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.
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.
Sano
Akira
A
Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
Nakamura
Hiroshi
H
D000469:Alkaline Phosphatase / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000737:chemistry; D010450:Endopeptidases / Q000737:chemistry; D006868:Hydrolysis; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D014025:Titanium / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry
D010748:Phosphopeptides; D011506:Proteins; D014025:Titanium; D000469:Alkaline Phosphatase; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin