Application of Malononitrile Derivatization Method for Structural Glycomics Study in Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

Since it is recognized that carbohydrates, as free oligosaccharides and constituents of glycoconjugates, play important roles in many biological functions of glycoproteins, a variety of analytical techniques have been developed for highly sensitive detection and analysis of complex oligosaccharides obtained from a variety of biological media [1]. Presently, among many analytical techniques, mass spectrometry has been recognized to be a highly powerful technique for the determination of molecular weights and for structural analysis for a variety of carbohydrates. Because of the low proton affinity and ionizability of free oligosaccharides, especially neutral oligosaccharides, the mass sensitivity and the detectable mass range of free oligosaccharides are generally poor in comparison with those of proteins and nucleic acids in positive ion mode. In order to solve these problems, a number of derivatization methods such as permethylation, peresterification, reductive amination and malononitrile addition have been developed [2-6]. Furthermore in the structural analyses of oligosaccharides by CID experiments [7, 8] with ESI mass spectrometry or by PSD experiments [9, 10] in MALDI-TOF mass spectrometry, these derivatization techniques have additional advantages. Firstly the mass shift of oligosaccharide derivatives, corresponding to the mass of an attached labeling group, facilitates the interpretation of the resulting peak patterns of fragment ions. Secondly the occurrence of a variety of fragmentation patterns, due to the direction of ion fragmentation in the oligosaccharide derivative, can give more information on the structure of the oligosaccharide. Among several derivatization techniques the malononitrile addition method has been developed recently by our group, and applied effectively for the separation and high-sensitivity detection of multi-component oligosaccharide mixture using on-line capillary liquid chromatography (LC)/ESI-MS in negative ion mode [11]. The malononitrile derivatization of oligosaccharide is accomplished easily under mildly basic aqueous conditions without any reagent except for malononitrile, which is a volatile compound with a small molecular weight, so that excess reagent is easily removed and/or does not interfere with the spectra of interest. Thus we could accomplish directly the high-sensitivity analyses of unpurified oligosaccharide derivatives using negative-ion ESI-MS without any sample pre-treatment procedure. In the present study we introduce the application of malononitrile derivatization method for structural analyses of oligosaccharides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.