Selective glycopeptide mapping of erythropoietin by on-line high-performance liquid chromatography-electrospray ionization mass spectrometry

Selective glycopeptide mapping of recombinant human erythropoietin (rhEPO) used as a model glycoprotein was successfully carried out by on-line high-p erformance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) using a Vydac C-18 column eluted in acetonitrile-1 mM ammonium acetate, pH 6.8. rhEPO expressed in a Chinese hamster ovary clone was exhau stively digested into four glycopeptides and nine peptides with endoprotein ase Glu-C. Both glycopeptides and peptides were eluted with trifluoroacetic acid as the eluent, whereas only glycopeptides were eluted selectively wit h ammonium acetate in the following order: N38, N24, O126, and N83. Further more, many glycoforms included in each glycopeptide were found to be separa ted by differences in the numbers of sialic acid and N-acetyllactosaminyl r epeats. Twenty, 16 and 22 different N-linked oligosaccharides were determin ed at Asn24, 38, and 83, respectively, and two different O-linked oligosacc harides were observed at Ser126. Our method is simple, rapid, and useful fo r determining the carbohydrate structures at each glycosylation site and fo r elucidating the site-specific carbohydrate heterogeneity. (C) 2001 Elsevi er Science B.V. All rights reserved.