Laboratory of Medical and Functional Glycomics, Graduate School of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
Anal Chem. 2011 Dec 1;83(23):9060-7. doi: 10.1021/ac2019848. Epub 2011 Nov 1.
Post-translational modifications (PTMs) of serine and threonine occur by diverse mechanisms, including phosphorylation, sulfation, and various types of sugar chain modifications, making characterization of the resulting structures very labor-intensive. Moreover, to fully understand the biological functions of PTMs, both the sites of modification and the modified structures must be analyzed. The present work describes a novel, versatile strategy in which the released O-glycan and the formerly glycosylated/phosphorylated peptide are labeled and thus amenable to further study. In this approach, glycopeptides/phosphopeptides are subjected to β-elimination in the presence of pyrazolone derivatives (BEP), which in the same reaction labels the formerly glycosylated/phosphorylated peptide. The reaction is essentially a β-elimination/Michael addition in which a carbon-carbon bond-forming Michael donor rather than a heteroatomic Michael donor is used. The O-glycans released upon BEP are recovered as bis-pyrazolone derivatives, without any detectable side reaction (peeling). Using this technique, the O-glycan profiles of model mucin-type glycoproteins were successfully analyzed. The BEP strategy discriminates between phosphorylated and GlcNAcylated peptides, since cleaved GlcNAc is detectable. In addition, both the released O-glycan and the formerly glycosylated peptide can be selectively labeled by different reagents via a β-elimination reaction performed in the presence of pyrazolone and the thiol Michael donor.
丝氨酸和苏氨酸的翻译后修饰（PTMs）通过多种机制发生，包括磷酸化、硫酸化和各种类型的糖链修饰，使得对所得结构的表征非常费力。此外，为了充分了解 PTM 的生物学功能，必须分析修饰部位和修饰结构。本工作描述了一种新颖的、通用的策略，其中释放的 O-聚糖和以前糖基化/磷酸化的肽被标记，因此可以进一步研究。在该方法中，糖肽/磷酸肽在吡唑啉酮衍生物（BEP）的存在下进行β-消除，该反应同时标记以前糖基化/磷酸化的肽。该反应本质上是β-消除/Michael 加成，其中使用形成碳-碳键的 Michael 供体而不是杂原子 Michael 供体。BEP 后释放的 O-聚糖作为双吡唑啉酮衍生物回收，没有任何可检测的副反应（剥落）。使用该技术，成功分析了模型粘蛋白型糖蛋白的 O-聚糖图谱。BEP 策略区分了磷酸化和 GlcNAc 化肽，因为可检测到裂解的 GlcNAc。此外，通过在吡唑啉酮和硫醇 Michael 供体存在下进行的β-消除反应，释放的 O-聚糖和以前糖基化的肽都可以通过不同的试剂选择性地标记。
