Structural analysis of glycoconjugates by on-target enzymatic digestion and MALDI-TOF-MS

Exoglycosidase digestion combined with matrix-assisted laser desorption/ion ization time-of-flight mass spectrometry (MALDI-TOF-MS) has been demonstrat ed to be an effective method for the structural characterization of glycoco njugates and oligosaccharides in picomolar amounts. A sample preparation me thod is described, in which 6-aza-2-thiothymine (ATT) in water is used as m atrix and enzymes are dialyzed before use against a low concentration of vo latile buffer such as ammonium acetate. Under these conditions, a series of sequential on-target exoglycosidase treatments was carried out in one sing le analyte spot in the presence of ATT matrix. Subsequent mass spectrometri c analysis of the resulting products yielded information on both the comple teness of the reaction and structural features of the glycoconjugates such as monosaccharide sequence, branching pattern, and anomeric configurations of the corresponding glycosidic linkages. The results show that all exoglyc osidases used retain their activity in the presence of ATT matrix. Hence, s tructural analysis of carbohydrates or mixtures thereof can be performed ve ry fast, without intermediate desalting steps or sample splitting. This app roach is illustrated by the analysis of underivatized glycans, oligosacchar ide derivatives, glycopeptides, and glycolipids. Depending on the analyte, amounts of sample required could be limited to a few picomoles.