title: Rapid and sensitive analyses of glycoprotein-derived oligosaccharides by liquid chromatography and laser-induced fluorometric detection capillary electrophoresis.
abstract: Asparagine-type oligosaccharides are released from core proteins as N-glycosylamines in the initial step of the action of the peptide N(4)-(N-acetyl-β-D-glucosaminyl)asparagine amidase F (PNGase F). The released N-glycosylamine-type oligosaccharides (which are exclusively present at least during the course of the enzyme reaction) could therefore be derivatized with amine-labeling reagents. Here we report a method using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a labeling reagent for glycosylamine-type oligosaccharides. We applied the method for the sensitive analysis of some oligosaccharide mixtures derived from well-characterized glycoproteins including human transferrin, α(1)-acid glycoprotein, bovine fetuin, and ribonuclease B. NBD-labeled oligosaccharides were successfully separated on an amide-bonded column or a diol-silica column. This labeling method included the release of oligosaccharides from glycoproteins and derivatization of oligosaccharides in a one-pot reaction and was completed within 3h. The method showed approximately fivefold higher sensitivity than that involving labeling with ethyl p-aminobenzoate (ABEE) in HPLC using fluorometric detection and a one order of magnitude higher response in ESI-LC/MS. We also applied this method for the sensitive analysis of glycoprotein-derived oligosaccharides by capillary electrophoresis with laser-induced fluorometric detection (LIF-CE). The limit of detection in HPLC and LIF-CE were 100fmol and 4fmol, respectively.
authors: Faculty of Pharmaceutical Sciences, Kinki University, Japan.
authors: Takehiro
authors: T
authors: Oyama
authors: Masahiro
authors: M
authors: Yodohsi
authors: Ayako
authors: A
authors: Yamane
authors: Kazuaki
authors: K
authors: Kakehi
authors: Takao
authors: T
authors: Hayakawa
authors: Shigeo
authors: S
authors: Suzuki
mesh_terms: D000818:Animals; D001216:Asparagine / Q000737:chemistry; D002236:Carbohydrate Conformation; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007834:Lasers; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D006023:Glycoproteins; D009844:Oligosaccharides; D001216:Asparagine; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase