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.

尽管蛋白质唾液酸化（protein sialylation）在细胞相互作用、癌症进展等生物学过程中发挥关键作用，但针对完整唾液酸化糖肽（sialylglycopeptides, SGPs）的简便高效分析方法仍十分有限。低丰度唾液酸化糖肽的分析通常需要在基于液相色谱-质谱（liquid chromatography–mass spectrometry, LC–MS）的综合分析前，先进行高效富集。本研究开发了一种结合温和高碘酸氧化、酰肼化学、铜催化叠氮/炔环加成（copper-catalyzed azide/alkyne cycloaddition, CuAAC）点击化学与动态共价交换的新型工作流程，用于选择性富集唾液酸化糖肽。完整的唾液酸化糖肽可轻松从蛋白质胰蛋白酶酶解物中分离，且特征离子可在LC-MS/MS分析过程中产生，从而实现明确的鉴定。通过同位素试剂验证了特征离子的结构及对应的动态共价交换过程。在优化后的实验条件下，采用牛胎球蛋白酶解物进行测试时，该方法的富集效率可达70%以上，并成功应用于小鼠肺组织提取物等复杂生物样本的分析。该方法兼具富集效率高、重现性好、操作简便且无需制备特殊材料等优势，有望成为唾液酸化糖肽分析领域极具应用前景的技术工具。