title: A strategy for the mapping of N-glycans by high-performance capillary electrophoresis.
abstract: We have evaluated high-performance capillary electrophoresis (HPCE) with respect to its suitability for use in establishing a carbohydrate-mapping database that would enable a carbohydrate structural analysis by mere comparison of migration times. The suitability of HPCE for carbohydrate structural assignments was ascertained by validation experiments. The migration times of distinct N-glycans, prepared and measured on different days, were shown to be highly reproducible, with a coefficient of variation of usually less than 0.20%, requiring only femtomoles of N-glycan per injection for reliable measurements. By including mesityl oxide and sialic acid as internal standards and a triple-correction method, HPCE fulfills the analytical requirements with respect to accuracy, precision, reproducibility, and sensitivity. The N-glycan-mapping database was established using a newly developed and optimized buffer system containing 1,5-diaminopentane as an organic modifier. Approximately 80 different sialylated N-glycans of known structure, which have thus far been measured and characterized, have been entered into our Lotus 1-2-3 mapping database. The database for structural determinations was tested using the N-linked carbohydrates released from recombinant human urinary erythropoietin (baby hamster kidney) by PNGase F treatment and from bovine serum fetuin and alpha 1-acid glycoprotein by automated and manual (large-scale) hydrazinolysis, respectively. The efficiency of the database and of the triple-correction method was further confirmed by HPCE measurements performed in a different laboratory and by a different analyst who used the HPCE system of a different manufacturer.
authors.affiliation: Research Laboratories of Behringwerke AG, Marburg, Federal Republic of Germany.
authors.forename: P
authors.initials: P
authors.lastname: Hermentin
authors.forename: R
authors.initials: R
authors.lastname: Doenges
authors.forename: R
authors.initials: R
authors.lastname: Witzel
authors.forename: C H
authors.initials: CH
authors.lastname: Hokke
authors.forename: J F
authors.initials: JF
authors.lastname: Vliegenthart
authors.forename: J P
authors.initials: JP
authors.lastname: Kamerling
authors.forename: H S
authors.initials: HS
authors.lastname: Conradt
authors.forename: M
authors.initials: M
authors.lastname: Nimtz
authors.forename: D
authors.initials: D
authors.lastname: Brazel
mesh_terms: D004586:Electrophoresis / Q000379:methods*; D004921:Erythropoietin / Q000737:chemistry; D007256:Information Systems; D009961:Orosomucoid / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D000509:alpha-Fetoproteins / Q000737:chemistry
chemical_list: D009961:Orosomucoid; D011134:Polysaccharides; D000509:alpha-Fetoproteins; D004921:Erythropoietin