title: Structural Characterization of Serum N-Glycans by Methylamidation, Fluorescent Labeling, and Analysis by Microchip Electrophoresis.
abstract: To characterize the structures of N-glycans derived from human serum, we report a strategy that combines microchip electrophoresis, standard addition, enzymatic digestion, and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). We compared (i) electrophoretic mobilities of known N-glycans from well-characterized (standard) glycoproteins through standard addition, (ii) the electrophoretic mobilities of N-glycans with their molecular weights determined by MALDI-MS, and (iii) electrophoretic profiles of N-glycans enzymatically treated with fucosidase. The key step to identify the sialylated N-glycans was to quantitatively neutralize the negative charge on both α2,3- and α2,6-linked sialic acids by covalent derivatization with methylamine. Both neutralized and nonsialylated N-glycans from these samples were then reacted with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) to provide a fluorescent label and a triple-negative charge, separated by microchip electrophoresis, and detected by laser-induced fluorescence. The methylamidation step leads to a 24% increase in the peak capacity of the separation and direct correlation of electrophoretic and MALDI-MS results. In total, 37 unique N-glycan structures were assigned to 52 different peaks recorded in the electropherograms of the serum samples. This strategy ensures the needed separation efficiency and detectability, easily resolves linkage and positional glycan isomers, and is highly reproducible.
authors: Department of Chemistry, Indiana University , Bloomington, Indiana 47405, United States.
authors: Indranil
authors: I
authors: Mitra
authors: Department of Chemistry, Indiana University , Bloomington, Indiana 47405, United States.
authors: Christa M
authors: CM
authors: Snyder
authors: Department of Chemistry, Indiana University , Bloomington, Indiana 47405, United States.
authors: Xiaomei
authors: X
authors: Zhou
authors: Department of Chemistry, Indiana University , Bloomington, Indiana 47405, United States.
authors: Margit I
authors: MI
authors: Campos
authors: Department of Chemistry, Indiana University , Bloomington, Indiana 47405, United States.
authors: William R
authors: WR
authors: Alley
authors: Department of Chemistry, Indiana University , Bloomington, Indiana 47405, United States.
authors: Milos V
authors: MV
authors: Novotny
authors: Department of Chemistry, Indiana University , Bloomington, Indiana 47405, United States.
authors: Stephen C
authors: SC
authors: Jacobson
mesh_terms: D046209:Electrophoresis, Microchip / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008744:Methylamines / Q000737:chemistry; D011134:Polysaccharides / Q000097:blood* / Q000737:chemistry*; D011721:Pyrenes / Q000737:chemistry; D012794:Sialic Acids / Q000032:analysis / Q000097:blood; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: C098210:1-aminopyrene-3,6,8-trisulfonic acid; D005456:Fluorescent Dyes; D008744:Methylamines; D011134:Polysaccharides; D011721:Pyrenes; D012794:Sialic Acids; C027451:methylamine