ABSTRACT
High pH anion-exchange chromatography (HPAEC) with pulsed amperometric detection has proven to be an invaluable tool for the characterization of carbohydrates [1,2]. Unfortunately, the primary information gained from this technique is retention time, and unless authentic standards are run, the identification of the separated oligosaccharides is tenuous. By combining mass spectrometry with this technique, the separated oligosac charides can be identified with greater certainty. Mass spectrometry can be used to obtain an exact mass of the oligosaccharide. Because most oligosac charides are composed of five unique monosaccharide units with different incremental masses (fucose, 146 Da; hexose, 162 Da; 7V-acetylhexosamine, 203 Da; TV-acetylneuraminic acid, 291 Da; 7V-glycolylneuraminic acid, 307 Da), knowledge of the molecular weight can be used to determine the potential composition of the oligosaccharide. Fast-atom bombardment (FAB) [3,4] and electrospray-ionization (ESI) [5,6] mass spectrometry have both been utilized successfully to this end. Matrix-assisted laser desor ption/ionization (MALDI) is the most suitable ionization method for the analysis of carbohydrates collected after HPAEC, because MALDI is 10100 times more sensitive than FAB for detection of underivatized oligosac charides [7] and is more tolerant of salts than either FAB or ESI [8]. This chapter will describe the implementation of MALDI time-of-flight (TOF) mass spectrometry for the analysis of HPAEC-separated fractions of TVlinked oligosaccharides released from recombinant tissue plasminogen acti vator (tPA).
