Characterization of glycosaminoglycans by liquid chromatography-tandem mass spectrometry

Carbohydrates have vital functions in the body; in particular, glycosylation serves to diversify protein functions. Despite their abundance, less information is known about carbohydrates, than nucleic acids and proteins, largely due to their non-template driven biosynthesis. The heterogeneity of glycosaminoglycan (GAG) structures renders them difficult to purify and hinders attempts to perform quantitative measurements and analyze their structural characteristics. Previously analytical methods for the characterization of GAGs focused on compositional analysis. The existing mass spectrometric methods for glycomics profiling did not suffice for small (0.1-10 micrograms, mug) biological samples. Mass spectrometry (MS) is a valuable tool for structural and quantitative analysis of biomolecules, such as GAGs, as it offers high analytical versatility, sensitivity and precision.; A sensitive and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform was developed for the quantification and structural characterization of GAGs from various biological samples. The LC-MS/MS platform allows for quantification of chondroitin sulfate/dermatan sulfate (CS/DS) oligosaccharide mixtures and obtains specific information about the positions of N-acetylgalactosamine sulfation and uronic acid epimerization.; It is advantageous to analyze GAGs from intact tissue as their structure can be related to various biological parameters such as aging and osteoarthritis. However, structural analysis of GAGs from connective tissues is challenging because of the amount and purity of starting material required. Furthermore, the available methods developed for chromatographic or electrophoretic analysis are not appropriate for the extraction and preparation of GAG oligosaccharides from tissue for MS analysis. Thus, a novel MS compatible GAG extraction method was introduced and applied to the LC-MS/MS platform for the study of healthy and diseased cartilage tissue.; Intact tissue samples are often only available in quantities containing less than 10 mug total GAG. In order to analyze such samples, a more sensitive LC-MS/MS platform was developed. Capillary amide-hydrophilic interaction (HILIC) chromatography was found to separate and recover GAG oligosaccharides at concentrations appropriate for intact tissue analysis Therefore, whole chain analysis for profiling CS/DS GAG expression as a function of spatial location in the joint (cartilage, tendon, ligament, muscle and synovial tissue) was demonstrated using an optimized amide-HILIC version of the original novel LC-MS/MS glycomics platform.