Chemical dephosphorylation for identification of multiply phosphorylated peptides and phosphorylation site determination

Yutaka Kyono, Naoyuki Sugiyama, Masaru Tomita, Yasushi Ishihama

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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 200mg 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.

Original languageEnglish
Pages (from-to)2277-2282
Number of pages6
JournalRapid Communications in Mass Spectrometry
Volume24
Issue number15
DOIs
Publication statusPublished - 2010 Aug 15

Fingerprint

Phosphopeptides
Phosphorylation
Peptides
Hydroxy Acids
Liquid chromatography
Chromatography
Liquid Chromatography
Oxides
Mass spectrometry
Mass Spectrometry
Metals
Phosphoproteins
Alkalies
Arabidopsis
Cultured Cells

ASJC Scopus subject areas

  • Spectroscopy
  • Analytical Chemistry
  • Organic Chemistry
  • Medicine(all)

Cite this

Chemical dephosphorylation for identification of multiply phosphorylated peptides and phosphorylation site determination. / Kyono, Yutaka; Sugiyama, Naoyuki; Tomita, Masaru; Ishihama, Yasushi.

In: Rapid Communications in Mass Spectrometry, Vol. 24, No. 15, 15.08.2010, p. 2277-2282.

Research output: Contribution to journalArticle

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