Precision Profiling of α‐1,4‐ Versus α‐1,6‐Glycosidase Activity Through Synthetic Linear and Branched Glycogen Tetramers Using Cyclic Ion Mobility Mass Spectrometry and High‐Performance Liquid Chromatography‐Mass Spectrometry
Abstract
Accurately defining glycosidase specificity is often limited by access to well‐defined oligosaccharide probes and analytical tools capable of resolving regioisomers at low concentrations. We synthesized two linear and one branched glycogen‐mimetic tetrasaccharides, bearing exclusively 1,2‐ cis , namely α‐1,4 or α‐1,6 linkages using a reagent‐controlled stereoselective glycosylation strategy. Their enzymatic degradation by Homo sapiens glycogen debranching enzyme ( Hs GDE) and two commercial α‐1,4‐glucosidases was assessed by hydrophilic interaction liquid chromatography‐mass spectrometry and high‐resolution cyclic ion mobility mass spectrometry (cIMS‐MS), enabling separation and quantification of substrates and products in the micromolar range. Hs GDE selectively cleaved the α‐1,6‐branch while showing low activity on terminal α‐1,6 linkages. The Saccharomyces cerevisiae α‐1,4‐glucosidase showed high α‐1,4 selectivity, whereas the rice enzyme displayed broad promiscuity toward all regioisomers. The cIMS‐MS provided rapid baseline separation of the synthetic tetrasaccharides and confirmed the absence of inhibition by nonsubstrate isomers. This integrated synthetic‐analytical workflow offers an efficient platform for rapid annotation of glycosidase specificity and mechanistic studies in glycogen metabolism.
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BibTeX
@article{carry2026precision,
title = {{Precision Profiling of α‐1,4‐ Versus α‐1,6‐Glycosidase Activity Through Synthetic Linear and Branched Glycogen Tetramers Using Cyclic Ion Mobility Mass Spectrometry and High‐Performance Liquid Chromatography‐Mass Spectrometry}},
author = {Delphine Carry and Ruben Ananian and Zineb Chaabi and Estelle Messé and Georg Niggemeyer and Benoit Keromnes and Jean‐françois Gallard and Cédric Przybylski and Peter Seeberger and Christian Roth and Salomé Poyer and Stéphanie Norsikian and Sébastien Vidal},
journal = {ChemistryEurope},
year = {2026},
month = oct,
doi = {10.1002/ceur.70381},
url = {https://hal.science/hal-05847101v1},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Precision Profiling of α‐1,4‐ Versus α‐1,6‐Glycosidase Activity Through Synthetic Linear and Branched Glycogen Tetramers Using Cyclic Ion Mobility Mass Spectrometry and High‐Performance Liquid Chromatography‐Mass Spectrometry
AU - Delphine Carry
AU - Ruben Ananian
AU - Zineb Chaabi
AU - Estelle Messé
AU - Georg Niggemeyer
AU - Benoit Keromnes
AU - Jean‐françois Gallard
AU - Cédric Przybylski
AU - Peter Seeberger
AU - Christian Roth
AU - Salomé Poyer
AU - Stéphanie Norsikian
AU - Sébastien Vidal
PY - 2026
DA - 2026/10/29
JO - ChemistryEurope
DO - 10.1002/ceur.70381
UR - https://hal.science/hal-05847101v1
AB - Accurately defining glycosidase specificity is often limited by access to well‐defined oligosaccharide probes and analytical tools capable of resolving regioisomers at low concentrations. We synthesized two linear and one branched glycogen‐mimetic tetrasaccharides, bearing exclusively 1,2‐ cis , namely α‐1,4 or α‐1,6 linkages using a reagent‐controlled stereoselective glycosylation strategy. Their enzymatic degradation by Homo sapiens glycogen debranching enzyme ( Hs GDE) and two commercial α‐1,4‐glucosidases was assessed by hydrophilic interaction liquid chromatography‐mass spectrometry and high‐resolution cyclic ion mobility mass spectrometry (cIMS‐MS), enabling separation and quantification of substrates and products in the micromolar range. Hs GDE selectively cleaved the α‐1,6‐branch while showing low activity on terminal α‐1,6 linkages. The Saccharomyces cerevisiae α‐1,4‐glucosidase showed high α‐1,4 selectivity, whereas the rice enzyme displayed broad promiscuity toward all regioisomers. The cIMS‐MS provided rapid baseline separation of the synthetic tetrasaccharides and confirmed the absence of inhibition by nonsubstrate isomers. This integrated synthetic‐analytical workflow offers an efficient platform for rapid annotation of glycosidase specificity and mechanistic studies in glycogen metabolism.
SN - 2751-4765
ER -