Journal
ANALYTICAL CHEMISTRY
Volume 89, Issue 17, Pages 8942-8950Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.7b01417
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Funding
- Medical Research Council (MRC)
- Engineering and Physical Sciences Research (EPSRC) Council UK (UoL/MRC-EPSRC)
- MRC [G117/423]
- Biotechnology and Biological Sciences Research Council [BB/I004343/1]
- Swedish Research Council [621-2013-5895]
- Kung Gustav V:s 80-ars foundation
- Petrus and Augusta Hedlund's foundation
- AFA insurance research fund
- Biotechnology and Biological Sciences Research Council [BB/I004343/1] Funding Source: researchfish
- Medical Research Council [G117/423] Funding Source: researchfish
- BBSRC [BB/I004343/1] Funding Source: UKRI
- MRC [G117/423] Funding Source: UKRI
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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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