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Abstract
Sulfated glycosaminoglycan (GAG) carbohydrates are linear, acidic polysaccharide chains abundant on the surface of mammalian cells that affect several biological processes through protein-binding interactions. Structural characterization of sulfated GAGs is challenging due to their non-template biosynthesis resulting in the production of heterogeneous mixtures with different chain lengths and varying modification patterns. 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 challenge. Using capillary zone electrophoresis tandem mass spectrometry (CZE-MS/MS), GAG mixtures were separated to reduce analyte heterogeneity before online tandem MS sequencing. This advance enables the determination of binding motifs responsible for the interaction of GAG chains with proteins, as these sequences typically range from at least tetrasaccharides to octasaccharides and greater in length.
For this work, GAG mixtures varying in extent and position of sulfation have been separated using reverse polarity CZE and detected in negative ion mode MS. Purified standards and various biological samples were analyzed using this platform. Compositional analysis was performed to determine the various components using accurate mass measurement, and the most intense species were analyzed using tandem mass spectrometry to sequence the GAG chains. Collision induced dissociation/high-energy collision dissociation (CID/HCD) and negative electron transfer dissociation (NETD) were utilized to fragment the isolated GAG precursors which enabled determination of sulfate position on GAGs post separation. GAGs extracted from healthy human urine and GAG chains removed from the proteoglycan bikunin were two biological samples analyzed.
For this work, GAG mixtures varying in extent and position of sulfation have been separated using reverse polarity CZE and detected in negative ion mode MS. Purified standards and various biological samples were analyzed using this platform. Compositional analysis was performed to determine the various components using accurate mass measurement, and the most intense species were analyzed using tandem mass spectrometry to sequence the GAG chains. Collision induced dissociation/high-energy collision dissociation (CID/HCD) and negative electron transfer dissociation (NETD) were utilized to fragment the isolated GAG precursors which enabled determination of sulfate position on GAGs post separation. GAGs extracted from healthy human urine and GAG chains removed from the proteoglycan bikunin were two biological samples analyzed.