期刊
PROTEOMICS
卷 16, 期 13, 页码 1858-1862出版社
WILEY-BLACKWELL
DOI: 10.1002/pmic.201500520
关键词
Extracellular phosphorylation; Glycosylation; Human serum; IMAC; MS; Technology
资金
- Hungarian Scientific Research Fund [105611]
- Janos Bolyai Fellowship of the Hungarian Academy of Sciences
Phosphorylation is the most widely studied posttranslational modification. Its role within the cell has been the focus of numerous large-scale studies. Recently there is growing evidence on the biological significance of extracellular phosphorylation. The analysis of these phosphopeptides is complicated by the abundance of glycosylation in the extracellular space, since glycopeptides are also enriched by the methods used for phosphopeptide isolation. Thus, we optimized IMAC for phosphorylation analysis of secreted proteins, specifically in human serum. Selectivity and efficiency of different enrichment conditions used in earlier large-scale phosphoproteomic studies were evaluated. We found that minimizing hydrophilic interactions in the enrichment allowed selective phosphopeptide isolation. Using a two-step IMAC enrichment protocol under these conditions led to the identification of similar to 100 phosphorylation sites from the tryptic digest of as little as 40 mu L human serum.
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