Automated Phosphoproteome Analysis for Cultured Cancer Cells by Two-Dimensional NanoLC-MS Using a Calcined Titania/C18 Biphasic Column

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  • IMAMI Koshi
    Institute for Advanced Biosciences, Keio University
  • SUGIYAMA Naoyuki
    Institute for Advanced Biosciences, Keio University Human Metabolome Technologies Inc.
  • KYONO Yutaka
    Institute for Advanced Biosciences, Keio University GL Sciences Inc.
  • TOMITA Masaru
    Institute for Advanced Biosciences, Keio University Human Metabolome Technologies Inc.
  • ISHIHAMA Yasushi
    Institute for Advanced Biosciences, Keio University PRESTO, Japan Science and Technology Agency

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We have developed an on-line automated system for phosphoproteome analysis using titania-based phosphopeptide enrichment followed by nanoLC-MS/MS. Titania beads were prepared by calcination of commercial chromatographic titania beads at 800°C to convert the crystalline structure. The obtained rutile-form titania exhibited higher selectivity in phosphopeptide enrichment than commercial titania, even in the absence of a competitive chelating reagent for non-phosphopeptides. For phosphoproteome analysis of human cervical cancer HeLa cells, tryptic digests of the cell extracts were directly injected into this on-line system, and 696 non-redundant phosphopeptides with 671 unambiguously determined phosphorylation sites, derived from 512 phosphoproteins, were successfully identified. This is the first successful application of an on-line automated phosphoproteome analysis system to complex biological samples.

収録刊行物

  • Analytical Sciences

    Analytical Sciences 24 (1), 161-166, 2008

    社団法人 日本分析化学会

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