Alterations in
oligosaccharides and types of
sialic acid (SA) attachments have been associated with different pathological states. Matrix-assisted
laser desorption mass spectrometry (MS) is commonly used for glycosylation studies. However, native sialylated
glycans are suppressed or not detected during MS experiments. Consequently, different approaches have been employed to neutralize the negative charge of the carboxyl group. In this study, we present the advantage of
phenylhydrazine (PHN) labeling for the detection and efficient discrimination of SA linkages when this derivatization follows alkyl esterification. As expected, PHN-labeled sialylated
oligosaccharides with the 2,6-linkage type can be easily recognized according to the additional shift in mass corresponding to the presence of a methyl or ethyl group. Surprisingly,
oligosaccharides with the 2,3-linked SA residue instead of a
lactone were detected carrying the second PHN unit. This was beneficial as no further processing after esterification was needed to stabilize the
lactone form. Moreover, during tandem mass experiments, all modified
glycans produced favorable fragmentation patterns with a coherent recognition of SA linkages. Although both types of esterification, herein called the EST-PHN approach, provided comparable results, methylation exhibited marginally higher linkage specificity than ethyl esterification. The simplicity and effectiveness of the methodology are demonstrated on the model compound,
sialyllactose, and its applicability for
biological studies is presented on N-
glycan profiling in the sera of
lung cancer patients.