Micro-Scale Analysis of N-Acetylneuraminic Acid

We have developed a micro-scale analytical method for the detection and quantification of Nacetylneuraminic acid (NANA)that does neither require any derivatization of NANAnoris it interfered by the presence of any monosaccharide component present on N-linked and O-linked carbohydrate chains of glycoproteins. The method involves acid hydrolysis of sub-nanomolar amounts of sialoglycoprotein (SGP) and subsequent neutralization, followed by high-pH anion-exchange chromatography (HPAE) with pulsed amperometric detection (PAD). The method wasvalidated, using a,-acid glycoprotein (AGP) (orosomucoid) as standard SGP. Six experimental series of decaplicates, each, were performed, varying i) the AGP concentration in PBS (i. e. 250, 500 and 750 ug/ml, respectively), ii) the sample amount used (i. e. 200, 100, 30, 15 and 10 ul, each, of defined AGP concentrations), iii) the mode of sample injection into the Dionex Bio-LC system (i. e. manual or automated injection by way of two different autoinjectors), iv) the sensitivity of the PAD detector(i. e. 1000 and 300 nAfull scale, respectively). Individual experimental series were repeated at different days and were occasionally performed by different persons. In each case, the NANA/AGP molar ratios were in the range of 15.0 +/- 0.4 mol/mol (corresponding with a standard deviation of <+/- 3 %), which is in excellent agreement with the NANA/AGP molar ratio described in theliterature

The patterns of the complex- and oligomannose-type glycans of uromodulin (Tamm-Horsfall glycoprotein) in the course of pregnancy

Uromodulin was isolated from urine of three pregnant women. Urine of each donor was collected at subsequent stages of their pregnancy and at one month after gestation. Each batch of uromodulin was enzymatically N-deglycosylated and the released N-glycans were isolated, quantified and profiled by high-pH anion-exchange chromatography. In the course of pregnancy no significant changes were detected in the negative charge distribution stemming from sialic acid and sulfate residues on the complex-type carbohydrate chains of uromodulin. Furthermore, no significant changes in the molar ratio between Man6GlcNAc2 and Man7GlcNAc2 were found in the course of pregnancy, only uromodulin from non-pregnant periods showed small differences

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 alpha1-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