A Versatile Method for Analysis of Serine/Threonine Posttranslational Modifications by 尾-Elimination in the Presence of Pyrazolone Analogues

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• 作者：Jun-ichi Furukawa ; Naoki Fujitani ; Kayo Araki ; Yasuhiro Takegawa ; Kota Kodama ; Yasuro Shinohara
• 刊名：Analytical Chemistry
• 出版年：2011
• 出版时间：December 1, 2011
• 年：2011
• 卷：83
• 期：23
• 页码：9060-9067
• 全文大小：860K
• 年卷期：v.83,no.23(December 1, 2011)
• ISSN：1520-6882

文摘

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 尾-elimination in the presence of pyrazolone derivatives (BEP), which in the same reaction labels the formerly glycosylated/phosphorylated peptide. The reaction is essentially a 尾-elimination/Michael addition in which a carbon鈥揷arbon 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 尾-elimination reaction performed in the presence of pyrazolone and the thiol Michael donor.