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.

POMGNT1 Is Glycosylated by Mucin-Type <i>O</i>-Glycans

“…Concentrated sPOMGNT1 (10 µL, 30 µg) was treated with 20 µL each of sodium hydroxide (0.4 M) and 0.5 M methanolic 1-phenyl-3-methyl-5-pyrazolone (PMP, Tokyo Chemical Industry, Tokyo, Japan) solution, followed by heating at 85°C for 16 h as described previously. 13) Ethanol precipitation was performed by the addition of a 4-fold volume of cold ethanol and incubation at −30°C for 3 h. Supernatants and precipitated proteins were separated by centrifugation at 15000 rpm for 10 min at 4°C. Supernatants were neutralized with 1.0 M hydrochloric acid and shaken vigorously with chloroform.…”

Protein O-glycosylation analysis

“…More recently, Zauner et al (2011) , Wang et al (2011) , and Furukawa et al (2011 have introduced new, one-pot combined methods for O-glycan release and labeling. These methods use dimethylamine (Zauner et al , 2011 ), ammonia (Wang et al , 2011 ), or sodium hydroxide (Furukawa et al , 2011 ) for β -elimination release and 1-phenyl-3-methyl-5-pyrazolone (PMP) for the concomitant labeling via a Michael addition.…”

“…These methods use dimethylamine (Zauner et al , 2011 ), ammonia (Wang et al , 2011 ), or sodium hydroxide (Furukawa et al , 2011 ) for β -elimination release and 1-phenyl-3-methyl-5-pyrazolone (PMP) for the concomitant labeling via a Michael addition.…”

“…Therefore, after one reaction, both the released/ labeled O-glycans and the remaining tagged peptide portion can be analyzed. This is another demonstration of the feasibility of being able to use the peptide portion for site specifi c analysis after β -elimination (Furukawa et al , 2011 ). Czeszak et al (2002) were likewise interested in analyzing O-glycosylation sites and aimed for a more potent way to chemically modify peptides as they were deglycosylated.…”

“…It is hoped that ways to reduce/ abolish this ' peeling ' reaction and/or combine the release of O-glycans with a suitable labeling technique will be identifi ed soon. Some recent papers have described the fi rst steps toward this goal (Maniatis et al , 2010 ;Furukawa et al , 2011 ;Wang et al , 2011 ;Zauner et al , 2011 ;Kozak et al , 2012 ). The effi cient blocking of the reducing end of the formerly O-linked oligosaccharide appears to be an important aspect in improving the stability of the O-glycans.…”