Offline coupling of low-pressure anion-exchange chromatography with MALDI-MS to determine the elution order of human milk oligosaccharides

Pooled human milk oligosaccharides were separated into neutral and several acidic oligosaccharide fractions by preparative anion-exchange chromatograp hy (AEC) using AG 1-X2. The oligosaccharides were eluted stepwise using dei onized water and three different concentrations of ammonium acetate buffer, pH 6.8. The elution order of the compounds was determined directly by matr ix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analys is of the AEC effluent without any cleanup or concentration steps. Up to a concentration of 500 mM ammonium acetate, the masses of acidic oligosacchar ides could be detected by screening the fractions in an automated mode. The combination of the improved chromatographic procedure, the applied MALDI m atrices, and operating parameters is suitable for the detection of neutral oligosaccharides as well as acidic oligosaccharides, The method provides hi gh sensitivity and mass accuracy, including for the high-molecular-weight m onosialylated oligosaccharides up to 2751.5 Da, The applied ionic strength of the anion-exchange eluents enables a rapid and an unambiguous compositio n assignment by MALDI-MS for neutral, monosialylated, and disialylated olig osaccharides from human milk The acidic fractions have to be desalted by el ectrodialysis and were finally analyzed by HPAEC-PAD to get a high-resoluti on "fingerprint" of structures present in each fraction. From these analyse s, it can be concluded that the isomeric variety of monosialylated oligosac charides occurring in human milk is higher than estimated before. (C) 2000 Academic Press.