Differentiation of α2-3- and α2-6-Linked Sialic Acid Linkages Using Esterification/Amidation Reactions and Tandem...

Among various oligosaccharides, sialylated oligosaccharides play crucial roles in both physiological and pathological processes, and sialic acid residues within these structures are often used as biomarkers. Mass spectrometry is a widely utilized tool for glycan structural analysis. However, sialic acid is prone to elimination during the sample preparation and the activation process in tandem mass spectra, resulting in a loss of information on sialylation. Consequently, the identification of sialic acid linkages presents a significant analytical challenge. One established method for differentiating α2 → 3- and α2 → 6-linked sialic acids involves esterification followed by ammonia treatment. In this process, the carboxyl group of α2 → 3-linked sialic acids is converted into an amide, while α2 → 6-linked sialic acids are transformed into esters, resulting in distinguishable molecular weights. However, partial conversion of α2 → 6-linked sialic acids to amides and α2 → 3-linked sialic acids to esters has been observed, which reduces the reliability of this differentiation approach.

A simple tandem mass spectrometry, based on the dissociation mechanisms of hexose and N-acetylhexosamine, is proposed to differentiate α2 → 3 and α2 → 6 sialic acid linkages after esterification and amidation.

Various oligosaccharides consisting of sialic acid with α2 → 3 or α2 → 6 linkages were studied. For some oligosaccharides, the amidation for α2 → 3 linked sialic acid and esterification for α2 → 6 linked sialic acid are nearly complete, while partial amidation for α2 → 6 linked sialic acid and partial esterification for α2 → 3 linked sialic acid for other oligosaccharides was found, suggesting differentiation cannot be solely dependent on the amidation and esterification (or molecular weights). We showed that the α2 → 3 and α2 → 6 linked sialic acids after esterification/amidation can be distinguished by a simple tandem mass spectrometry.

The proposed tandem mass spectrometry enables accurate differentiation of α2 → 3 and α2 → 6 sialic acid linkages, even when both of them undergo amidation or esterification.