Depletion of Acidic Phosphopeptides by SAX To Improve the Coverage for the Detection of Basophilic Kinase Substrates

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• 作者：Mingming Dong ; Mingliang Ye ; Kai Cheng ; Chunxia Song ; Yanbo Pan ; Chunli Wang ; Yangyang Bian ; Hanfa Zou
• 刊名：Journal of Proteome Research
• 出版年：2012
• 出版时间：September 7, 2012
• 年：2012
• 卷：11
• 期：9
• 页码：4673-4681
• 全文大小：496K
• 年卷期：v.11,no.9(September 7, 2012)
• ISSN：1535-3907

文摘

The Ser/Thr protein kinases fall into three major subgroups, pro-directed, basophilic, and acidophilic, on the basis of the types of substrate sequences that they preferred. Despite many phosphoproteomics efforts that have been taken for global profiling of phosphopeptides, methodologies focusing on analyzing a particular type of kinase substrates have seldom been reported. Selective enrichment of phosphopeptides from basophilic kinase substrates is difficult because basophilic motifs are cleaved by trypsin during digestion. In this study, we develop a negative enrichment strategy to enhance the identification of basophilic kinase substrates. This method is based on an observation that high pH strong anion exchange (SAX) chromatography can separate tryptic phosphopeptides according to the number of acidic amino acidic residues that they have. Thus, SAX was applied to deplete acidic phosphopeptides from the phosphopeptide mixture, which improved the coverage for the detection of basophilic kinase substrates. The SAX depletion approach was further combined with online SCX-RP separation for large-scale analysis of mouse liver phosphoproteome, which resulted in the identification of 6944 phosphorylated sites. It was found that motifs associated with basophilic kinases prevail for these identified phosphorylated sites.

Keywords:

phosphoproteome analysis; SAX; depletion of acidic phosphopeptides; basophilic kinase substrates