Titania as a chemo-affinity support for the column-switching HPLC analysis of phosphopeptides: Application to the characterization of phosphorylation sites in proteins by combination with protease digestion and electrospray ionization mass Spectrometry

被引：83

作者：

Sano, A ^{[1
]}

Nakamura, H ^{[1
]}

机构：

[1] Sci Univ Tokyo, Fac Pharmaceut Sci, Noda, Chiba 2788510, Japan

来源：

ANALYTICAL SCIENCES | 2004年 / 20卷 / 05期

关键词：

D O I：

10.2116/analsci.20.861

中图分类号：

O65 [分析化学];

学科分类号：

070302 ; 081704 ;

摘要：

A method for the determination of phosphorylation sites in phosphoproteins based on column-switching high-performance liquid chromatography (HPLC) has been developed. The HPLC system consisted of a titania precolumn for the selective adsorption of phosphopeptides, an anion-exchange analytical column and a UV detector (215 nm). Rabbit muscle phosphorylase a (RPa) and porcine stomach pepsin (PSP) were tested as model phosphoproteins. After protease digestion, the resulting phosphopeptides were successfully isolated by column-switching HPLC. The phosphopeptide fractions were analyzed by electrospray ionization mass spectrometry with a positive or negative ion mode after purification by reversed-phase HPLC. Pseudo-molecular ion peaks corresponding to Gln-Ile-Ser(p)-Val-Arg (MW 681.7) and Glu-Ala-Thr-Ser(p)-Gln-Glu-Leu (MW 856.8) were detected from the tryptic digest of RPa and chymotryptic digest of PSP, respectively, which agreed with the theoretically expected phosphopeptide fragments.

引用

页码：861 / 864

页数：4

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