A STRATEGY FOR THE MAPPING OF N-GLYCANS BY HIGH-PERFORMANCE CAPILLARYELECTROPHORESIS

We have evaluated high-performance capillary electrophoresis (HPCE) wi th respect to its suitability for use in establishing a carbohydrate-m apping database that would enable a carbohydrate structural analysis b y mere comparison of migration times. The suitability of HPCE for carb ohydrate structural assignments was ascertained by validation experime nts. The migration times of distinct N-glycans, prepared and measured on different days, were shown to be highly reproducible, with a coeffi cient of variation of usually less than 0.20%, requiring only femtomol es of N-glycan per injection for reliable measurements. By including m esityl oxide and sialic acid as internal standards and a triple-correc tion method, HPCE fulfills the analytical requirements with respect to accuracy, precision, reproducibility, and sensitivity. The N-glycan-m apping database was established using a newly developed and optimized buffer system containing 1,5-diaminopentane as an organic modifier. Ap proximately 80 different sialylated N-glycans of known structure, whic h have thus far been measured and characterized, have been entered int o our Lotus 1-2-3 mapping database. The database for structural determ inations was tested using the N-linked carbohydrates released from rec ombinant human urinary erythropoietin (baby hamster kidney) by PNGase F treatment and from bovine serum fetuin and (alpha 1)-acid glycoprote in by automated and manual (large-scale) hydrazinolysis, respectively. The efficiency of the database and of the triple-correction method wa s further confirmed by HPCE measurements performed in a different labo ratory and by a different analyst who used the HPCE system of a differ ent manufacturer. (C) 1994 Academic Press,Inc.