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

Abstract

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.

Shotgun ion mobility mass spectrometry sequencing of heparan sulfate saccharides

“…However, this analysis does not provide insight into larger bioactive motifs, typically consisting of 4-10 monosaccharide units. Partial digestion and isolation of larger bioactive oligosaccharide structures (<dp10) is possible with considerable effort, but heterogeneity often prevents complete characterization 4,5 . A handful of tetrasaccharides (out of around 5000 theoretical structures) have been confidently characterized since the number of isomeric structures is limited at this size [6][7][8][9] .…”

Analysis of the Glycosaminoglycan Chains of Proteoglycans

“…The method was effective and allowed the separation of oligosaccharide from tetrasaccharide to octasaccharide, enabling more confident interpretation of the MS/MS data. 57…”

Salt-free fractionation of complex isomeric mixtures of glycosaminoglycan oligosaccharides compatible with ESI-MS and microarray analysis

“…The vast diversity of potential structures (5.3 million octasaccharides) makes complete coverage by synthetic methods impractical 11 . Therefore, the need for efficient separation technique prior to MS or microarray printing that is readily compatible with these techniques is clear 12,13 .…”

“…Compared with SAX, IPRP is more easily coupled with other separation methods and MS. Hyphenation of IPRP with SEC and time of flight mass spectrometry have greatly improved separation and provided more complete and important structural information of low molecular weight heparin (LMWH) 22 . But the ion-pair agents will easily induce in-source contamination and cause signal suppression when coupled with MS 13 . Some IPRP agents can also interfere with microarray immobilization schemes 23 .…”