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Versatile Separation and Analysis of Heparan Sulfate Oligosaccharides Using Graphitized Carbon Liquid Chromatography and Electrospray Mass Spectrometry

Journal article
Authors R. L. Miller
S. E. Guimond
M. Prescott
J. E. Turnbull
Niclas G. Karlsson
Published in Analytical Chemistry
Volume 89
Issue 17
Pages 8942-8950
ISSN 0003-2700
Publication year 2017
Published at Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 8942-8950
Language en
Links 10.1021/acs.analchem.7b01417
Keywords glycosaminoglycan tetrasaccharides, detachment dissociation, compositional analysis, chondroitin sulfate, lc-ms, performance, glycomics, acid, quantification, proteins
Subject categories Analytical Chemistry


Heparin and heparan sulfate (HS) by nature contain multiple isomeric structures, which are fundamental for the regulation of biological processes. Here we report the use of a porous graphitized carbon (PGC) LC-MS method with effective separation and sensitivity to separate mixtures of digested HS oligosaccharides. Application of this method allowed the separation of oligosaccharide mixtures with various degree of polymerization (dp) ranging from dp4 to dp8, two dp4 isomers that were baseline resolved, four dp6 isomers, and the observation of a dp3 oligosaccharide. PGC LC-MS of complex mixtures demonstrated that compounds eluted from the column in decreasing order of hydrophilicity, with the more highly sulfated structures eluting first. Our data indicate that sulfation levels, chain length, and conformation all effect elution order. We found that PGC's resolving capabilities for the dp4 and dp6 isomeric structures makes this methodology particularly useful for the sequencing of HS saccharides, because the lack of contaminating isomeric structures provides unambiguous structural assignments from the MS/MS data. Collectively this work demonstrates that PGC column-based methods are powerful tools for enhanced separation and analysis of heterogeneous mixtures of HS saccharide species.

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