Premium
Characterization of derivatization of sialic acid with 2‐aminoacridone and determination of sialic acid content in glycoproteins by capillary electrophoresis and high performance liquid chromatography ‐ ion trap mass spectrometry
Author(s) -
Che FaYun,
Shao XiaoXia,
Wang KeYi,
Xia QiChang
Publication year - 1999
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(19991001)20:14<2930::aid-elps2930>3.0.co;2-x
Subject(s) - chromatography , sialic acid , chemistry , derivatization , mass spectrometry , capillary electrophoresis , glycoprotein , n acetylneuraminic acid , biochemistry
A simple and highly sensitive capillary electrophoresis (CE) method for determining the content of N ‐acetylneuraminic acid (Neu5Ac) in glycoproteins was developed. Neu5Ac was derivatized with 2‐aminoacridone (AMAC) by reductive amination, and the AMAC‐Neu5Ac adduct could be readily separated from the other 11 AMAC‐derivatized neutral and acidic monosaccharides usually present in glycoproteins by CE in a 0.3 mol/L borate buffer, pH 10.5, and detected at 260 nm. The derivatization of Neu5Ac was achieved at 55°C for 4 h. AMAC‐Neu5Ac was stable at 20°C in the dark for at least 12 h while at room temperature it spontaneously converted into another substance with a lower electrophoretic mobility, which was identified as decarboxylated AMAC‐Neu5Ac by high performance liquid chromatography — ion trap mass spectrometry (HPLC‐ITMS). Concentration and mass of Neu5Ac as low as 1 μmol/L and 35 fmol could be detected. The linear correlation coefficient between the ratio of peak area to migration time of AMAC‐Neu5Ac and the concentration of Neu5Ac ranging from 10 to 120 μmol/L was 0.9978 ( n =8). This method was successfully applied to the analysis of sialic acid in human urinary trypsin inhibitor (hu‐UTI), bovine α 1 ‐acid glycoprotein (α 1 ‐AGP) and recombinant human erythropoietin (rhu‐EPO). By combination of CE and HPLC‐ITMS we found that N ‐glycolylneuraminic acid (Neu5Gc) was present in bovine α 1 ‐AGP in addition to Neu5Ac, with a quantity comparable to that of the latter.