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

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Abstract

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.

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Authors and Affiliations

  1. Institute for Advanced Biosciences, Keio University, Tsuruoka, 997-0017, Japan

    Koshi Imami, Naoyuki Sugiyama, Yutaka Kyono, Masaru Tomita & Yasushi Ishihama

  2. Human Metabolome Technologies Inc., Kakuganji, Tsuruoka, 997-0052, Japan

    Naoyuki Sugiyama & Masaru Tomita

  3. GL Sciences Inc., 237-2 Sayamagahara, Iruma, Saitama, 358-0032, Japan

    Yutaka Kyono

  4. PRESTO, Japan Science and Technology Agency, Sanbancho Bldg., 5-Sanbancho, Chiyoda, Tokyo, 102-0075, Japan

    Yasushi Ishihama

Authors
  1. Koshi Imami
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  2. Naoyuki Sugiyama
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  3. Yutaka Kyono
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  4. Masaru Tomita
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  5. Yasushi Ishihama
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Correspondence to Yasushi Ishihama.

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Imami, K., Sugiyama, N., Kyono, Y. et al. Automated Phosphoproteome Analysis for Cultured Cancer Cells by Two-Dimensional NanoLC-MS Using a Calcined Titania/C18 Biphasic Column. ANAL. SCI. 24, 161–166 (2008). https://doi.org/10.2116/analsci.24.161

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  • DOI: https://doi.org/10.2116/analsci.24.161

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