Data in support for the measurement of heparan sulfate and dermatan sulfate by LC-MS/MS analysis.
ABSTRACT: This article provides supplementary data for the paper "LC-MS/MS method for simultaneous quantification of heparan sulfate and dermatan sulfate in urine by butanolysis derivatization" (Forni et al., 2018). Several parameters were tested to optimize sample preparation by butanolysis in order to carry out simultaneous quantifications of HS and DS by tandem mass spectrometry. Here we describe step-by-step instructions to perform HS and DS analysis in urine samples using external calibration curves of standards of known concentration. Sample are quantified by interpolation from the calibration curve and reported in µg/mL. Then, HS and DS are normalized to creatinine concentration and reported as mg/g uCr.
Project description:Mucopolysaccharidoses (MPSs) are lysosomal storage disorders caused by deficiencies of enzymes involved in the catabolism of glycosaminoglycans (GAGs). Various treatments such as enzyme replacement therapy and/or hematopoietic stem cell transplant are available for MPSs. Early initiation of treatment improves the outcome and delays the onset of symptoms, highlighting the need for newborn screening for MPSs. The main objective of this project was to devise and validate a multiplex urine filter paper method for GAG analysis using a tandem mass spectrometry (MS/MS) approach to screen newborns for MPSs. Eluted urine samples from 21-day-old newborns were evaporated and a methanolysis reaction was performed. Samples were resuspended and analyzed using a UPLC-MS/MS system. A one-minute chromatographic method allowed the absolute quantification of heparan sulfate (HS), dermatan sulfate (DS), and creatinine. Method validation revealed high precision (< 9% relative standard deviation) and accuracy (< 7% bias) for all analytes. The reference values normalized to creatinine obtained by the analysis of five hundred 21-day-old newborn urine samples were 34.6 +/-6.2 mg/mmol of creatinine and 17.3 +/-3.9 mg/mmol of creatinine for HS and DS, respectively. We present a rapid and efficient method for populational newborn urine screening using MS/MS, which could also be applied to high-risk screening.
Project description:Chemokines play diverse roles in human pathophysiology, ranging from trafficking leukocytes and immunosurveillance to the regulation of metabolism and neural function. Chemokine function is intimately coupled to binding tissue glycosaminoglycans (GAGs), heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS). Currently, very little is known about how the structural features and sequences of a given chemokine, the structure and sulfation pattern of a given GAG, and structural differences among GAGs and among chemokines impact binding interactions. In this study, we used solution NMR spectroscopy to characterize the binding interactions of two related neutrophil-activating chemokines, CXCL1 and CXCL5, with HS, CS, and DS. For both chemokines, the dimer bound all three GAGs with higher affinity than did the monomer, and affinities of the chemokines for CS and DS were lower than for HS. NMR-based structural models reveal diverse binding geometries and show that the binding surfaces for each of the three GAGs were different between the two chemokines. However, a given chemokine had similar binding interactions with CS and DS that were different from HS. Considering the fact that CXCL1 and CXCL5 activate the same CXCR2 receptor, we conclude that GAG interactions play a role in determining the nature of chemokine gradients, levels of free chemokine available for receptor activation, how chemokines bind their receptors, and that differences in these interactions determine chemokine-specific function.
Project description:Heparan sulfate (HS) mediates a wide range of protein binding interactions key to normal and pathological physiology. Though liquid chromatography coupled with mass spectrometry (LC-MS) based disaccharide composition analysis is able to profile changes in HS composition, the heterogeneity of modifications and the labile sulfate group present major challenges for liquid chromatography tandem mass spectrometry (LC-MS/MS) sequencing of the HS oligosaccharides that represent protein binding determinants. Here, we report online LC-MS/MS sequencing of HS oligosaccharides using hydrophilic interaction liquid chromatography (HILIC) and negative electron transfer dissociation (NETD). A series of synthetic HS oligosaccharides varying in chain length (tetramers and hexamers), number of sulfate groups (3-7), sulfate patterns (sulfate positional isomers), and uronic acid epimerization (epimers) were separated and sequenced. The LC elution order of isomeric compounds was associated with their fine structure. The application of an online cation exchange device (ion suppressor) enhanced the precursor charge states, and the subsequent NETD produced abundant glycosidic fragments, allowing the characterization of both lowly sulfated and highly sulfated HS oligosaccharides. Furthermore, the diagnostic cross-ring ions differentiated the 6-O sulfation and 3-O sulfation, allowing unambiguous structural assignment. Collectively, this LC-NETD-MS/MS method is a powerful tool for sequencing of heterogeneous HS mixtures and is applicable for the differentiation of both isomers and epimers, for the characterization of saccharide mixtures with a varying extent of sulfation and even for the determination of both predominant and rare modification motifs. Thus, LC-NETD-MS/MS has great potential for further application to biological studies.
Project description:Chondroitin/dermatan sulfate (CS/DS) proteoglycans consist of unbranched sulfated polysaccharide chains of repeating GalNAc-GlcA/IdoA disaccharide units, attached to serine residues on specific proteins. The CS/DS proteoglycans are abundant in the extracellular matrix where they have essential functions in tissue development and homeostasis. In this report a phylogenetic analysis of vertebrate genes coding for the enzymes that modify CS/DS is presented. We identify single orthologous genes in the zebrafish genome for the sulfotransferases chst7, chst11, chst13, chst14, chst15 and ust and the epimerase dse. In contrast, two copies were found for mammalian sulfotransferases CHST3 and CHST12 and the epimerase DSEL, named chst3a and chst3b, chst12a and chst12b, dsela and dselb, respectively. Expression of CS/DS modification enzymes is spatially and temporally regulated with a large variation between different genes. We found that CS/DS 4-O-sulfotransferases and 6-O-sulfotransferases as well as CS/DS epimerases show a strong and partly overlapping expression, whereas the expression is restricted for enzymes with ability to synthesize di-sulfated disaccharides. A structural analysis further showed that CS/DS sulfation increases during embryonic development mainly due to synthesis of 4-O-sulfated GalNAc while the proportion of 6-O-sulfated GalNAc increases in later developmental stages. Di-sulfated GalNAc synthesized by Chst15 and 2-O-sulfated GlcA/IdoA synthesized by Ust are rare, in accordance with the restricted expression of these enzymes. We also compared CS/DS composition with that of heparan sulfate (HS). Notably, CS/DS biosynthesis in early zebrafish development is more dynamic than HS biosynthesis. Furthermore, HS contains disaccharides with more than one sulfate group, which are virtually absent in CS/DS.
Project description:Glycosaminoglycans (GAGs) play important roles on the regulation of extracellular signaling, neuronal development, and cartilage maintenance. The extracellular concentration of total GAGs has been used as an established measure for the diagnosis of mucopolysaccharidoses (MPSs). Heparan sulfate (HS), Dermatan sulfate (DS) and chondroitin sulfate are known to be elevated in the GAGs under pathological conditions associated with MPS. Furthermore, the selective accumulation of disease-specific one of, or a combination of, them has also been used for the estimation of subtypes of MPS. A previously developed method [Auray-Blais C et al. Molecular Genetics and Metabolism 102 (2011) 49-56.] measures the concentration of GAGs using liquid chromatography with tandem mass spectrometry (LC-MS/MS) with higher precision. To ask whether the selective accumulation of HS and DS in the urine of MPS II patients discriminate the attenuated and severe type of MPS II, we examined the concentrations of HS and DS by this methodology. Compared to the healthy controls, we found a marked elevation of HS and DS in all of the MPS II-affected patients. Among patients who received ERT with confirmed elevation of antibody titer, the concentrations of HS in the urine of patients with attenuated type were lower than those with severe type of MPS II. In these patients, the concentrations of DS by LC-MS/MS and of total GAG by DMB failed to depend on the accumulation of antibody. These results suggest that the LC-MS/MS method employed in this study might discriminate the subtypes of MPS II in different clinical background.
Project description:Mucopolysaccharidosis (MPS) is caused by the deficiency of a specific hydrolytic enzyme that catalyzes the step-wise degradation of glycosaminoglycans (GAGs). In this study, we propose an empirical method to calculate levels of GAG-derived disaccharides based on the quantity (peak areas) of chondroitin sulfate (CS) with the aim of making a diagnosis of MPS more accurate and reducing the occurrence of false positive and false negative results. In this study, levels of urinary GAG-derived disaccharides were measured in 67 patients with different types of MPS and 165 controls without MPS using a tandem mass spectrometry assay. Two different methods of reporting GAG-derived disaccharides were assessed; normalization to urinary CS (in ?g/mL), and normalization to ?g/mg creatinine. CS-normalization yielded more consistent values than creatinine-normalization. In particular, levels of urinary dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) significantly varied because of changes in urine creatinine levels, which were proportional to age but inversely proportional to DS, HS, and KS measurements. Using CS-normalization revealed the actual status of DS, HS, and KS without the influence of factors such as age, urine creatinine, and other physiological conditions. It could discriminate between the patients with MPS and controls without MPS, and also to evaluate changes in GAG levels pre- and post-enzyme replacement therapy.
Project description:Microfluidic chip-based hydrophilic interaction chromatography (HILIC) is a useful separation system for liquid chromatography-mass spectrometry (LC-MS) in compositional profiling of heparan sulfate (HS) oligosaccharides; however, ions observed using HILIC LC-MS are low in charge. Tandem MS of HS oligosaccharide ions with low charge results in undesirable losses of SO(3) from precursor ions during collision induced dissociation. One solution is to add metal cations to stabilize sulfate groups. Another is to add a nonvolatile, polar compound such as sulfolane, a molecule known to supercharge proteins, to produce a similar effect for oligosaccharides. We demonstrate use of a novel pulsed makeup flow (MUF) HPLC-chip. The chip enables controlled application of additives during specified chromatographic time windows and thus minimizes the extent to which nonvolatile additives build up in the ion source. The pulsed MUF system was applied to LC-MS/MS of HS oligosaccharides. Metal cations and sulfolane were tested as additives. The most promising results were obtained for sulfolane, for which supercharging of the oligosaccharide ions increased their signal strengths relative to controls. Tandem MS of these supercharged precursor ions showed decreased abundances of product ions from sulfate losses yet more abundant product ions from backbone cleavages.
Project description:The biosynthesis of heparan sulfate proteoglycans is tightly regulated by multiple feedback mechanisms, which support robust developmental systems. One of the regulatory network systems controlling heparan sulfate (HS) biosynthesis is sulfation compensation. A previous study using Drosophila HS 2-O- and 6-O-sulfotransferase (Hs2st and Hs6st) mutants showed that loss of sulfation at one position is compensated by increased sulfation at other positions, supporting normal FGF signaling. Here, we show that HS sulfation compensation rescues both Decapentaplegic and Wingless signaling, suggesting a universal role of this regulatory system in multiple pathways in Drosophila. Furthermore, we identified Sulf1, extracellular HS 6-O-endosulfatase, as a novel component of HS sulfation compensation. Simultaneous loss of Hs2st and Sulf1 led to 6-O-oversulfation, leading to patterning defects, overgrowth, and lethality. These phenotypes are caused at least partly by abnormal up-regulation of Hedgehog signaling. Thus, sulfation compensation depends on the coordinated activities of Hs2st, Hs6st, and Sulf1.
Project description:Diagnosis of the mucopolysaccharidoses (MPSs) generally relies on an initial analysis of total glycosaminoglycan (GAG) excretion in urine. Often the dimethylmethylene blue dye-binding (DMB) assay is used, although false-negative results have been reported. We report a multiplexed diagnostic test with a high sensitivity for all MPSs and with the potential to identify patients with I-cell disease (ML II) and mucolipidosis III (ML III).Urine samples of 100 treatment naive MPS patients were collected and analyzed by the conventional DMB assay and a multiplex assay based on enzymatic digestion of heparan sulfate (HS), dermatan sulfate (DS) and keratan sulfate (KS) followed by quantification by LC-MS/MS. Specificity was calculated by analyzing urine samples from a cohort of 39 patients suspected for an inborn error of metabolism, including MPSs.The MPS cohort consisted of 18 MPS I, 16 MPS II, 34 MPS III, 10 MPS IVA, 3 MPS IVB, 17 MPS VI and 2 MPS VII patients. All 100 patients were identified by the LC-MS/MS assay with typical patterns of elevation of HS, DS and KS, respectively (sensitivity 100%). DMB analysis of the urine was found to be in the normal range in 10 of the 100 patients (sensitivity 90%). Three out of the 39 patients were identified as false-positive, resulting in a specificity of the LS-MS/MS assay of 92%. For the DMB this was 97%. All three patients with MLII/MLIII had elevated GAGs in the LC-MS/MS assay while the DMB test was normal in 2 of them.The multiplex LC-MS/MS assay provides a robust and very sensitive assay for the diagnosis of the complete spectrum of MPSs and has the potential to identify MPS related disorders such as MLII/MLIII. Its performance is superior to that of the conventional DMB assay.
Project description:Biosynthesis of heparan sulfate (HS) involves conversion of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) units catalyzed by glucuronyl C5-epimerase (Hsepi). IdoA units are the favored substrate for 2-O-sulfotransferase (2OST). We used HEK293 cells as a model to investigate the effects of overexpression of these enzymes on HS structure. Overexpression of Hsepi alone resulted in an unexpected increase in HS chain length. A Hsepi point-mutant (Y168A), devoid of catalytic activity, failed to affect chain length. Moreover, the effect of Hsepi overexpression on HS chain length was abolished by simultaneous overexpression of 2OST. These findings raise novel aspects on regulation of HS biosynthesis. We propose a hypothetical enzyme-binding protein (EBP) with distinct, specific and partly overlapping binding sites, the interactions of which will determine levels of enzymes available to the biosynthetic process.