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Chemical dephosphorylation for identification of multiply phosphorylated peptides and phosphorylation site determination
Author(s) -
Kyono Yutaka,
Sugiyama Naoyuki,
Tomita Masaru,
Ishihama Yasushi
Publication year - 2010
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.4627
Subject(s) - chemistry , dephosphorylation , phosphorylation , protein phosphorylation , identification (biology) , biochemistry , chromatography , phosphatase , protein kinase a , botany , biology
We have developed a novel strategy to improve the efficiency of identification of multiply phosphorylated peptides isolated by hydroxy acid modified metal oxide chromatography (HAMMOC). This strategy consists of alkali‐induced chemical dephosphorylation (beta‐elimination reaction) of phosphopeptides isolated by HAMMOC prior to analysis by liquid chromatography/mass spectrometry (LC/MS). This approach identified 1.9‐fold more multiply phosphorylated peptides than the conventional approach without beta‐elimination from a digested mixture of three standard phosphoproteins. In addition, the accuracy of phosphorylation site determination in synthetic phosphopeptides was significantly improved. Finally, we applied this approach to a cell lysate. By combining this dephosphorylation approach with the conventional approach, we successfully identified 1649 unique phosphopeptides, including 325 multiply phosphorylated phosphopeptides, from 200 µg of cultured Arabidopsis cells. These results indicate that chemical dephosphorylation prior to LC/MS analysis increases the efficiency of identification of multiply phosphorylated peptides, as well as the accuracy of phosphorylation site determination. Copyright © 2010 John Wiley & Sons, Ltd.