Analytical Sciences

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

Koshi IMAMI,*¹ Naoyuki SUGIYAMA,*^1,*² Yutaka KYONO,*^1,*³ Masaru TOMITA,*^1,*² and Yasushi ISHIHAMA*^1,*⁴

*1 Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan
*2 Human Metabolome Technologies Inc., Kakuganji, Tsuruoka 997-0052, Japan
*3 GL Sciences Inc., 237-2 Sayamagahara, Iruma, Saitama 358-0032, Japan
*4 PRESTO, Japan Science and Technology Agency, Sanbancho Bldg., 5-Sanbancho, Chiyoda, Tokyo 102-0075, Japan

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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