Selective Enrichment of Sialylglycopeptides Enabled by Click Chemistry and Dynamic Covalent Exchange

Despite the important roles of protein
sialylation in biological
processes such as cellular interaction and cancer progression, simple
and effective methods for the analysis of intact sialylglycopeptides
(SGPs) are still limited. Analyses of low-abundance SGPs typically
require efficient enrichment prior to comprehensive liquid chromatography–mass
spectrometry (LC–MS)-based analysis. Here, a novel workflow
combining mild periodate oxidation, hydrazide chemistry, copper-catalyzed
azide/alkyne cycloaddition (CuAAC) click chemistry, and dynamic covalent
exchange has been developed for selective enrichment of SGPs. The
intact SGPs could be separated easily from protein tryptic digests,
and the signature ions were produced during LC–MS/MS for unambiguous
identification. The structure of the signature ions and corresponding
dynamic covalent exchange were confirmed by using an isotopic reagent.
Under the optimized condition, over 70% enrichment efficiency of SGPs
was achieved using bovine fetuin digests, and the method was successfully
applied to complex biological samples, such as a mouse lung tissue
extract. The high enrichment efficiency, good reproducibility, and
easily adopted procedure without the need to generate specialized
materials make this method a promising tool for broad applications
in SGP analysis.