A strategy for the mapping of N-glycans by high-pH anion-exchange chromatography with pulsed amperometric detection.
We have evaluated the high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) with respect to its suitability to establish a carbohydrate mapping database that would enable carbohydrate structural analysis by mere comparison of retention times. The suitability of HPAE-PAD for carbohydrate structural analysis was ascertained by validation experiments. The retention times of distinct N-glycans, prepared and measured on different days, were shown to be highly reproducible, with a coefficient of variation (CV) of less than 0.5%, requiring less than 100 pmol of N-glycan per injection for reliable measurements. Including appropriate internal chromatographic standards, such as (Neu5Ac)1, (Neu5Ac)2, (Neu5Ac)3, and Neu5Gc, the HPAE-PAD method fulfills the analytical requirements with respect to accuracy, precision, reproducibility, and sensitivity. The N-glycan mapping database was established, using two optimized linear gradients "S" and "A" for sialylated and asialo N-glycans, respectively. Approximately 100 different N-glycans of known structure, which have thus far been measured and characterized, have entered our Lotus 1-2-3 mapping database. The efficiency of the database for structural determinations was tested, using the N-linked carbohydrates isolated from rhuEPO, expressed in BHK cells. Nine different sialylated N-glycans of rhuEPO (BHK) could be assigned with a deviation of less than +/- 0.5%, using gradient S, and six of the eight asialo N-glycans of rhuEPO (BHK) detected with gradient A could be assigned with an accuracy of less than +/- 1%, three of them even with an accuracy of less than 0.1%, providing the reliability of the established HPAE-PAD mapping database.
Hermentin
P
P
Research Laboratories of Behringwerke AG, Marburg, Germany.
Witzel
R
R
Vliegenthart
J F
JF
Kamerling
J P
JP
Nimtz
M
M
Conradt
H S
HS
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002852:Chromatography, Ion Exchange; D016208:Databases, Factual; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000302:isolation & purification; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry*; D009626:Terminology as Topic
D009844:Oligosaccharides; D011134:Polysaccharides