Characterization of Sulfated Oligosaccharides in

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Anal. Chem. 2006, 78, 4534-4542

Characterization of Sulfated Oligosaccharides in Mucopolysaccharidosis Type IIIA by Electrospray Ionization Mass Spectrometry Kerryn E. Mason, Peter J. Meikle, John J. Hopwood, and Maria Fuller*

Lysosomal Diseases Research Unit, Department of Genetic Medicine, Children, Youth and Women’s Health Service, 72 King William Road, North Adelaide, South Australia, 5006 Australia, and Department of Pediatrics, University of Adelaide, Adelaide, South Australia, 5005 Australia

Heparan sulfate is a linear glycosaminoglycan with considerable structural diversity that binds a myriad of growth factors and proteins that play pivotal roles in a variety of biological processes. We have investigated the structural complexity of partially degraded fragments of heparan sulfate in mucopolysaccharidosis type IIIA in which there is a defect in heparan sulfate catabolism. Mono- to hexadecasaccharides were isolated from the urine of a mucopolysaccharidosis IIIA patient and shown to have non-reducing end glucosamine N-sulfate residues, reflecting the catabolic deficiency in heparan Nsulfatase (sulfamidase) activity. The use of nitrous acid digestion (pH 1.5) combined with separation by reversephase high-performance liquid chromatography and analysis by electrospray ionization-mass spectrometry identified multiple forms of these oligosaccharides with some N-acetylated glucosamine residues and one to three sulfates per disaccharide. Furthermore, we demonstrated that each oligosaccharide existed in multiple sulfated forms. Many structural isomers were present, suggesting a complex mixture of oligosaccharides present in the urine as a consequence of a defect in heparan sulfate degradation.

HS degradation begins with endo-degradation of the long-chain polymer to HS oligosaccharides that are further degraded from their non-reducing end by the sequential action of eight lysosomal exoenzymes. These exoenzymes convert the oligosaccharides to monosaccharides and inorganic sulfate for transport out of the lysosome and reutilization by the cell. In mucopolysaccharidosis (MPS) type IIIA, the catabolism of HS is blocked due to a deficiency of the exoenzyme heparan N-sulfatase (sulfamidase).2 An inability to hydrolyze non-reducing end HS N-sulfate esters leads to the accumulation of partially degraded HS oligosaccharide fragments in the lysosomes of affected cells and their excretion in the urine. Although HS is involved in a plethora of cellular processes,3-6 the association between fragments of HS that accumulate in MPS IIIA and the pathology of this disorder is unclear. There have been a number of studies to characterize HS in MPS patients. HS has been isolated from the urine of MPS patients by strong anion exchange chromatography and characterized by polyacrylamide gel electrophoresis, demonstrating a range of oligosaccharide structures from tetrasaccharides to structures with more than 20 disaccharide repeats.7 HS has also been analyzed by depolymerization with bacterial heparin lyases to disaccharides followed by capillary electrophoresis8-10 or strong anion-exchange highperformance liquid chromatography (HPLC).11-13 The bacterial

Heparan sulfate (HS) is a linear glycosaminoglycan (GAG) composed of a variable number of repeating disaccharide units consisting of alternating uronic acid (UA) and R-linked (1,4) glucosamine residues (GlcN). The UA may be R-linked (1,4) L-iduronic acid (IdoA) or β-linked (1,4) D-glucuronic acid (GlcA), which may be unsulfated or O-sulfated (S) on the C2-hydroxyl. The amino group of the GlcN may be N-sulfated (GlcNS) or N-acetylated (GlcNAc), with sulfate on the C6-hydroxyl, and to a lesser extent on the C3-hydroxyl. HS forms domain structures with areas of low sulfation (GlcA-GlcNAc disaccharide, NA domain) and high sulfation (IdoA-GlcNS disaccharide, NS domain) with mixed sequences of N-acetylated and N-sulfated disaccharides separating the two domains. Such variability gives rise to a large number of complex sequences.1

(2) Neufeld, E. F.; Muenzer, J. The Mucopolysaccharidoses, 8th ed.; McGrawHill: New York, 2001. (3) Wu, Z. L.; Zhang, L.; Beeler, D. L.; Kuberan, B.; Rosenberg, R. D. FASEB J. 2002, 16, 539-545. (4) Liu, J.; Shriver, Z.; Pope, R. M.; Thorp, S. C.; Duncan, M. B.; Copeland, R. J.; Raska, C. S.; Yoshida, K.; Eisenberg, R. J.; Cohen, G.; Linhardt, R. J.; Sasisekharan, R. J. Biol. Chem. 2002, 277, 33456-33467. (5) Wu, Z. L.; Zhang, L.; Yabe, T.; Kuberan, B.; Beeler, D. L.; Love, A.; Rosenberg, R. D. J. Biol. Chem. 2003, 278, 17121-17129. (6) Ashikari-Hada, S.; Habuchi, H.; Kariya, Y.; Itoh, N.; Reddi, A. H.; Kimata, K. J. Biol. Chem. 2004, 279, 12346-12354. (7) Byers, S.; Rozaklis, T.; Brumfield, L. K.; Ranieri, E.; Hopwood, J. J. Mol. Genet. Metab. 1998, 65, 282-290. (8) Desai, U. R.; Wang, H.; Ampofo, S. A.; Linhardt, R. J. Anal. Biochem. 1993, 15, 120-127. (9) Mao, W. J.; Thanawiroon, C.; Linhardt, R. J. Biomed. Chromatogr. 2002, 16, 77-94. (10) Militsopoulou, M.; Lamari, F. N.; Hjerpe, A.; Karamanos, N. K. Electrophoresis 2002, 23, 1104-1109. (11) Lee, G. J.; Lui, D. W.; Pav, J. W.; Tieckelmann, H. J. Chromatogr. 1981, 212, 65-73. (12) Vives, R. R.; Pye, D. A.; Salmivirta, M.; Hopwood, J. J.; Lindahl, U.; Gallagher, J. T. Biochem. J. 1999, 339, 767-773.

* Corresponding author. Telephone: +61 8 8161 6741. Fax: +61 8 8161 7100. E-mail: [email protected]. (1) Esko, J. D.; Selleck, S. B. Annu. Rev. Biochem. 2002, 71, 435-471.

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polysaccharide lyases cleave HS chains by an eliminase mechanism and generate an unsaturated product that can be monitored at a wavelength of 232 nm.14 This approach results in complete digestion of the HS and provides detail on the disaccharide composition; however, it is not possible to determine the order of these disaccharides. Recent developments in reverse-phase ion-pair HPLC using volatile mobile phases has enabled a transition from ultraviolet (UV) to mass spectrometric detection15,16 with the identification of heparin-derived oligosaccharides from di- to tetradecasaccharides.17 Electrospray ionization-mass spectrometry (ESIMS) has become a prominent analytical tool for the identification, structural characterization, and quantification of HS-derived oligosaccharides.18-22 However, minimizing the loss of sulfate from the oligosaccharides in ESI is challenging, with the level of desulfation depending on ion source conditions, pH, and solvents.23 Recently, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to identify di- to pentadecasaccharides from the urine of MPS I and II patients. These oligosaccharides were composed of N-acetylated and unsubstituted hexosamine repeating disaccharides with various levels of sulfation.24,25 In these earlier studies, ESI-MS/MS alone was unable to confirm the order of the disaccharides within these oligosaccharides or the existence of different sulfated structures. In this study, we have isolated oligosaccharides from MPS IIIA urine and used a combination of nitrous acid digestion and reverse-phase HPLC ESI-MS to characterize these oligosaccharides. EXPERIMENTAL SECTION Materials. Recombinant human heparan N-sulfatase was prepared from a Chinese hamster ovary-K1 expression system as previously described.26 Heparin (porcine intestinal mucosa) was supplied by Sigma-Aldrich (Castle Hill, NSW, Australia). Acetonitrile and chloroform were HPLC grade from Ajax FineChem (Seven Hills, NSW, Australia). Solid-phase extraction columns containing 50 mg of copolymeric-hydrophobic and aminopropyl phase were from United Chemical Technologies (Bristol, PA), and 1-phenyl-3-methyl-5-pyrazolone (PMP) was purchased from Tokyo Kasei Kogyo (Tokyo, Japan). Size exclusion Bio-Gel P2, P4, and P6 were obtained from Bio-Rad (Hercules, CA). A 5 µm Alltima C18-LL column (150 × 2.1 mm) from Alltech Associates (Deerfield, IL) with a 2 mm EXSIL ODS 5 µm guard column from SGE (Austin, TX) was used for HPLC separation. (13) Turnbull, J. E. Methods Mol. Biol. 2001, 171, 141-147. (14) Linhardt, R. J.; Rice, K. G.; Merchant, Z. M.; Kim, Y. S.; Lohse, D. L. J. Biol. Chem. 1986, 261, 14448-14454. (15) Kuberan, B.; Lech, M.; Zhang, L.; Wu, Z. L.; Beeler, D. L.; Rosenberg, R. D. J. Am. Chem. Soc. 2002, 124, 8707-8718. (16) Thanawiroon, C.; Linhardt, R. J. J. Chromatogr. A 2003, 1014, 215-223. (17) Thanawiroon, C.; Rice, K. G.; Toida, T.; Linhardt, R. J. J. Biol. Chem. 2004, 279, 2608-2615. (18) Chai, W.; Luo, J.; Lim, C. K.; Lawson, A. M. Anal. Chem. 1998, 70, 20602066. (19) Zaia, J.; McClellan, J. E.; Costello, C. E. Anal. Chem. 2001, 73, 6030-6039. (20) Pope, R. M.; Raska, C. S.; Thorp, S. C.; Liu, J. Glycobiology 2001, 11, 505513. (21) Saad, O. M.; Leary, J. A. Anal. Chem. 2003, 75, 2985-2995. (22) Behr, J. R.; Matsumoto, Y.; White, F. M.; Sasisekharan, R. Rapid Commun. Mass Spectrom. 2005, 19, 2553-2562. (23) Naggar, E. F.; Costello, C. E.; Zaia, J. J. Am. Soc. Mass Spectrom. 2004, 15, 1534-1544. (24) Fuller, M.; Meikle, P. J.; Hopwood, J. J. Glycobiology 2004, 14, 443-450. (25) Fuller, M.; Chau, A.; Nowak, R. C.; Hopwood, J. J.; Meikle, P. J. Glycobiology 2006, 16, 318-325.

Isolation of Oligosaccharides. MPS IIIA and control urine (500 mL of each) were adjusted to pH 5 with glacial acetic acid and then centrifuged at 9000g for 10 min to remove debris. The supernatant was diluted 1 to 2 with H2O to adjust the chloride concentration to below 100 mM to maximize binding of sulfated oligosaccharides. GAG were partially purified using anion exchange as described by Fuller et al. with minor modifications.24 Briefly, the urine was applied to a DEAE Sephacel column (2.5 × 7 cm) at a flow rate of 50 mL/h. The column was washed with 100 mM CH3COONH4, pH 5 (150 mL); then eluted with 100 mM CH3COONH4 and 1.2 M LiCl, pH 5 (165 mL); followed by 100 mM CH3COONH4 and 2 M LiCl, pH 5 (150 mL). All eluates were assayed for UA,27 and the UA-containing fractions were pooled and then size-fractionated on either Bio-Gel P2, P4, or P6 columns (1.5 × 170 cm) in 0.5 M HCOONH4. These eluant fractions were also assayed for UA. Derivatization of Oligosaccharides. Fractions from the BioGel P2, P4, and P6 columns (10-100 µg of UA equivalents) were lyophilized and then derivatized using PMP.25,28 Derivatization with PMP results in the addition of two PMP groups to the reducing end monosaccharide with subsequent elimination of H2O.29 To remove excess derivatizing reagent, samples were either extracted three times with 500 µL of chloroform or desalted on a copolymeric-hydrophobic and aminopropyl (50 mg) solid-phase extraction column and lyophilized.25 Heparan N-Sulfatase Digestion. Fractions from the Bio-Gel P4 and P6 columns containing 10 µg of UA equivalents were lyophilized and digested with 1 µg of recombinant human heparan N-sulfatase in 50 mM CH3COONa, pH 5.4, containing 0.5 mg/ mL bovine serum albumin (50 µL) by incubation at 37 °C for 16 h. Samples were then lyophilized, derivatized, and analyzed by mass spectrometry. Nitrous Acid (pH 1.5) Treatment. Heparin and fractions from the Bio-Gel P2, P4, and P6 columns were subjected to pH 1.5 nitrous acid deamination.30 Briefly, nitrous acid was prepared by mixing equal volumes of cold 0.5 M sulfuric acid and 0.5 M BaN2O4, and BaSO4 was removed by centrifugation at 13000g for 5 min. Heparin (porcine intestinal mucosa, 200 mg) was dissolved in 725 µL of H2O and digested with an equal volume of nitrous acid at room temperature for 30 min. The solution was neutralized with an equal volume of 4 M ammonium sulfamate. Oligosaccharides (10-100 µg of UA equivalents) were dissolved in 40 µL of H2O and digested with 100 µL of supernatant at room temperature for 30 min. Samples were neutralized with 15 µL of 2 M Na2CO3. Nitrous acid treated heparin was then size-fractionated on a BioGel P2 column (1.5 × 170 cm) in 0.5 M HCOONH4. All samples were lyophilized, derivatized, and analyzed by HPLC mass spectrometry. HPLC Separation. Reverse-phase HPLC separation of oligosaccharides was performed on a 5 µm Alltima C18-LL column (150 × 2.1 mm) with a 2 mm EXSIL ODS 5 µm guard column. Mobile phases A (acetonitrile:H2O, 1:99 v/v) and B (acetonitrile: (26) Bielicki, J.; Hopwood, J. J.; Melville, E. L.; Anson, D. S. Biochem. J. 1998, 329, 145-150. (27) Blumenkrantz, N.; Asboe-Hansen, G. Anal. Biochem. 1973, 54, 484-489. (28) Honda, S.; Akao, E.; Suzuki, S.; Okuda, M.; Kakehi, K.; Nakamura, J. Anal. Biochem. 1989, 180, 351-357. (29) Ramsay, S. L.; Meikle, P. J.; Hopwood, J. J. Mol. Genet. Metab. 2003, 78, 193-204. (30) Shively, J. E.; Conrad, H. E. Biochemistry 1976, 15, 3932-3942.

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H2O, 80:20 v/v) both contained 16 mM CH3COONH4, 24 mM acetic acid, and 0.5 mM triethylamine, pH 4.5. The addition of an ion-pair reagent to the mobile phase was required to provide the best retention and separation for the mono- and disaccharides. A stepped gradient from 0 to 60% B was run over a period of 70 min at 0.2 mL/min. The gradient program was 0 to 5 min 0% B, 5.01 to 15 min 3% B, 15.01 to 25 min 11% B, 25.01 to 35 min 18% B, 35.01 to 45 min 25% B, 45.01 to 55 min 37% B, 55.01 to 65 min 55% B, and 65.01 to 70 min 0% B. The liquid chromatography system consisted of two Agilent series 1100 pumps and an Agilent series 1100 UV detector. The elution profiles in the HPLC separations were monitored by absorbance at 245 nm and by ESI-MS. Mass Spectrometry. Mass spectrometric analysis of the oligosaccharides was performed in negative ion mode using a PE Sciex API 3000 triple quadrupole mass spectrometer with a turboionspray source. Nitrogen was used as the auxiliary, curtain and collision gas. Ion source temperature was set at 200 °C and spray voltage to -4500 V. Declustering potential and focusing potential were optimized for each oligosaccharide. Samples were either infused at 5 µL/min using a Harvard syringe pump or a 20 µL volume was injected manually. For ESI-MS/MS (product ion analysis), the collision energy was ramped from -130 to -5 in 4-V increments with cell exit potential set at -15 V, while Q3 was scanned from 50 to 1500 amu in 2 s. For ESI-MS, scans were acquired from 250 to 2000 amu in 2 s. Data processing was performed on Analyst 1.3 software. RESULTS AND DISCUSSION Isolation and Identification of Sulfated Oligosaccharides in MPS IIIA Urine. Anion exchange of MPS IIIA and control human urine resulted in the isolation of a total of 25 and 3.6 mg of UA equivalents, respectively. The chromatograms of patient and control oligosaccharides on Bio-Gel P4 (Figure 1a) show high molecular weight oligosaccharides in the void volume (75-100 mL) corresponding to approximately 40% of the total oligosaccharide for both urines. Nona- to monosaccharides eluted between 130 and 275 mL. To obtain larger oligosaccharides, a urine sample from a MPS IIIA patient was applied to a Bio-Gel P2 column, and the void volume was concentrated and reapplied to a Bio-Gel P6 column. The chromatogram of oligosaccharides on the Bio-Gel P6 (Figure 1b) shows 34% of the total oligosaccharides in the void volume (96-120 mL). Hexadeca- to octasaccharides eluted between 124 and 208 mL. Urinary oligosaccharides within the fractionation range from both columns were subsequently characterized by ESI-MS and identified by the presence of characteristic multiply charged ions from [M - H]-1 to [M - 6H]-6 where M includes the addition of 2 PMP moieties to the reducing end monosaccharide.29 ESI-MS analysis enabled the identification of 36 different oligosaccharide compositions ranging in size from mono- to hexadecasaccharide, with variable numbers of sulfates. No corresponding HS-sulfated oligosaccharides were observed in the control urine. Interpretation of ESI mass spectra in this work can be complex, as the conditions used result in the observation of losses of sulfate and glycosidic cleavages. Representative mass spectra are shown in Figure 2. ESI-MS of the oligosaccharides eluting at 175 mL from Bio-Gel P4 (Figure 1a) shows four oligosaccharide compositions; a trisaccharide, GlcN-UA-GlcNAc with 2 sulfate groups (2S), indicated by the ions at m/z 1047.4 4536 Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

Figure 1. Elution profile of control and MPS IIIA urine from BioGel P4 and P6 columns. Sulfated oligosaccharides were isolated from control and MPS IIIA human urine by anion exchange chromatography. The oligosaccharides were then size fractionated on a Bio-Gel P4 column (1.5 × 170 cm) and fractions assayed for UA. Panel a shows the UA profile for the MPS IIIA (-) and control (- -) urine. A second aliquot of MPS IIIA urine was fractionated on a Bio-Gel P2 and the void volume concentrated then further fractionated on a BioGel P6 column (1.5 × 170 cm). Fractions were assayed for UA. Panel b shows the Bio-Gel P6 UA elution profile for human MPS IIIA urine with di- to hexasaccharides removed.

[M - H]-1 and m/z 523.5 [M - 2H]-2; a tetrasaccharide, GlcNUA-GlcNAc-UA (2S), indicated by the ions at m/z 1223.8 [M H]-1, m/z 611.5 [M - 2H]-2 and m/z 524.5 [M - PMP - 2H]-2; a tetrasaccharide, GlcN-UA-GlcN-UA (4S), indicated by the ion at m/z 446.1 [M - 3H]-3; and a pentasaccharide, GlcN-UA-GlcNUA-GlcNAc with 3-5 sulfate groups (3-5S). The ions indicating the pentasaccharide composition are [M - 3H]-3 at m/z 541.0, 514.5, and 487.9 and [M - 2H]-2 at m/z 812.5, 772.0, and 732.0 corresponding to the 5, 4, and 3S forms, respectively (Figure 2a). It is noteworthy that there are a number of ions observed below m/z 400 present in both control and MPS III urine that do not correspond to HS-derived oligosaccharides. ESI-MS of the oligosaccharides eluting at 148 mL from Bio-Gel P6 (Figure 1b) shows two dodecasaccharides. The dodecasaccharide composition GlcN-[UA-GlcN]/[UA-GlcNAc]4-UA (2-5S) is indicated by the ions at m/z 979.0, 952.2, 925.1, and 899.2 [M - H]-3 and m/z 734.1, 714.0, 694.0, and 674.1 [M - 4H]-4 corresponding to the 5, 4, 3, and 2S forms, respectively. Another dodecasaccharide composition GlcN-[UA-GlcN]2/[UA-GlcNAc]3-UA (3-6S) is indicated by the ions at m/z 991.9, 964.7, 938.6, and 911.6 [M - H]-3 and m/z 743.7, 723.6, 703.6, and 683.5 [M - 4H]-4 corresponding to the 6, 5, 4, and 3S forms, respectively (Figure 2b). It has been reported that oligosaccharides derivatized with PMP undergo a chemical loss from the reducing end to give a characteristic product ion at m/z 256 for a GlcNAc and at m/z 331 for a UA by ESI-MS/MS.24,29 Using this we were able to identify the reducing end for the di- to decasaccharide compositions as either GlcNAc or UA. Figure 3 shows the collision spectra

Figure 2. ESI-MS of selected oligosaccharides from MPS IIIA urine. Oligosaccharides eluting from the Bio-Gel P4 at 175 mL and Bio-Gel P6 at 148 mL were lyophilized, derivatized with PMP, and analyzed by ESI-MS. The mass spectrum of oligosaccharides eluting from the Bio-Gel P4 at 175 mL (panel a) indicates the presence of four oligosaccharides: a trisaccharide, GlcN-UA-GlcNAc (2S), indicated by the ions at m/z 1047.4 [M - H]-1 and m/z 523.5 [M - 2H]-2; a tetrasaccharide, GlcN-UA-GlcNAc-UA (2S), indicated by the ions at m/z 1223.8 [M - H]-1, m/z 611.5 [M - 2H]-2 and 524.5 [M - 2H]-2 corresponding to the loss of one PMP from the parent ion m/z 611.5; a tetrasaccharide, GlcN-UAGlcN-UA (4S) indicated by the ion at m/z 446.1 [M - 3H]-3; and a pentasaccharide, GlcN-UA-GlcN-UA-GlcNAc (3-5S). The ions indicating the pentasaccharide are m/z 541.0, 514.5, and 487.9 [M - 3H]-3 and m/z 812.5, 772.0, and 732.0 [M - 2H]-2 for the 5, 4, and 3S forms, respectively. The mass spectrum of the oligosaccharides eluting at 148 mL from Bio-Gel P6 (panel b) shows two dodecasaccharides. The dodecasaccharide GlcN-[UA-GlcN]/[UA-GlcNAc]4-UA (2-5S) is indicated by the ions at m/z 979.0, 952.2, 925.1, and 899.2 [M - H]-3 and m/z 734.1, 714.0, 694.0, and 674.1 [M - 4H]-4 corresponding to the 5, 4, 3, and 2S forms, respectively. Another dodecasaccharide GlcN-[UA-GlcN]2/[UA-GlcNAc]3-UA (3-6S) is indicated by the ions at m/z 991.9, 964.7, 938.6, and 911.6 [M - H]-3 and m/z 743.7, 723.6, 703.6, and 683.5 [M - 4H]-4 corresponding to the 6, 5, 4, and 3S forms, respectively.

and characteristic product ions for a tetrasaccharide and a pentasaccharide composition (eluted at 175 mL from Bio-Gel P4; Figure 1a). The collision spectra of the m/z 611.3 [M - 2H]-2 ion from the tetrasaccharide GlcN-UA-GlcNAc-UA (2S) (Figure 3a) shows ions at m/z 173.1 [PMP - H]-1, m/z 331.0 corresponding to internal glycosidic backbone cleavage of reducing end UA with one PMP moiety, and m/z 269.0 corresponding to a PMP molecule with a fragmented UA, confirming UA at the reducing end. Ions corresponding to m/z 484.0 [M - PMP - S - 2H]-2, m/z 524.2 [M - PMP - 2H]-2, and the internal glycosidic backbone fragment GlcN-UA-GlcNAc (1S) [M - H]-1 at m/z 637.3 were also observed. The collision spectra of the m/z 514.3 [M -

3H]-3 ion from the pentasaccharide composition GlcN-UA-GlcNUA-GlcNAc (4S) (Figure 3b) show ions at m/z 172.9 [PMP - H]-1, m/z 487.8 [M - S - 3H]-3, and m/z 256.3 corresponding to a PMP molecule with a fragmented GlcNAc. Larger structures consistent with an extended series of disaccharides were also observed (data not shown). Therefore from the urine of a MPS IIIA patient, we identified a series of “even” numbered oligosaccharides from di- to hexadecasaccharides. These oligosaccharide compositions had increasing numbers of GlcN-UA disaccharides (1 to 4 repeats) and GlcNAc-UA disaccharides (0 to 5 repeats) with reducing end UA; proposed structures are detailed in Table 1. We also observed a Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

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Figure 3. Product ion spectra of oligosaccharides from MPS IIIA urine. Oligosaccharides eluting from the Bio-Gel P4 at 175 mL were lyophilized, derivatized with PMP, and analyzed by ESI-MS/MS. Panel a shows the product ion spectra of a tetrasaccharide, GlcN-UAGlcNAc-UA (2S), for the [M - 2H]-2 ion at m/z 611.3. Major product ions were observed at m/z 173.1 [PMP - H]-1, m/z 331.0 corresponding to internal glycosidic backbone cleavage of reducing end UA with one PMP moiety and m/z 269.0 corresponding to a PMP molecule with a fragmented UA. Major product ions were observed at m/z 484.0 [M - PMP - S - 2H]-2, m/z 524.2 [M - PMP - 2H]-2 and m/z 637.3 corresponding to the internal glycosidic backbone fragment GlcN-UA-GlcNAc (1S) [M - H]-1. Panel b shows the product ion spectra of a pentasaccharide, GlcN-UA-GlcN-UA-GlcNAc (4S), for the [M - 3H]-3 ion at m/z 514.3. Major product ions were observed at m/z 172.9 [PMP - H]-1, m/z 487.8 [M - S - 3H]-3 and m/z 256.3 corresponding to a PMP molecule with a fragmented GlcNAc.

series of “odd” numbered oligosaccharides from mono- to pentadecasaccharides. These oligosaccharide compositions had a similar structure to the even oligosaccharides in relation to repeating disaccharides of GlcN-UA and GlcNAc-UA; however, they have reducing end GlcNAc (Table 2). These proposed structures are consistent with the composition of HS GAG.31 Identification of the Non-reducing End. The oligosaccharides identified in the MPS IIIA urine would be expected to have non-reducing GlcNS residues reflective of the enzyme deficiency. To confirm this, di- to decasaccharides were treated with heparan N-sulfatase, analyzed by ESI-MS and shown to have N-sulfated glucosamine at the non-reducing end. Figure 4 shows a hexasaccharide composition (eluted between 150 and 165 mL from Bio-Gel P4; Figure 1a) treated with recombinant heparan N(31) Maccarana, M.; Sakura, Y.; Tawada, A.; Yoshida, K.; Lindahl, U. J. Biol. Chem. 1996, 271, 17804-17810.

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sulfatase. An observed mass decrease of 80, 40, and 27 amu from [M - H]-1, [M - 2H]-2, and [M - 3H]-3 charged ions respectively indicates the loss of a sulfate group (SO3). The hexasaccharide composition GlcN-[UA-GlcN]/[UA-GlcNAc]-UA (2-5S) is indicated by the [M - 2H]-2 ions at m/z 779.8, 820.7, 859.8, and 899.8 and with 3-5S as indicated by the [M - 3H]-3 ions at m/z 546.0, 573.2, and 599.7 (Figure 4a). Following heparan N-sulfatase digestion, the ions m/z 899.8 and 599.7 of the hexasaccharide (5S) have lost SO3 and thus were not observed. At the same time we also noted the appearance of a signal at m/z 740.0 corresponding to the [M - 2H]-2 ion of the hexasaccharide (1S) (Figure 4b). These results support the loss of sulfate from N-sulfated glucosamine at the non-reducing end of each sulfated oligosaccharide. Position of N-Acetylated Glucosamine in Sulfated Oligosaccharides. We have identified a range of oligosaccharides in the urine of an MPS IIIA patient consisting of repeating disaccharides of GlcN-UA and GlcNAc-UA with reducing end UA or GlcNAc (Tables 1 and 2). The order of the disaccharides within these oligosaccharide compositions could not be determined from the ESI-MS or ESI-MS/MS data. Nitrous acid (pH 1.5) reacts with glucosamine N-sulfate residues producing reducing end anhydromannose (AnM).30 We have used nitrous acid (pH 1.5) digestion in combination with HPLC ESI-MS to determine the position of the GlcNAc residue within the sulfated oligosaccharide compositions. This analytical strategy has been applied to oligosaccharide compositions from penta- to decasaccharide (Tables 1 and 2). It is apparent that the position of the GlcNAc residues in these oligosaccharides is not fixed at any one point but are at multiple positions, thereby generating a large number of oligosaccharide structures. Figure 5 shows a mixture of a penta- and hexasaccharide compositions (elution volume 160 mL from Bio-Gel P4; Figure 1a) treated with nitrous acid (pH 1.5). In this mixture the pentasaccharide structure is clear, but the hexasaccharide composition GlcN-[UA-GlcN]/[UA-GlcNAc]-UA (2-5S) could equally exist as GlcN-UA-GlcN-UA-GlcNAc-UA (2-5S) or GlcN-UA-GlcNAcUA-GlcN-UA (2-5S). The predicted nitrous acid (pH 1.5) digestion products for the pentasaccharide and two hexasaccharide structures are detailed in Table 3, and the observation of four key products will identify the two alternate structures. Figure 5 shows extracted ion chromatograms (XIC) with a ( 0.5 amu window, corresponding to the four key structures. An ion at m/z 982.5 was observed at 38.9 min, corresponding to the [M - H]-1 for UA-GlcNAc-UA-PMP (1S), which confirms the hexasaccharide GlcNS-UA-GlcNS-UA-GlcNAc-UA (3S) (Figure 5a); an ion at m/z 562.8 [M - 2H]-2 observed at 38.7 min, with a corresponding ion at m/z 1127 [M - H]-1 (not shown), is UA-GlcNAc-UA-AnMPMP (1S); and the m/z 603.3 ion at 42.7 min is the [M - H]-1 of UA-PMP (1S) (Figure 5b,c). The presence of both digest products confirms the hexasaccharide structure GlcNS-UA-GlcNAc-UAGlcNS-UA (3S). The ion at m/z 806.3 observed at 40.0 min corresponds to the [M - H]-1 of UA-GlcNAc-PMP (1S) from the pentasaccharide GlcNS-UA-GlcNS-UA-GlcNAc (1-3S) (Figure 5d). Thus, we were able to confirm the presence of both hexasaccharide structures in our sulfated oligosaccharide preparation from the urine of a MPS IIIA patient. Identification of Sulfates in Oligosaccharides. The reported structure of HS indicates that up to three sulfates can be located on the GlcN residue and one on the UA monosaccharides;1

Table 1. Proposed Structures of Oligosaccharides in MPS IIIA with UA Reducing End proposed structurea

no. of sulfate groups

GlcN GlcNAc

2-1 2-1

GlcN-UA GlcN-UA-GlcNAc-UA GlcN-[UA-GlcNAc]2-UA GlcN-[UA-GlcNAc]3-UA

1 2-1 2-1 2-1

GlcN-UA-GlcN-UA GlcN-[UA-GlcN]/[UA-GlcNAc]-UA GlcN-[UA-GlcN]/[UA-GlcNAc]2-UA GlcN-[UA-GlcN]/[UA-GlcNAc]3-UA GlcN-[UA-GlcN]/[UA-GlcNAc]4-UA GlcN-[UA-GlcN]/[UA-GlcNAc]5-UA

4-3 5-3 5-2 5-1 5-3 5-2

GlcN-[UA-GlcN]2/[UA-GlcNAc]-UA GlcN-[UA-GlcN]2/[UA-GlcNAc]2-UA GlcN-[UA-GlcN]2/[UA-GlcNAc]3-UA GlcN-[UA-GlcN]2/[UA-GlcNAc]4-UA GlcN-[UA-GlcN]2/[UA-GlcNAc]5-UA

7-4 7-3 7-3 6-3 6-3

GlcN-[UA-GlcN]3/[UA-GlcNAc]-UA GlcN-[UA-GlcN]3/[UA-GlcNAc]2-UA GlcN-[UA-GlcN]3/[UA-GlcNAc]3-UA GlcN-[UA-GlcN]3/[UA-GlcNAc]4-UA

9-6 8-6 8-4 8-4

GlcN-[UA-GlcN]4/[UA-GlcNAc]2-UA GlcN-[UA-GlcN]4/[UA-GlcNAc]3-UA

14-11 14-11

charge states observed

Series 1

Series 2

Series 3

Series 4

Series 5

a

elution on P4 (mL)

-1 -1

220-245 220-275

-1 -1, -2 -2 -2

215-230 175-180 145-150 205

-2, -3 -2, -3, -4 -2, -3, -4 -3, -4 -3, -4, -5 -3, -4, -5

180-190 150-165 135

-3,-4 -3,-4 -3, -4, -5 -4, -5 -4, -5

145

elution on P6 (mL)

176-180 152-160 140-152 132-136 180-208 156-168 148-152 136-140 132

-4, -5 -4, -5 -4, -5 -4, -5

164 148-156 124-144 132

-4, -5, -6 -4, -5, -6

124 124

UA, uronic acid; GlcN, glucosamine; GlcNAc, N-acetylglucosamine.

Table 2. Proposed Structures of Oligosaccharides in MPS IIIA with GlcNAc Reducing End proposed structurea

no. of sulfate groups

charge states observed

Series 6 GlcN-UA-GlcNAc GlcN-[UA-GlcNAc]2 GlcN-[UA-GlcNAc]3

2 2 2-1

GlcN-UA-GlcN-UA-GlcNAc GlcN-[UA-GlcN]/[UA-GlcNAc]-UA-GlcNAc GlcN-[UA-GlcN]/[UA-GlcNAc]2-UA-GlcNAc GlcN-[UA-GlcN]/[UA-GlcNAc]3-UA-GlcNAc GlcN-[UA-GlcN]/[UA-GlcNAc]4-UA-GlcNAc

5-3 5-3 6-2 5-3 3-2

Series 7

Series 8

a

elution on P4 (mL)

-1, -2 -2, -3 -3, -4

195-210 165 140

-2, -3 -3, -4 -3, -4,-5 -3, -4 -3, -4, -5

150-180 140-145 130

200

GlcN-[UA-GlcN]2-UA-GlcNAc GlcN-[UA-GlcN]2/[UA-GlcNAc]-UA-GlcNAc GlcN-[UA-GlcN]2/[UA-GlcNAc]2-UA-GlcNAc GlcN-[UA-GlcN]2/[UA-GlcNAc]3-UA-GlcNAc

7-4 6-3 6-4 6-3

-3, -4 -4,-5,-6 -4, -5,-6 -4, -5

GlcN-[UA-GlcN]4/[UA-GlcNAc]2-UA-GlcNAc

Series 9 14-12

-4, -5, -6

elution on P6 (mL)

156-168 148-160

136 184-208 160-172 152-160 136-140 120

UA, uronic acid; GlcN, glucosamine; GlcNAc, N-acetylglucosamine.

therefore, it is possible that the HS-derived oligosaccharides in MPS IIIA would be expected to contain multiple sulfate residues. We have identified a range of oligosaccharides in the urine of a MPS IIIA patient consisting of mono- to hexadecasaccharide compositions showing the presence of multiple sulfates (Tables 1 and 2). Interpretation of the ESI-MS data is complicated due to the presence of in-source loss of sulfate where the magnitude of sulfate loss may be influenced by many parameters.23 We used

HPLC ESI-MS to separate different sulfated species and to assess the degree of in-source loss of sulfate for sulfated oligosaccharides derived from heparin and the urine of a MPS IIIA patient. Using reverse-phase HPLC, we were able to separate mono- to octasaccharides isolated from MPS IIIA urine. We confirmed that multiple sulfated species of mono- to octasaccharides in the MPS IIIA urine were real, with only a minor proportion of their signal due to insource loss of sulfate. Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

4539

Figure 4. Mass spectra of oligosaccharides following recombinant heparan N-sulfatase digestion. Oligosaccharides eluting from the BioGel P4 between 150 and 165 mL were lyophilized, derivatized with PMP, and analyzed by ESI-MS. Panel a shows the ESI-MS of a hexasaccharide, GlcN-[UA-GlcN]/[UA-GlcNAc]-UA (2-5S) producing [M - 2H]-2 ions at m/z 779.8, 820.7, 859.8, and 899.8, and with 3-5S producing [M - 3H]-3 ions at m/z 546.0, 573.2, and 599.7. Panel b shows the same hexasaccharide from MPS IIIA human urine following treatment with recombinant heparan N-sulfatase.

A tetrasaccharide UA-GlcN-UA-AnM (3-5S), produced from partial nitrous acid digestion (pH 1.5) of heparin, was used to establish HPLC ESI-MS conditions for maximum separation of sulfated species. Figure 6 shows the XIC with a ( 0.5 amu window, corresponding to the three sulfated tetrasaccharide structures. Two peaks at 10.1 and 10.4 min were observed for m/z 701.8, corresponding to UA-GlcN-UA-AnM (5S) (Figure 6a). These peaks represent different structural isomers resulting from the position of the sulfates on the sugar residues. Comparison of Figure 6a with Figure 6b shows that the same two peaks at 10.1 and 10.4 min are present for the signal at m/z 662.0. This corresponds to the same tetrasaccharide with four sulfates, and the elution of these peaks coincident with the UA-GlcN-UA-AnM (5S) indicates the loss of sulfate in the ESI source. Additional peaks in Figure 6b at 11.3, 12.3, and 13.6 min correspond to positional isomers of the tetrasaccharide with four sulfates. In Figure 6c the signal from m/z 621.8 corresponds to UA-GlcN-UA-AnM (3S). However, the only peaks observed coelute with the four and five sulfate forms of the tetrasaccharide indicating that they result from sulfate loss and that no UA-GlcN-UA-AnM (3S) was present in the original oligosaccharide mixture. Figure 7 shows the XIC (with a ( 0.5 amu window) for the four sulfated species of the hexasaccharide GlcNS-[UA-GlcN]/ 4540 Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

Figure 5. Extracted ion chromatograms of nitrous acid (pH 1.5) digest products from selected oligosaccharides. Oligosaccharides eluting from the Bio-Gel P4 at 160 mL were lyophilized, treated with nitrous acid, derivatized with PMP, and analyzed by reverse-phase HPLC ESI-MS. The data were searched using extracted ion chromatograms (XIC, with a ( 0.5 amu window) corresponding to four structures: Panel a shows the XIC of m/z 982.5 with a peak at 38.9 min, corresponding to the [M - H]-1 for UA-GlcNAc-UA-PMP (1S); Panel b shows the XIC of m/z 562.8 with a peak at 38.7 min, corresponding to the [M - 2H]-2 for UA-GlcNAc-UA-AnM-PMP (1S); Panel c shows the XIC of m/z 603.3 with a peak at 42.7 min, corresponding to the [M - H]-1 of UA-PMP (1S); The presence of the first three digest products confirms the hexasaccharide is present as GlcNSUA-GlcNAc-UA-GlcNS-UA (3S) and GlcNS-UA-GlcNS-UA-GlcNAcUA (3S); Panel d shows the XIC of m/z 806.3 with a peak at 40.0 min, corresponding to the [M-H]-1 of UA-GlcNAc-PMP (1S), further confirming the structure of the pentasaccharide as GlcNS-UA-GlcNSUA-GlcNAc (3S).

[UA-GlcNAc]-UA (2-5S), isolated from MPS IIIA urine (elution volume 160 mL from Bio-Gel P4, Figure 1a). The oligosaccharide composition of the hexasaccharide has been previously determined from Q1 scans (Figure 4a), from collision spectra (data not shown), and by susceptibility to digestion with recombinant heparan N-sulfatase (Figure 4b). Ion signals were observed at m/z 899.8, 859.7, 820.0, and 779.8 with major peaks at retention times of 43.8, 45.0, 46.3, and 47.7 min, respectively (Figure 7a-d), confirming the presence of 5, 4, 3, and 2 sulfated forms of this hexasaccharide. The hexasaccharide (5S) peak at 43.8 min for m/z 899.8 (Figure 7a) shows loss of one sulfate with a peak at 43.8 min for m/z 859.7 (4S) (Figure 7b) and a further loss of sulfate with a peak at the same time for m/z 820.0 (Figure 7c). Similar losses of sulfate were observed for the hexasaccharide (4S) and (3S) leading to (3S) and (2S) forms of the hexasaccharide, respectively, indicating in-source loss of sulfate for this hexasaccharide. However, the major peak in each ion scan had a unique elution time and, thereby, was not the result of in-source sulfate loss. Therefore, we were able to demonstrate that each of the 2,

Table 3. Nitrous Acid Digest Products for Penta- and Hexasaccharide proposed structurea

nitrous acid productsa

MW

GlcNS-UA-GlcNS-UA-GlcNAc-UA 3S

AnM-PMP UA-AnM-PMP (1S) UA-AnM-PMP (2S) UA-GlcNAc-UA-PMP* UA-GlcNAc-UA-PMP (1S)* AnM-PMP UA-GlcNAc-UA-AnM-PMP* UA-GlcNAc-UA-AnM-PMP (1S)* UA-PMP* UA-PMP 1S* AnM-PMP UA-AnM-PMP (1S) UA-AnM-PMP (2S) UA-GlcNAc-PMP* UA-GlcNAc-PMP (1S)*

492 748 828 903 983 492 1047 1127 524 604 492 748 828 727 807

GlcNS-UA-GlcNAc-UA-GlcNS-UA 3S

GlcNS-UA-GlcNS-UA-GlcNAc 3S

a UA, uronic acid; GlcNS, glucosamine N-sulfate; GlcNAc, N-acetylglucosamine; S, sulfate; AnM-anhydromannose. An asterisk (*) indicates key oligosaccharides for determination of the different structures.

Figure 6. Extracted ion chromatograms for the sulfated forms of the tetrasaccharide, UA-GlcN-UA-AnM (3-5S). The tetrasaccharide was lyophilized, derivatized with PMP and analyzed by reverse-phase HPLC ESI-MS. The data were searched using XIC (with a ( 0.5 amu window) corresponding to the three sulfated species: Panel a shows the XIC of m/z 701.8 with a peak at 10.1 and 10.4 min, corresponding to the [M-H]-2 for UA-GlcN-UA-AnM (5S); Panel b shows the XIC of m/z 662.0 with a peaks from 10 to 14 min, corresponding to the [M-H]-2 for UA-GlcN-UA-AnM (4S); and Panel c shows the XIC of m/z 621.8 with peaks from 10 to 14 min, corresponding to the [M-H]-2 for UA-GlcN-UA-AnM (3S).

3, 4, and 5 sulfated forms of the hexasaccharide composition was present in the original oligosaccharide mixture and that only a small proportion was derived from the loss of sulfate in the electrospray source. The difference between the degree of sulfate loss in the tetrasaccharide produced from nitrous acid digestion (Figure 6) and the MPS IIIA hexasaccharide (Figure 7) may reflect the number of sulfates per monosaccharide and the position of the sulfates on these structures.

Figure 7. Extracted ion chromatograms for the sulfated forms of the hexasaccharide, GlcNS-UA-GlcNAc-UA-GlcN-UA (2-5S). Oligosaccharides eluting from the Bio-Gel P4 column at 160 mL were lyophilized, derivatized with PMP and analyzed by reverse-phase HPLC ESI-MS. The data were searched using XIC (with a ( 0.5 amu window) corresponding to the four sulfated species: Panel a shows the XIC of m/z 899.8 with a peak at 43.8 min, corresponding to the [M-H]-2 for GlcNS-[UA-GlcNAc]/[UA-GlcN]-UA (5S); Panel b shows the XIC of m/z 859.7 with a peak at 45.0 min, corresponding to the [M-H]-2 for GlcNS-[UA-GlcNAc]/[UA-GlcN]-UA (4S); Panel c shows the XIC of m/z 819.0 with a peak at 46.3 min, corresponding to the [M-H]-2 for GlcNS-[UA-GlcNAc]/[UA-GlcN]-UA (3S); and Panel d shows the XIC of m/z 779.8 with a peak at 47.7 min, corresponding to the [M-H]-2 for GlcNS-[UA-GlcNAc]/[UA-GlcN]-UA (2S).

CONCLUSION HPLC ESI-MS/MS is a powerful technique for the structural characterization of HS oligosaccharides. When combined with Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

4541

nitrous acid deamination and recombinant enzyme treatment this approach was able to provide structural detail of HS-derived oligosaccharides isolated from the urine of a MPS IIIA patient. A series of di- to hexadecasaccharides with non-reducing end GlcNS and increasing numbers of GlcN-UA and GlcNAc-UA disaccharides with reducing end UA (“even” numbered) and reducing end GlcNAc (“odd” numbered) were identified. Structural isomers of these oligosaccharides were present representing a complex mixture of oligosaccharides in the urine of MPS IIIA. The number of sulfates on these oligosaccharides varied from one to three per disaccharide unit, and although a minor degree of sulfate loss was observed, multiple sulfate forms were clearly identified. The array of oligosaccharide structures present in these patients has

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Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

the potential to influence a range of cellular processes which may be involved in the pathogenesis of MPS IIIA. ACKNOWLEDGMENT We acknowledge the contribution of the MPS patients and their families. This research was supported by the University of Adelaide (Australia), the National Health and Medical Research Council (Australia), and the Wellcome Trust (United Kingdom) Grant No. 060104Z/00/Z. Received for review November 24, 2005. Accepted April 12, 2006. AC052083D