One‐pot nonreductive O‐glycan release and labeling with 1‐phenyl‐3‐methyl‐5‐pyrazolone followed by ESI‐MS analysis

Abstract

A novel one-pot procedure for the nonreductive release of O-linked glycans from glycoproteins and the simultaneous derivatization of released glycans with 1-phenyl-3-methyl-5-pyrazolone (PMP) is described. Unlike the traditional reductive β-elimination, which produces alditols, this new method employs PMP/ammonia aqueous solution as the reaction medium. The O-glycans are released from glycoproteins and derivatized with PMP nonreductively, specifically, and quantitatively. Samples can be easily purified from ammonia, excess PMP, and peptide residues by evaporation, chloroform extraction, and solid-phase extraction (SPE) column fractionation for HPLC, CE, or MS analysis. The procedure has been elaborated with two purified glycoproteins, porcine stomach mucin and bovine fetuin, and successfully applied to O-glycan profiling of a challenging biological specimen, healthy human plasma. This new procedure has shown methodological significance in O-glycan analysis.

Isolation and structural characterization of the polysaccharide LRGP1 from Lycium ruthenicum

“…The oligosaccharides were eluted with 3 mL of deionized water, and the eluted fractions were collected and diluted to 1000 times with MeOH for ESI-MS analysis (Wang, Fan, Zhang, Wang, & Huang, 2011). The ESI-MS data were acquired on a Thermo Scientific LTQ XL ion trap mass spectrometer (USA) in positive ion mode.…”

Toward a Platform for Comprehensive Glycan Sequencing

“…Early success with mild base catalyzed derivatization of monosaccharides and more recently with larger oligomers; N-and O-glycans (11,12) support this interest. The synchrony of PMP condensation and glycan elimination prompted Wang et al (13) (Northwest University, Xian, China) to bring this into a one-pot reaction. The catalyzed ␤-elimination and rapid Michael addition to the reducing terminus seems to diminish the chance of peeling, an important consideration for select glycans.…”

Protein O-glycosylation analysis

“…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.…”

“…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.…”