Chapter title |
Variable Digestion Strategies for Phosphoproteomics Analysis.
|
---|---|
Chapter number | 15 |
Book title |
Phospho-Proteomics
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3049-4_15 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3048-7, 978-1-4939-3049-4
|
Authors |
Humberto Gonczarowska-Jorge, Margherita Dell’Aica, Clarissa Dickhut, René P. Zahedi |
Editors |
Louise von Stechow |
Abstract |
In recent years, mass spectrometry-based phosphoproteomics has propelled our knowledge about the regulation of cellular pathways. Nevertheless, typically applied bottom-up strategies have several limitations. Trypsin, the preferentially used proteolytic enzyme shows impaired cleavage efficiency in the vicinity of phosphorylation sites. Moreover, depending on the frequency and distribution of tryptic cleavage sites (Arg/Lys), generated peptides can be either too short or too long for confident identification using standard LC-MS approaches. To overcome these limitations, we introduce an alternative and simple approach based on the usage of the nonspecific serine protease subtilisin, which enables a fast and reproducible digestion and provides access to "hidden" areas of the proteome. Thus, in a single LC-MS experiment >1800 phosphopeptides were confidently identified and localized from 125 μg of HeLa digest, compared to >2100 sites after tryptic digestion. While the overlap was less than 20 %, subtilisin allowed the identification of many phosphorylation sites that are theoretically not accessible via tryptic digestion, thus considerably increasing the coverage of the phosphoproteome. |
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