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Evaluation of the Low-Specificity Protease Elastase for Large-Scale Phosphoproteome Analysis

Wang, Bin and Malik, Rainer and Nigg, Erich and Körner, Roman. (2008) Evaluation of the Low-Specificity Protease Elastase for Large-Scale Phosphoproteome Analysis. Analytical chemistry, Vol. 80, no. 24. pp. 9526-9533.

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Official URL: http://edoc.unibas.ch/dok/A5249328

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Abstract

Comprehensive phosphorylation site mapping is the central goal of phosphoproteome studies, but complete protein sequence coverage is rarely obtained using one single protease. In this study, we have evaluated the use of elastase, in comparison to trypsin, to increase phosphorylation site coverage of mitotic spindle proteins enriched from cultured human cells. We took advantage of the high mass accuracy of Orbitrap mass spectrometers and optimized the database search specificity by analyzing both elastase cleavage preferences and employing a dedicated two-step database search strategy. Through this approach, we have approximately doubled the number of detectable phosphorylation sites from elastase digested samples. Remarkably, phosphorylation sites detected by trypsin and elastase were highly complementary with an overlap of less than 10%. In total, we identified 1068 phosphorylation sites using trypsin and 467 phosphorylation sites using elastase. Approximately 30% of the phosphorylation sites were exclusively identified after digestion by elastase, demonstrating the value of this enzyme for phosphoproteome studies.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum
05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Cell Biology (Nigg)
UniBasel Contributors:Nigg, Erich A.
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
ISSN:0003-2700
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:22 Mar 2012 14:22
Deposited On:22 Mar 2012 13:31

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