Abstract
Glycosaminoglycans (GAGs) are distributed in the whole body and play a variety of important physiological roles associated with inflammation, growth, coagulation, fibrinolysis, lipolysis, and cell-matrix biology. Accumulation of undegraded GAGs in lysosomes gives rise to a distinct clinical syndrome, mucopolysaccharidoses. Measurement of each specific GAG in a variety of specimens is urgently required to understand GAG interaction with other molecules, physiological status of patients, and prognosis and pathogenesis of the disease.We established a highly sensitive and accurate tandem mass spectrometry (LC-MS/MS) method for measurements of disaccharides derived from four specific GAGs [dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and chondroitin sulfate (CS)]. Disaccharides were produced by specific enzyme digestion of each GAG, and quantified by negative ion mode of multiple reaction monitoring. Subclasses of HS and GAGs with identical molecular weights can be separated using a Hypercarbcolumn (2.0 mm×50 mm, 5 μm) with an aectonitrile gradient in ammonium acetate (pH 11.0).We also developed a GAG assay by RapidFire with tandem mass spectrometry (RF-MS/MS). The RF system consists of an integrated solid phase extraction robot that binds and de-salts samples from assay plates and directly injects them into a MS/MS detector, reducing sample processing time to ten seconds. RF-MS/MS consequently yields much faster throughput than conventional LC-MS/MS-based methods.However, the RF system does not have a chromatographic step, and therefore, cannot distinguish GAGs that have identical molecular weights. Both methods can be applied to analysis of dried blood spots, blood, and urine specimens.In this article, we compare the assay methods for GAGs and describe their potential applications.
Highlights
Glycosaminoglycans (GAGs) comprise chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and hyaluronan
Disaccharide GAGs assayed by LC-MS/MS have been used for diagnosis, screening, prognosis of clinical severity, and assessment of therapeutic efficacy in human patients and animal models with MPS [2,9,10,11,12,22,24,25,26,27]
Our studies have demonstrated that an RF-MS/MS platform is capable of detecting disaccharides of ΔDiHS-0S, ΔDiHS-NS, ΔDi-4S/6S, and KS with similar reproducibility, sensitivity, and specificity as LCMS/MS [3,4,5,6,7,8,9,10], suggesting that the LC component may not be essential for detection and identification of these disaccharides
Summary
Glycosaminoglycans (GAGs) comprise chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and hyaluronan. The intra-assay precision values/coefficient of variation (CV) determined from analysis of ΔDiHS-0S, ΔDiHS-NS, ΔDiHS-6S, ΔDi-6S, and ΔDi-4S for control serum are less than 8.9, 1.7, 11.7, 11.9, and 12.1%, respectively. The intra-assay precision values/CVs for Galβ1-4GlcNAc(6S) and Gal(6S)β1-4GlcNAc(6S) in control serum were less than 8.2 and 5.3% andinter-assay precision values/CVs were less than 6.6 and 5.7% These results demonstrate the reproducibility and accuracy of the method. The moderate correlation in serum ΔDiHS-NS and ΔDiHS-0S measurements of control subjects between LC-MS/MS and RF-MS/MS indicates that results from each assay are comparable (Figure 9). Disaccharide GAGs assayed by LC-MS/MS have been used for diagnosis, screening, prognosis of clinical severity, and assessment of therapeutic efficacy in human patients and animal models with MPS [2,9,10,11,12,22,24,25,26,27]. A clear difference in Di-4S level between a rat with MPS VI and a control rat was observed (Figure 11)
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