A strategy for the mapping of N-glycans by high-performance capillary electrophoresis.
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.
Hermentin
P
P
Research Laboratories of Behringwerke AG, Marburg, Federal Republic of Germany.
Doenges
R
R
Witzel
R
R
Hokke
C H
CH
Vliegenthart
J F
JF
Kamerling
J P
JP
Conradt
H S
HS
Nimtz
M
M
Brazel
D
D
D004586:Electrophoresis / Q000379:methods*; D004921:Erythropoietin / Q000737:chemistry; D007256:Information Systems; D009961:Orosomucoid / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D000509:alpha-Fetoproteins / Q000737:chemistry
D009961:Orosomucoid; D011134:Polysaccharides; D000509:alpha-Fetoproteins; D004921:Erythropoietin