TY - JOUR
T1 - Oxidative Release of O-Glycans under Neutral Conditions for Analysis of Glycoconjugates Having Base-Sensitive Substituents
AU - Vos, Gaël M
AU - Weber, Julia
AU - Sweet, Igor R
AU - Hooijschuur, Kevin C
AU - Sastre Toraño, Javier
AU - Boons, Geert-Jan
N1 - Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society.
PY - 2023/6/13
Y1 - 2023/6/13
N2 - Protein
O-glycosylation is one of the most diverse post-translational modifications. A critical step in the analysis of
O-glycomes is the release of glycans from glycoconjugates. Current release methods rely mainly on β-elimination, which can result in peeling reactions and loss of base-sensitive functionalities leading to misidentification of glycans. To address this challenge, well-defined synthetic glycopeptides were used to establish a robust workflow for the oxidative release of
O-glycans suitable for glycomics. Treatment of
O-glycopeptides with neutralized hypochlorite resulted in the selective formation of lactic/glycolic acid glycosides, thereby retaining unique information of the parent amino acid (serine/threonine) that is lost by β-elimination. It locks the glycan in a closed ring configuration, thereby preventing peeling, and furthermore, the carboxylate of the anomeric tag promotes ionization in negative ion mode mass spectrometry, thereby increasing signal intensities. Labile modifications such as sialic acids, sulfates, and acetyl esters are maintained during the release procedure. The promise of the approach was demonstrated by the analysis of
O-glycans from bovine submaxillary mucin, which identified mono- and di-
O-acetylated sialoglycans as well as previously undetected tri-
O-acetylated and sulfated glycans. The use of well-defined glycopeptide standards made it also possible to identify reaction intermediates, which in turn allowed us to postulate a reaction mechanism for oxidative
O-glycan release under neutral conditions.
AB - Protein
O-glycosylation is one of the most diverse post-translational modifications. A critical step in the analysis of
O-glycomes is the release of glycans from glycoconjugates. Current release methods rely mainly on β-elimination, which can result in peeling reactions and loss of base-sensitive functionalities leading to misidentification of glycans. To address this challenge, well-defined synthetic glycopeptides were used to establish a robust workflow for the oxidative release of
O-glycans suitable for glycomics. Treatment of
O-glycopeptides with neutralized hypochlorite resulted in the selective formation of lactic/glycolic acid glycosides, thereby retaining unique information of the parent amino acid (serine/threonine) that is lost by β-elimination. It locks the glycan in a closed ring configuration, thereby preventing peeling, and furthermore, the carboxylate of the anomeric tag promotes ionization in negative ion mode mass spectrometry, thereby increasing signal intensities. Labile modifications such as sialic acids, sulfates, and acetyl esters are maintained during the release procedure. The promise of the approach was demonstrated by the analysis of
O-glycans from bovine submaxillary mucin, which identified mono- and di-
O-acetylated sialoglycans as well as previously undetected tri-
O-acetylated and sulfated glycans. The use of well-defined glycopeptide standards made it also possible to identify reaction intermediates, which in turn allowed us to postulate a reaction mechanism for oxidative
O-glycan release under neutral conditions.
KW - Animals
KW - Cattle
KW - Glycoproteins/chemistry
KW - Polysaccharides/chemistry
KW - Glycosylation
KW - Glycopeptides/chemistry
KW - Oxidative Stress
UR - http://www.scopus.com/inward/record.url?scp=85163956749&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.3c00127
DO - 10.1021/acs.analchem.3c00127
M3 - Article
C2 - 37259796
SN - 0019-7866
VL - 95
SP - 8825
EP - 8833
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 23
ER -