Phosphopeptide-selective column-switching RP-HPLC with a titania precolumn.
A methodology of phosphopeptide-selective analysis coupled with column-switching HPLC utilizing titania as precolumn media is presented. Phosphopeptides were selectively enriched on titania packing within a protein/peptide mixture without any additional procedure, and analyzed by column-switching high-performance liquid chromatography. First, phospho-compounds were separated from complex mixtures by trapping them under acidic conditions on a titania packing, where non-phosphorylated compounds were effused out of the precolumn. Subsequently, phospho-compounds were desorbed from the titania column under a specific condition and analyzed. The behavior of phospho-compounds on a titania surface, especially adsorption/desorption, was precisely examined and optimized. A phosphoric buffer was successively employed for the elution of phosphopeptides on a titania surface by competition with the free phosphate group. From the successes of a selective concentration/analysis of phosphopeptides with column-switching HPLC with a titania precolumn, a novel phosphopeptide-selective RP-HPLC analysis has been shown to have an application possibility as a tool for phosphoproteomics.
Kuroda
Ikuma
I
GL Sciences Inc., 237-2 Sayamagahara, Iruma, Saitama 358-0032, Japan. i-kuroda@gls.co.jp
Shintani
Yukihiro
Y
Motokawa
Masanori
M
Abe
Shuzo
S
Furuno
Masahiro
M
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D010748:Phosphopeptides / Q000032:analysis*; D014025:Titanium / Q000737:chemistry*
D010748:Phosphopeptides; C009495:titanium dioxide; D014025:Titanium