Analytical Sciences
Online ISSN : 1348-2246
Print ISSN : 0910-6340
ISSN-L : 0910-6340
Original Papers
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
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2004 Volume 20 Issue 5 Pages 861-864


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.

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© 2004 by The Japan Society for Analytical Chemistry
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