title: A versatile method for analysis of serine/threonine posttranslational modifications by β-elimination in the presence of pyrazolone analogues.
abstract: 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.
authors: Laboratory of Medical and Functional Glycomics, Graduate School of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
authors: Jun-ichi
authors: J
authors: Furukawa
authors: Naoki
authors: N
authors: Fujitani
authors: Kayo
authors: K
authors: Araki
authors: Yasuhiro
authors: Y
authors: Takegawa
authors: Kota
authors: K
authors: Kodama
authors: Yasuro
authors: Y
authors: Shinohara
mesh_terms: D006020:Glycopeptides / Q000032:analysis; D010748:Phosphopeptides / Q000032:analysis; D011499:Protein Processing, Post-Translational; D047069:Pyrazolones / Q000737:chemistry*; D012694:Serine / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013912:Threonine / Q000737:chemistry*
chemical_list: D006020:Glycopeptides; D010748:Phosphopeptides; D047069:Pyrazolones; D013912:Threonine; C038362:pyrazolone; D012694:Serine