A Method for Characterising Human Intervertebral Disc Glycosaminoglycan Disaccharides using Liquid Chromatography-Mass Spectrometry with Multiple Reaction Monitoring

Intervertebral disc (IVD) degeneration results in the depletion of proteoglycans and glycosaminoglycans (GAGs), which can lead to structural and mechanical loss of IVD function, ingrowth of nociceptive nerve fbres and eventually discogenic pain. Specifc GAG types as well as their disaccharide paterns can be predictive of disease and degeneration in several tissues but have not been comprehensively studied within the IVD. A highly sensitive mass spectrometry based technique with multiple reaction monitoring (MRM) was used to provide characterisation of chondroitin sulphate (CS), hyaluronic acid (HA), heparan sulphate (HS) and their disaccharide sulphation paterns across diferent anatomical regions of human IVDs. Principal component analysis further distinguished important regional variations and proposed potential ageing variations in GAG profles. CS was the GAG in greatest abundance in the IVD followed by HA and HS. Principal component analysis identifIed clear separation of GAG profiles between nucleus pulposus and annulus fibrosus in young and old specimens. Distinct patterns of predominantly expressed disaccharides of CS and HS between young and old IVD samples, provided preliminary evidence that important alterations in disaccharides occur within IVDs during ageing. This technique offered a novel approach to identify and quantify specific GAG disaccharides in human IVDs and the data presented were the first to offer insight into the spatial distribution as well as association with ageing of GAGs and GAG disaccharide sulphation patterns across the human IVD.