Automated phosphoproteome analysis for cultured cancer cells by two-dimensional nanoLC-MS using a calcined titania/C18 biphasic column.
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 degrees 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.
Imami
Koshi
K
Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan.
Sugiyama
Naoyuki
N
Kyono
Yutaka
Y
Tomita
Masaru
M
Ishihama
Yasushi
Y
D002851:Chromatography, High Pressure Liquid; D030541:Databases, Genetic; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008863:Microspheres; D009862:Online Systems; D010750:Phosphoproteins / Q000235:genetics*; D010766:Phosphorylation; D020543:Proteome / Q000235:genetics*; D014025:Titanium / Q000737:chemistry*; D014357:Trypsin
D010750:Phosphoproteins; D020543:Proteome; D014025:Titanium; D014357:Trypsin