a single reversed phase separation dimension without the need for time-consuming prefractionation steps. Compared with conventional DDA LC-MS/MS experiments, a substantially higher number of peptides could be identified from a sample, and this increase was more pronounced for low intensity precursor ions.

Abstract

LC-MS/MS has emerged as the method of choice for the identification and quantification of protein sample mixtures. For very complex samples such as complete proteomes, the most commonly used LC-MS/MS method, data-dependent acquisition (DDA) precursor selection, is of limited utility. The limited scan speed of current mass spectrometers along with the highly redundant selection of the most intense precursor ions generates a bias in the pool of identified proteins toward those of higher abundance. A directed LC-MS/MS approach that alleviates the limitations of DDA precursor ion selection by decoupling peak detection and sequencing of selected precursor ions is presented. In the first stage of the strategy, all detectable peptide ion signals are extracted from high resolution LC-MS feature maps or aligned sets of feature maps. The selected features or a subset thereof are subsequently sequenced in sequential, non-redundant directed LC-MS/MS experiments, and the MS/MS data are mapped back to the original LC-MS feature map in a fully automated manner. The strategy, implemented on an LTQ-FT MS platform, allowed the specific sequencing of 2,000 features per analysis and enabled the identification of more than 1,600 phosphorylation sites using a single reversed phase separation dimension without the need for time-consuming prefractionation steps. Compared with conventional DDA LC-MS/MS experiments, a substantially higher number of peptides could be identified from a sample, and this increase was more pronounced for low intensity precursor ions.

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Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Institute of Molecular Life Sciences
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2008
Deposited On:24 Apr 2013 11:20
Last Modified:21 Aug 2017 15:03
Publisher:American Society for Biochemistry and Molecular Biology
ISSN:1535-9476
Additional Information:This research was originally published in: Schmidt, Alexander; Gehlenborg, Nils; Bodenmiller, Bernd; Mueller, Lukas N; Campbell, Dave; Mueller, Markus; Aebersold, Ruedi; Domon, Bruno (2008). An integrated, directed mass spectrometric approach for in-depth characterization of complex peptide mixtures. Molecular & Cellular Proteomics, 7(11):2138-2150. © the American Society for Biochemistry and Molecular Biology.
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1074/mcp.M700498-MCP200
PubMed ID:18511481

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