Investigation of different combinations of derivatization, separation methods and electrospray ionization mass spectrometry for standard oligosaccharides and glycans from ovalbumin

被引:60
作者
Saba, JA [1 ]
Shen, XD [1 ]
Jamieson, JC [1 ]
Perreault, H [1 ]
机构
[1] Univ Manitoba, Dept Chem, Winnipeg, MB R3T 2N2, Canada
来源
JOURNAL OF MASS SPECTROMETRY | 2001年 / 36卷 / 05期
关键词
glycoprotein; ovalbumin; oligosaccharide; derivatization; electrospray; high-performance liquid chromatography/electrospray ionization mass spectrometry;
D O I
10.1002/jms.158
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Derivatization procedures using 1-phenyl-3-methyl-5-pyrazolone (PMP) and 2-aminonaphthalene trisulfone (ANTS) were selected among a number of well known methods for labelling carbohydrates. PMP derivatives were selected owing to our laboratory's previous high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) experience with these, whereas the ANTS-labelled compounds were prepared for fluorophore-assisted carbohydrate electrophoresis (FACE) separation. ANTS-oligosaccharide standards were characterized to study their ionization patterns. Reversed-phase and normal-phase HPLC systems were coupled on-line with ESI-MS. Each necessitated its own mobile phase system which, in turn, imposed some important changes in the ionization conditions used and/or on the ionization patterns and spectra obtained. Following characterization of the intact glycoprotein ovalbumin with ESI-MS, its glycans were detached using the enzyme PNGase-F. The glycans were subjected to PMP and ANTS derivatization. It was very difficult to separate ANTS derivatives by reversed-phase HPLC owing to lack of retention, and normal-phase HPLC offered reasonable retention with limited separation. PMP compounds overall yielded better normal- and reversed-phase separations and improved sensitivity over the ANTS-labelled sugars, for which negative mode ESI had to be used. The combination of ESI of intact ovalbumin and ESI of PMP-glycans gave rise to the detection of over 20 different glycoforms, excluding the possible presence of structural isomers for each sugar composition detected. Copyright (C) 2001 John Wiley & Sons, Ltd.
引用
收藏
页码:563 / 574
页数:12
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