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.
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.
Kurihara
Takamasa
T
Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
Min
Jun Zhe
JZ
Hirata
Asuka
A
Toyo'oka
Toshimasa
T
Inagaki
Shinsuke
S
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000191:economics / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D009844:Oligosaccharides / Q000032:analysis* / Q000378:metabolism; D010047:Ovalbumin / Q000737:chemistry* / Q000378:metabolism; D011402:Pronase / Q000378:metabolism; D011721:Pyrenes / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization / Q000191:economics / Q000379:methods*; D013997:Time Factors; D000509:alpha-Fetoproteins / Q000737:chemistry* / Q000378:metabolism
D005456:Fluorescent Dyes; D009844:Oligosaccharides; D011721:Pyrenes; D000509:alpha-Fetoproteins; C104379:pyrene sulfonyl chloride; D010047:Ovalbumin; D012260:Ribonucleases; C029600:ribonuclease B; D011402:Pronase