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Rapid analysis of N ‐linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) by reversed‐phase ultra‐performance liquid chromatography with fluorescence detection and electrospray ionization time‐of‐flight mass spectrometry
Author(s) -
Kurihara Takamasa,
Min Jun Zhe,
Hirata Asuka,
Toyo'oka Toshimasa,
Inagaki Shinsuke
Publication year - 2009
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1147
Subject(s) - chemistry , fetuin , chromatography , oligosaccharide , ovalbumin , electrospray ionization , mass spectrometry , high performance liquid chromatography , glycoprotein , ribonuclease , monosaccharide , biochemistry , antigen , biology , genetics , rna , gene
Rapid, selective and sensitive determination of N ‐linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) was performed by ultra‐performance liquid chromatography (UPLC) with fluorescence (FL) and electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOF‐MS). The asparaginyl‐oligosaccharide moiety was first liberated from each glycoprotein by pronase E (a proteolitic enzyme). The oligosaccharide fractions separated by gel‐permeation chromatography were labeled with 1‐pyrenesulfonyl chloride (PSC, a fluorescence reagent), separated by UPLC in a short run time, and then detected by FL and TOF‐MS. The PSC‐labeled oligosaccharides were selectively identified from the FL detection and then sensitively determined by ESI‐TOF‐MS. As the results, 15, eight and four kinds of N ‐linked oligosaccharides were detected from ovalbumin, ribonuclease B and fetuin, respectively. Because the present method is rapid (within 9 min), selective and sensitive (approximate 60 fmol, S/N = 5), the determination of N ‐linked oligosaccharides in various glycoproteins seems to be possible. Copyright © 2008 John Wiley & Sons, Ltd.