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Heparan sulfate is synthesized by most animal cells and interacts with numerous proteins via specific sulfation motifs to regulate various physiological processes. Various 3-O-sulfated motifs are considered to be key in controlling the binding specificities to the functional proteins. One such motif synthesized by 3-O-sulfotransferase-1 (3OST-1) serves as a binding site for antithrombin (AT) and has been thoroughly studied because of its pharmacological importance. However, the physiological roles of 3-O-sulfates produced by other 3OST isoforms, which do not bind AT, remain obscure, in part due to the lack of a standard method to analyze this rare modification. This study aims to establish a method for quantifying 3-O-sulfated components of heparan sulfate, focusing on non-AT-binding units. We previously examined the reaction products of human 3OST isoforms and identified five 3-O-sulfated components, including three non-AT-binding disaccharides and two AT-binding tetrasaccharides, as digestion products of heparin lyases. In this study, we prepared these five components as a standard saccharide for HPLC analysis. Together with eight non-3-O-sulfated disaccharides, a standard mixture of 13 units was prepared. Using reverse-phase ion-pair HPLC with a postcolumn fluorescent labeling system, the separation conditions were optimized to quantify the 13 units. Finally, we analyzed the compositional changes of 3-O-sulfated units in heparan sulfate from P19 cells before and after neuronal differentiation. We successfully detected the 3-O-sulfated units specifically expressed in the differentiated neurons. This is the first report that shows the quantification of three non-AT-binding 3-O-sulfated units and establishes a new approach to explore the physiological functions of 3-O-sulfate., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Mucopolysaccharidoses (MPS) are caused by deficiency of lysosomal enzyme activities needed to degrade glycosaminoglycans (GAGs), which are long unbranched polysaccharides consisting of repeating disaccharides. GAGs include: chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and hyaluronan. Their catabolism may be blocked singly or in combination depending on the specific enzyme deficiency. There are 11 known enzyme deficiencies, resulting in seven distinct forms of MPS with a collective incidence of higher than 1 in 25,000 live births. Accumulation of undegraded metabolites in lysosomes gives rise to distinct clinical syndromes. Generally, the clinical conditions progress if untreated, leading to developmental delay, systemic skeletal deformities, and early death. MPS disorders are potentially treatable with enzyme replacement therapy or hematopoietic stem cell transplantation. For maximum benefit of available therapies, early detection and intervention are critical. We recently developed a novel high-throughput multiplex method to assay DS, HS, and KS simultaneously in blood samples by using high performance liquid chromatography/tandem mass spectrometry for MPS. The overall performance metrics of HS and DS values on MPS I, II, and VII patients vs. healthy controls at newborns were as follows using a given set of cut-off values: sensitivity, 100%; specificity, 98.5-99.4%; positive predictive value, 54.5-75%; false positive rate, 0.62-1.54%; and false negative rate, 0%. These findings show that the combined measurements of these three GAGs are sensitive and specific for detecting all types of MPS with acceptable false negative/positive rates. In addition, this method will also be used for monitoring therapeutic efficacy. We review the history of GAG assay and application to diagnosis for MPS., (Copyright © 2013 Elsevier Inc. All rights reserved.)