Investigation of different combinations of derivatization, separation methods and electrospray ionization mass spectrometry for standard oligosaccharides and glycans from ovalbumin.
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.
Saba
J A
JA
Department of Chemistry, University of Manitoba, 144 Dysart Road, Winnipeg, Manitoba R3T 2N2, Canada.
Shen
X
X
Jamieson
J C
JC
Perreault
H
H
D000581:Amidohydrolases; D000818:Animals; D000983:Antipyrine / Q000031:analogs & derivatives; D002240:Carbohydrate Sequence; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D000077553:Edaravone; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D009281:Naphthalenes; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D010047:Ovalbumin / Q000737:chemistry*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization
D005456:Fluorescent Dyes; D009281:Naphthalenes; D009844:Oligosaccharides; D011134:Polysaccharides; D010047:Ovalbumin; C046465:8-amino-1,3,6-naphthalenetrisulfonic acid; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D000077553:Edaravone; D000983:Antipyrine