Glycobiology Advance Access originally published online on April 20, 2005
Glycobiology 2005 15(8):818-826; doi:10.1093/glycob/cwi064
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Compositional profiling of heparin/heparan sulfate using mass spectrometry: assay for specificity of a novel extracellular human endosulfatase
3 Department of Chemistry, University of California, Berkeley, CA 94720; 4 Department of Anatomy, University of California, San Francisco, CA 94143; 5 UCSF Comprehensive Cancer Center, University of California, San Francisco, CA 94143; 6 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; 7 Howard Hughes Medical Institute, University of California, Berkeley, CA 94720
1 To whom correspondence should be addressed; e-mail: jaleary{at}ucdavis.edu
2 Present address: Department of Chemistry and Division of Molecular and Cellular Biology, Genome Center, University of California Davis, One Shields Road, Davis, CA 95616
Received on February 4, 2005; revised on April 13, 2005; accepted on April 14, 2005
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Abstract |
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An important class of carbohydrates studied within the field of glycobiology, heparin and heparan sulfate (HS) have been implicated in a diverse array of biological functions. Changes in their sulfation pattern and domain organization have been associated with different pathological situations such as viral infectivity, tumor growth, and metastasis. To obtain structural information about these biomolecules, and the modifications they may undergo during different stages of cell growth and development, a mass spectrometry-based method was developed and used to obtain unambiguous structural information on the glycosaminoglycans (GAGs) that comprise heparin/HS. The method was applied to assay for the heparin substrate specificity of a newly discovered human extracellular endosulfatase, HSulf-2, which has been implicated in tumorigenesis. This new protocol incorporates 12 known heparin disaccharides, including three sets of isomers. A unique response factor (R) is determined for each disaccharide, whereas a multiplexed and data processing method is incorporated for faster data acquisition and quantification purposes. Proof of principle was performed by using various heparin/HS samples isolated from bovine and porcine tissues.
Key words: glycosaminoglycans / heparan sulfate / heparin / mass spectrometry / sulfatase assay
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary materialAcknowledgmentsReferences |
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The glycosaminoglycans (GAGs), heparin and heparan sulfate (HS), are highly charged, sulfated polysaccharides, which can be found on the cell surface and in the extracellular matrix. There is a recent, rising interest in studying the structure and function of heparin/HS GAGs, as potential tumor growth suppressors (Liu et al., 2002
), and for optimizing the therapeutic use of low molecular weight heparin (LMWH) oligosaccharides for cardiovascular disease and thrombosis (Shriver et al., 2000). Current methodologies to determine disaccharide composition of these GAGs focus on the enzymatic depolymerization of heparin/HS, followed by the separation of the disaccharide constituents by high performance liquid chromatography (HPLC) (Kerns and Linhardt, 1995; Murata et al., 1995; Volpi et al., 1995; Karamanos et al., 1997; Kinoshita and Sugahara, 1999; Vives et al., 1999) or capillary electrophoresis (CE) (Desai et al., 1993a; Ruiz-Calero et al., 1998; Mao et al., 2002; Militsopoulou et al., 2002), and detection and quantification by ultraviolet (UV) absorption. This process can sometimes be time-consuming, and occasionally requires an additional derivatization step before separation. Therefore, it remains of interest to have a fast, accurate, and complementary method for determining the compositional analysis of any heparin/HS sample. Such samples can include smaller heparin oligosaccharides, where the composition information can be used toward complete sequence analysis, or for larger species, to characterize LMWH or unfractionated heparin/HS from various lot preparations or tissue sources.
Herein, we describe a method by using electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) to incorporate the compositional analysis of 12 known heparin disaccharides, including three sets of isomers. This method serves as an initial characterization step for profiling heparin/HS from different sources, for assaying new enzymes with putative glycosaminoglycan substrates, as well as a tool to provide a quick estimate of how much heparin/HS material is available. The method was applied to several biological sources of these saccharides including unfractionated heparin/HS from various porcine and bovine tissues, and it was also utilized to assay for the activity of a novel extracellular human sulfatase, HSulf-2, against different heparin substrates. The expression levels of this recently cloned endoglucosamine 6-sulfatase were found to be higher in certain cancers (breast, central nervous system, and colon), and it has been implicated in the regulation of growth during tumorigenesis, making it a very interesting target for further characterization (Morimoto-Tomita et al., 2003).
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary materialAcknowledgmentsReferences |
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Heparin/HS GAGs are highly negatively charged, linear polysaccharides composed of the variably sulfated disaccharide units [-4HexAß(1,4)-GlcNS
1-]n (Varki et al., 1999). Depolymerization of these polysaccharides, using a combination of the bacterially derived enzymes, heparin lyases I, II, and III, from flavobacterium heparinum, yields C4-C5 unsaturated uronic acid–glucosamine disaccharides, which may be modified by N- and O-sulfation (6-O- and 3-O-sulfation of the glucosamine and 2-O-sulfation of the uronic acid) (Figure 1). Heparin/HS have considerable heterogeneity in molecular size, disaccharide composition, and sulfate content, all which can vary according to the tissue and species of origin, as well as being continuously regulated to control the binding of various growth and differentiation factors during cell growth and development (Esko and Selleck, 2002). Consequently, analysis of the unsaturated heparin disaccharide composition can give specific, quantitative results for the characterization of any changes in sulfation patterns and may provide evidence indicative of tissue and cell status. A combination of ESI-MS and MS/MS was used in this study for the analysis of the constituent heparin disaccharides.
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Heparin isomeric disaccharides are differentiated and mixtures quantified by ESI-MS/MS
At least 12 heparin disaccharide structures with varying degrees of sulfation have been identified as constituents of heparin/HS. The structures of these disaccharides, which are used in the developed method, are shown in Figure 1. Four of the twelve disaccharides, IA, IVA, IS, and IVH, are distinguished directly by their mass-to-charge ratios. The three sets of isomers, which comprise the remaining eight disaccharides (IIA/IIIA, IIS/IIIS/IH, and IIH/IIIH/IVS), can also be differentiated, from the resulting product ion spectra generated upon collision-induced dissociation (CID) of each molecular ion (Saad and Leary, 2004). A summary of these product ion spectra is provided in Table I. Ions shown in bold are those determined to be diagnostic for the presence of each particular disaccharide, and their contributions in the MS2 spectrum are directly monitored for quantification purposes.
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For each disaccharide isomer, reproducibility was one of the criteria used to identify ideal diagnostic ions or combinations thereof (Supplementary Table I). Relative abundances of these diagnostic ions when incorporated into a system of equations could then be used to determine the relative amounts of each component in any given mixture (Saad and Leary, 2003). To demonstrate the utility of this quantification method for the heparin isomeric disaccharides, a series of two- and three-component mixtures were prepared for each set of isomers, with variable concentrations of each isomer. These were analyzed in triplicate, and the percent of each disaccharide in the mixture was calculated (Supplementary Table II). The error for all of the samples was small (
3.3%), establishing the utility of the method for this system of heparin disaccharides.
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Complete quantification of 12 heparin disaccharides using four ESI-MS/MS experiments
For the compositional analysis of any mixture of the 12 heparin disaccharides, the concentration of each nonisomeric disaccharide is calculated based on a single-point response factor (R) relating the ratio of the peak area of a disaccharide’s extracted ion chromatogram (XIC) to that of an internal standard, 
UA2S-GlcNCOEt6S (I-P), in the solution. For the isomers, a second step for determining the relative amounts of each isomer is also incorporated utilizing the contributions of diagnostic ions from three MS2 experiments. To optimize this approach for speed and throughput, the four experiments required for complete quantification of all 12 heparin disaccharides were integrated into one instrument method by using ThermoElectron’s Xcalibur 1.2 software (San Jose, CA). This was accomplished by using a combination of four scan events, which include an MS1 profile experiment, followed by three MS2 experiments on precursor ions at m/z 247.7, 416.1, and 458.1, respectively (Figure 2). After combining this information, the final step normalizes the composition data to 100% (see also Supplementary methods).
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The complete quantification procedure was shown to work quite effectively over a broad range of sample compositions, and the accuracy of the method was tested by using several mock mixtures. Two representative examples of the results are summarized in Table II. Each value reported in Table II was determined by using the series of one-point R factors for the disaccharides and represents the average of three measurements. The compositions of the mock mixtures shown correspond to compositional profiles similar to that expected from HS (mixture 1) and heparin digests (mixture 2), respectively (Militsopoulou et al., 2002), and the largest deviation in the determination of the amount of any of the disaccharides was 2.6%.
Application to the quantification of heparin/HS samples
Having demonstrated the effectiveness of the quantification method on mock mixtures, it was then applied to the analysis of the following commercially available samples from five different biological sources: heparin from bovine intestinal mucosa and porcine intestinal mucosa, LMWH from porcine intestine, and heparin from bovine lung, as well as HS from bovine kidney. To determine disaccharide composition, the exhaustive enzymatic digestions of these samples were carried out overnight (16 h) in 20 mM ammonium acetate, 2 mM Ca(OAc)2 with 0.01 unit of each heparin lyases I, II, and III (Saad and Leary, 2003). Under these conditions, the digestions worked effectively, and no purification was required before mass spectrometric analysis. The resulting digests were quantitatively analyzed, and the disaccharide compositional profiles determined for the five samples as summarized in Table III. As a representative example, the mass spectra obtained for the HS sample from bovine kidney are shown in Figure 3.
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); (c) MS2 spectrum (m/z 458.1
HS is generally less sulfated than heparin and with lower l-iduronic acid (IdoA) content, thus presenting greater structural variability (Gallagher, 1997). For instance, the unsulfated disaccharide, IVA (m/z 378.1), is present in the largest amount of the total disaccharide composition, 50.1%, whereas the trisulfated disaccharide, IS (m/z 191.4), is very low in concentration, 6.4% (Figure 3a and Table III). Also, there are notable ion contributions at m/z 247.7, 458.1, and 416.1, which can correspond to any mixture of the disaccharide isomers. Collisional activation of the ion at m/z 247.7, isomeric for the disaccharides IIS, IIIS, and IH (Figure 3b), produces product ions at m/z 97, 168.6, and 218. Each of these ions is diagnostic (Table I), indicating that a mixture of the three disulfated disaccharide isomers must be present, and specifically at a ratio of 22.3:12.5:1, for IIS, IIIS, and IH, respectively. Similarly, the consideration of the MS2 spectrum of m/z 458.1 (Figure 3c) shows a diagnostic product ion at m/z 357, but no ion at m/z 237, the diagnostic ion for the IIIA disaccharide. Therefore, we can conclude that no disaccharide IIIA is present in bovine kidney HS, only disaccharide IIA contributes to the ion intensity at m/z 458.1 in Figure 3a. In contrast, the product ion spectrum obtained upon CID of m/z 416.1 (Figure 3d) indicates that the major monosulfated disaccharide species present is disaccharide IVS, based on the presence of the diagnostic product ion at m/z 138. However, the ion contribution at m/z 258 also indicates the presence of disaccharide IIH, whereas the lack of an ion at m/z 237 suggests that no disaccharide IIIH is present in the sample (see Table I for diagnostic ions). Disaccharide compositional analyses were similarly evaluated for all other heparin samples summarized in Table III.
Assay for HSulf-2 activity: a mammalian extracellular heparin-degrading endosulfatase
Sulfatases are a group of enzymes catalyzing the hydrolysis of sulfate ester bonds from a wide variety of substrates, ranging from small molecule steroid sulfates to GAG. The protein-designated HSulf-2 was recently cloned by Morimoto-Tomita et al. and found to be endoproteolytically processed in the secretory pathway and secreted into the extracellular condition medium of transfected Chinese hamster ovary cells (Morimoto-Tomita et al., 2002). Furthermore, its expression is up-regulated in MCF-7 breast cancer cells, where it is also secreted into the condition medium (Morimoto-Tomita, M., Uchimura, K., Bistrup, A., Lum, D.H., Egeblad, M., Boudreau, N., Werb, Z., Rosen, S.D., manuscript submitted for publication). This enzyme’s activity was assayed and found to exhibit both arylsulfatase activity and highly specific endoglucosamine-6-sulfatase activity against intact heparin.
Herein, the MS methodology developed was applied to assay for the activity of the sulfatase, HSulf-2, against various heparin substrates. The procedure for the assay was modified from the method published by Morimoto-Tomita et al. (2002), for use with our MS approach. Final conditions of 20 mM triethanolamine buffer at pH 8.0 with 0.625 M Mg(OAc)2 were found optimal for direct MS analysis without sacrificing enzyme activity. Under these conditions, three important features should be noted: (1) the HSulf-2 enzyme’s activity remained the same in comparison with previous conditions in N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES) buffer with MgCl2, as measured against the model substrate 4-methylumbelliferyl sulfate (4-MUS), (2) the heparin lyases used in the final depolymerization step of heparin substrates were not impaired by the presence of the triethanolamine or Mg(OAc)2 from the first reaction step, and (3) the mass spectra acquired of mixtures of heparin disaccharides obtained under these reaction conditions did not show significant suppression of ionization by higher salt concentrations or matrix effects. Substrate samples were first treated with HSulf-2, followed by digestion with heparin lyases I, II, and III. Optimal MS results were achieved when reactions were quenched into 1:1 acetonitrile/water solutions containing 10 mM ammonium hydroxide.
To assay for HSulf-2 activity, intact heparin was first treated with conditioned medium (CM) from MCF-7 cells. The described MS method was then used for disaccharide compositional analysis to evaluate the enzyme’s activity on specific sulfation modifications. As seen from the results in Figure 4, HSulf-2-conditioned media produced an obvious decrease in the amount of the trisulfated disaccharide unit, IS, and a corresponding increase in the ion at m/z 247.7. Collision-induced dissociation of m/z 247.7 confirmed that this increase was specifically from the IIIS disaccharide isomer, formed from having lost the 6-O-sulfate ester. The quantitative results of the assay are shown in Figure 4c. By using the MS-based method to assay for HSulf-2 activity, it was shown that HSulf-2 mainly acts endolytically on the trisulfated disaccharide species within heparin, reducing the amount of disaccharide, IS, from 49 to 31% of the heparin composition. This confirms data obtained previously by using an indirect HPLC-UV assay (Morimoto-Tomita et al., 2002). Evidence was observed that the enzyme may also act to a much lesser extent on another disaccharide motif within the heparin molecule, the 6-N-sulfated disaccharide. This is indicated by the small reduction in the amount of IIS disaccharide seen and the increase in the amount of IVS disaccharide present (Figure 4c).
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The endolytic activity of HSulf-2 on several smaller substrates was also investigated as summarized in Table IV. These include LMWH (consisting of 10–12 disaccharide subunits), an isolated fully sulfated, unsaturated decasaccharide from heparin (MW 2861), as well as a fully sulfated tetrasaccharide of structure UA2S-GlcNS6S-Ido2S-GlcNS6S (MW 1145), and the disaccharide IS. No desulfation product was observed with the disaccharide IS unit upon extended reaction times (>15 h). For LMWH, there was a 36% decrease of IS subunits upon desulfation, very similar to heparin’s 37% decrease. The decasaccharide substrate showed an even greater decrease of 47% in IS subunits, and in both cases, a corresponding increase in disaccharide IIIS was observed. Interestingly, the smallest substrate of HSulf-2 was found to be the tetrasaccharide substrate, and this was desulfated to a much lesser extent (14% decrease of IS).
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Discussion |
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Although heparin has been used clinically as an anticoagulant for >60 years, it has been shown only recently that specific structures within heparin and HS proteoglycans regulate the activities of many enzymes, growth factors, and other extracellular matrix proteins (Bernfield et al., 1999
). Structural characterization of these GAGs is of particular interest. It usually involves enzymatic depolymerization by bacterial heparin lyases and analysis of the products using various analytical techniques such as HPLC, electrophoresis, nuclear magnetic resonance, or MS (Desai et al., 1993a; Kerns and Linhardt, 1995; Murata et al., 1995; Volpi et al., 1995; Karamanos et al., 1997; Ruiz-Calero et al., 1998; Kinoshita and Sugahara, 1999; Vives et al., 1999; Ruiz-Calero et al., 2001; Kuberan et al., 2002; Militsopoulou et al., 2002; Ruiz-Calero et al., 2002). Each of these cases generally requires a liquid chromatography separation step whose resolution is optimized at the expense of longer run times, as well as the equilibration times usually required pre- and postruns. Our group and others have sought to exploit the use of MS with its sensitivity and speed by avoiding separation steps for the analysis of GAGs (Desaire and Leary, 2000a; Saad and Leary, 2003; Zaia and Costello, 2003; Zamfir et al., 2002, 2003; Saad and Leary, 2004). Herein, we have described a convenient, sensitive, and accurate method to apply for the compositional analysis of several different heparin/HS samples after enzymatic depolymerization. Incorporating all 12 heparin disaccharides, the determination of R factors and normalization factors required for quantification is routinely accomplished from only four samples, and run within minutes utilizing the described multiscan event instrument method. This is done directly before the analysis of biological samples, whereas traditionally, a series of calibration curves would require considerably more sample consumption and time to run. Furthermore, any additional sample then requires only a few minutes analysis time and provides accurate and precise results.
With the growing interest in the study of heparin/HS structure and function, the applications of this method are wide ranging. For example, the reported compositional analysis of disaccharides is a useful means to evaluate the structure of different LMWH species, which are generated and evaluated for clinical use as well as their possible anticancer properties (Sasisekharan et al., 2002). The usefulness of this ESI-MS method is underlined by the wide availability of the ion trap MS, reproducibility of the CID spectra obtained, as well as the advantages of speed and sensitivity of the MS analysis. After validating the presented method by analyzing various heparin samples, it was modified to develop a direct assay for the recently discovered endosulfatase, HSulf-2. Desulfation of different substrates, ranging from heparin to molecules as small as a tetrasaccharide, was monitored. As expression levels of this extracellular sulfatase are higher in certain tumor cells, the enzyme might function in the regulation of growth factors during tumor formation, and therefore, as a therapeutic target of high interest (Morimoto-Tomita et al., 2003). The described MS assay could serve as a diagnostic tool to detect tumors at an early stage.
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Materials and methods |
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General materials and methods
All heparin disaccharide standards were purchased from Calbiochem (La Jolla, CA) or Sigma Chemical Co. (St. Louis, MO). Heparin lyases I (EC 4.2.2.7 [EC] ) and II (no EC number) were obtained from Sigma, and heparin lyase III (EC 4.2.2.8 [EC] ) from Calbiochem. Each enzyme lot was first tested to ensure no contaminating sulfatase activity was present, by overnight reaction with the trisulfated heparin disaccharide,
UA2S-GlcNS6S (IS). Heparin (ammonium salt) from porcine intestinal mucosa, HS (sodium salt) from bovine kidney, and heparin (sodium salt) from bovine intestinal mucosa were purchased from Sigma. Heparin from bovine lung and LMWH from porcine intestinal mucosa were both purchased from Calbiochem. The heparin decasaccharide was a kind gift from Dr. Zachary Shriver and Dr. Ganesh Venkataraman (Momenta Pharmaceuticals, Cambridge, MA). A mixture of unsaturated tetrasaccharides obtained by heparin lyase I cleavage, followed by gel permeation chromatography was purchased from Dextra Laboratories (Reading, UK), and from this, the fully sulfated heparin tetrasaccharide substrate was isolated by analytical SAX-HPLC using a linear gradient of 0–1 M NaCl at pH 3.5 and desalted by using 1 kDa Dispo-Biodialyzer (The Nest Group, Southborough, MA). Solvents used were of HPLC grade and purchased from Fisher (Santa Clara, CA). Unfractionated heparin/HS samples were desalted by using a 3-kDa molecular weight centrifugal filter and washed three times with ultrapure Milli-Q water (Millipore, Billerica, MA). Heparin disaccharide stock solutions were prepared at concentrations of 2 mM on the basis of their absorbance at 232 nm in 0.03 M HCl (
232 = 5500 M–1 cm–1) (Desai et al., 1993b).
Expression of HSulf-2 in MCF-7 cells
MCF-7 cells (breast cancer cell line) were cultured in 10-cm dishes in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin (P/S), and 1 µg/mL insulin. For collecting the CM, the medium was replaced by OptiMEM (Invitrogen, Carlsbad, CA) supplemented with P/S. Cells were incubated for an additional 3 days at 37°C before CM was collected. CM was 150-fold concentrated on an Amicon® Ultra PL-30 centrifugal filter device (Millipore), dialyzed into 50 mM N(EtOH)3, pH 8, stabilized with 10% glycerol, and stored at –20°C.
MS
Mass spectra were obtained by using an ESI source on a quadrupole ion trap instrument (ThermoFinnigan LCQ, San Jose, CA). The data acquisition software used was Xcalibur, Version 1.2. Disaccharide standards and mock mixtures were sprayed at a concentration of 50–100 pmol/µL, from a 1:1 MeOH/H2O solution with 10 mM NH4OH. Samples were introduced by flow injection analysis at 20 µL/min by using a Harvard syringe pump and 1:1 MeOH/ H2O solvent. Spectra were obtained in negative ion mode by using a spray voltage at 3.8 kV and a capillary temperature of 200°C for all experiments. For MS2 experiments, the selection of each precursor ion was achieved by using an isolation width of 3 Da, and the ion was activated at 0.6–0.8 V (29% normalized collision energy) for 100 ms, and the qz value was maintained at 0.25. Each mass spectrum obtained consists of an average of 10–20 scans.
Heparinase digestions for compositional analysis
Digestions of 100 µg samples of heparin/HS were carried out in 75 µL of 20 mM ammonium acetate buffer, pH 7.5, containing 2 mM Ca(OAc)2 and 0.01 unit each of heparin lyases I, II, and III, and incubated at 37°C for 16 h. One international unit (1U) produces 1 µmol of unsaturated uronic acid per min at 37°C. The enzymatic digestion was quenched by adding 200 µL of MeOH, followed by 20 µL of an aqueous solution (0.2 M) of ammonium hydroxide, and 105 µL of water to make the solution 1:1 MeOH/H2O. A 10 µL aliquot of the sample was diluted further 10-fold into final solution of 1:1 MeOH/H2O, 10 mM ammonium hydroxide and containing 5 µM of the internal standard, disaccharide IP, for a total concentration of 50 µM heparin disaccharides. Samples were analyzed by ESI-MSn without further purification.
Endoglucosamine-6-sulfatase assays of HSulf-2
The procedure for the assays is modified from the method published by Morimoto-Tomita et al. (2002). For the MS assay, 5 µL of the 150-fold concentrated CM in 50 mM N(EtOH)3 was mixed with 625 µM Mg(OAc)2 and 5 µg of the heparin substrate in a total volume of 12.5 µL. After incubation for 15 h at 37°C, the reactions were terminated by heating for 8 min at 100°C. A mixture of 1 mU, each of heparin lyases I, II, and III in 2 µL of 100 mM NH4OAc, pH 7.5, 1 µL of 20 mM Ca(OAc)2, and 4.5 µL water was added to the reaction mixture and incubated for 5 h at 37°C. The digestion was stopped by heating for 8 min at 100°C. Composition analysis of the resulting disaccharide mixture was determined by MS; 10 µL of the reaction mixture was diluted to obtain a final volume of 250 µL of 1:1 CH3CN/water containing 10 mM NH4OH and the internal standard (I-P), 5 µM. The samples were then analyzed by ESI-MSn without further purification.
Quantification method of heparin disaccharides
After complete digestion of heparin/HS by the heparin lyases, a combination of ESI-MS and MS/MS experiments allows the identification and quantification of the 12 disaccharide constituents. The MS method involves two steps for the complete quantitative analysis of the disaccharide composition. The first step is the direct quantitative analysis of the amount of the four nonisomeric disaccharides from a profile MS1 spectrum by using an internal standard. In this step, we also obtain information about the sum of the amounts of each set of isomers, but not the relative amounts of each individual disaccharide isomer. The second step of our methodology allows for completing the compositional analysis by determining the relative amounts of each isomer contributing to the signal for the group in the MS1 spectrum as shown in Figure 2. This step involves the use of diagnostic ions’ intensities in the MS2 spectra described above and a method previously developed in our laboratory for the quantitative analysis of mixtures of isomers (Desaire and Leary, 2000a,b; Saad and Leary, 2003). For further detailed description of this method, see Supplementary methods.
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Supplementary material |
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Supplementary Table I. Reproducibility of diagnostic ion contributions for disaccharide standards from MS2 spectra. Supplementary Table II. Quantitative analysis of mock mixtures of isomeric disaccharides. Supplementary data are available at Glycobiology online (http://glycob.oupjournals.org).
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary materialAcknowledgmentsReferences |
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O.M.S. and J.A.L gratefully acknowledge the NIH for funding this research (Grant No. GM47356). This work was also supported by a postdoctoral fellowship of the German Academic Exchange Service (DAAD) to H.E. and by grants from the Mizutani Foundation (S.D.R.) and an UCSF Comprehensive Cancer Center Intramural Award from the Alexander and Margaret Stewart Trust (S.D.R.).
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Abbreviations |
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CID, collision-induced dissociation; CM, conditioned medium; ESI, electrospray ionization; GAG, glycosaminoglycans; GlcN, glucosamine; HexA, hexuronic acid; HPLC, high performance liquid chromatography; HS, heparan sulfate; LMWH, low molecular weight heparin; MS, mass spectrometry; MS/MS, tandem MS; NAc, N-acetylation of the glucosamine; NS, N-sulfation of the glucosamine; 2S, 2-O-sulfation; 6S, 6-O-sulfation;
UA, a 4,5 unsaturated uronic acid; IA, UA2S-GlcNAc6S; IIA, UA-GlcNAc6S; IIIA, UA2S-GlcNAc; IVA, UA-GlcNAc; IH, UA2S-GlcN6S; IIH, UA-GlcN6S; IIIH, UA2S-GlcN; IVH, UA-GlcN; IS, UA2S-GlcNS6S; IIS, UA-GlcNS6S; IIIS, UA2S-GlcNS; IVS, UA-GlcNS |
References |
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