Tandem mass spectrometry methods have been developed for the structural analysis of purified oligomers to determine sites of sulfo-modification involved in these binding relationships, but the analysis of mixtures remains a significant challeng. Using capillary electrophoresis tandem mass spectrometry (CE-MS/MS), GAG mixtures can be separated to reduce analyte heterogeneity before online tandem MS sequencing. Capillary electrophoresis can take a mixture of GAGs with identical mass/elemental composition and separate them based on their shape and charge distribution before they reach the mass spectrometer. The separated GAGs can then be broken down into informative fragments which can be used to assign unique molecular structures using established tandem mass spectrometry techniques. Tetrasaccharide mixtures varying in extent and position of sulfation have been baseline resolved via reverse polarity CE and detected in negative ion mode MS. Current efforts are directed to longer GAGs, threshold and electron-based activation methods for MS/MS, and the analysis more complex mixtures of GAGs derived from human serum and animal cartilage. Fig. 1 Separation of positional isomer heparan sulfate tetrasaccharides using capillary electrophoresis-mass spectrometry. Fig. 2 Annotated structures of positional isomers fragmented using higher energy collision induced dissociation post separation. Fig. 3: Extracted ion chromatograms of depolymerized chondroitin sulfate molecules that were extracted from cartilage.
