A method for characterising human intervertebral disc glycosaminoglycan disaccharides using liquid chromatography-mass spectrometry with multiple reaction monitoring

Abstract

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 fibres and eventually discogenic pain. Specific GAG types as well as their disaccharide patterns 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 patterns across different anatomical regions of human IVDs. Principal component analysis further distinguished important regional variations and proposed potential ageing variations in GAG profiles. 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.

Fig. 1

Flow chart for human intervertebral disc (IVD) specimen treatment for glycosaminoglycan disaccharide composition analyses. (a) 7 regions tested in each IVD: outer anterior annulus (AO), inner anterior annulus (AI), nucleus pulposus (NP), inner posterior annulus (PI), outer posterior annulus (PO), inner posterior-lateral annulus (LI), and outer posterior-lateral annulus (LO). (b) Glycosaminoglycans purified from the IVD regions, digested by the heparinases and chondroitinase ABC. (c) Structures of the disaccharides after adding heparinase I/II/III and chondroitinase ABC. ΔUA2X (1,3) GalNAc4X6X is generated from chondroitin sulphate (CS). ΔUA2X (1,4) GlcNY6X is generated from heparan sulphate (HS). ΔUA (1,3) GlcNAc is generated from hyaluronic acid (HA). X = H or SO₃H. Y = H or SO₃H or COCH₃. (d) Extracted ion chromatograms (EICs) of the disaccharides.

Fig. 2

Composition of GAGs in different regions of the IVDs. (a) Percentage of each GAG type out of total GAG content. Percent of Chondroitin sulphate (CS), Hyaluronic acid (HA), and Heparan sulphate (HS) in the outer anterior annulus (AO), inner anterior annulus (AI), nucleus pulposus (NP), inner posterior annulus (PI), outer posterior annulus (PO), inner posterior-lateral annulus (LI), and outer posterior-lateral annulus (LO). (b) Total GAG composition quantified as ng GAG/mg of dry tissue weight. Data are averaged across all 6 IVDs (n = 6) for each anatomical region of interest. One-Way ANOVA between GAG types within each region shows significance *compared to HA and HS if p < 0.05 and ^#compared to HS if p < 0.05. Mean ± SD.

Fig. 3

Concentrations of CS, HA and HS, in ng GAG/mg of dry tissue weight, across the 7 regions of the disc. The grey doted line represents the GAG composition of the young (9 year old) specimen (n = 1), mean of biological replicates for each region; the red line represents the average of all IVD specimens, mean ± SD (n = 6); the black solid line represents the average of all specimens except the one young (9 year old) IVD, mean ± SD (n = 5 mature specimens) for each region.

Fig. 4

(a-c) Percentage of CS, HA and HS out of total GAG in the NP and outer AF regions (AO, PO and LO) in each of 6 IVD specimens. (d-f) Total CS, HA and HS represented as ng GAG/mg dry tissue weight in NP and outer AF regions (AO,PO,LO) in each IVD. For the 9 year old IVD, NP measurements are averaged across n = 4 biological replicates and AF measurements are averaged across n = 2 biological replicates. For all other IVDs NP and AF measurements are averaged across n = 4 biological replicates. Students t-test between NP and AF indicate significance if *p < 0.05. The average % GAG and GAG by dry weight of all 6 specimens is represented in the last two bars of each graph, labelled as “All”.

Fig. 5

CS disaccharides, C4S and C6S, in the (a) NP and (b) AF. HS disaccharides, H0S and H2S, in the (c) NP (d) AF. Grey doted line represents the GAG composition of the young specimen, n = 1. Red line represents the average of all IVDs, n = 6. The black solid line represents the average of all IVDs except the young 9 year old IVD, n = 5. Data are represented as mean ± SD for the GAG content in ng GAG/mg dry tissue weight for the red and black lines. The grey doted line represents the average of the biological replicates for the one 9 year old IVD in the NP and AF.

Fig. 6

Principal component analysis (PCA) of all the IVD samples from different patients based on their total GAG composition, HS disaccharide composition and CS disaccharide composition. (a) PCA loadings indicating the coefficients of principal component (PC) 1 and PC2 for the GAG components and the disaccharide components. (b) PCA scores of the IVD samples on principal component (PC) 1 and PC2. Samples were grouped by patients. (c) PCA scores of the IVD samples on PC 1 and PC2. Samples were grouped for different regions by colour considering anterior / lateral / posterior AF regions or nucleus pulposus (NP) regions. (d) PCA scores of the IVD samples on PC 1 and PC2, samples were grouped by inner / outer AF regions or the NP regions.

Fig. 7

Principal component analysis (PCA) of the IVD samples from different IVD regions based on the total GAG compositions. Panel (a) and (b) are the same set of results distinguished by different groups. (a) Posterior, anterior and lateral AF regions were highlighted. (b) Inner and outer AF regions were highlighted.