Phosphopeptide shotgun analysis using mass spectrometry and Phos-tag agarose beads

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  • Kimura Yayoi
    Graduate School of Nanobioscience, Yokohama City University
  • Nomura Ayako
    Advanced Medical Research Center, Yokohama City University
  • Ino Yoko
    Graduate School of Nanobioscience, Yokohama City University
  • Ohara Osamu
    Laboratory for Immunogenomics, RIKEN Research Center for Allergy and Immunology Kazusa DNA Research Institute
  • Hirano Hisashi
    Graduate School of Nanobioscience, Yokohama City University Advanced Medical Research Center, Yokohama City University

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  • Ｐｈｏｓ－ｔａｇアガロースおよび質量分析装置を用いたリン酸化ペプチドのショットガン分析
  • Phos-tag アガロース オヨビ シツリョウ ブンセキ ソウチ オ モチイタ リン サンカ ペプチド ノ ショットガン ブンセキ

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Abstract

Enrichment of phosphopeptides is an important process for mass spectrometric analysis. Here, we used the enrichment method using the biphasic Phos-tag/C18 tip, which consists of Phos-tag agarose beads overlaid on the C18 disk filter in a micropipet tip. In the cell lysates of GIST882 cells, 3187 phosphopeptides (1688 unique phosphopeptides) were identified by nano LC-MS/MS analysis coupled with phosphopeptide enrichment using this method. This method was comparable to another phosphopeptide enrichment method using Titansphere Phos-TiO Kit in numbers of peptides detected. A total number of unique phosphopeptides detected by the two methods was 3202, and the overlap between phosphopeptides enriched by two methods was 36%. Therefore, it is concluded that Phos-tag agarose method is an alternative method for identification of phosphoproteins.<br>

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