6S Sulfation

The present work demonstrates the use of negative ion electrospray Q-oTOF mass spectrometry for the 4S/6S sequence analysis of CS oligosaccharides up ...
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Anal. Chem. 2001, 73, 6030-6039

Tandem Mass Spectrometric Determination of the 4S/6S Sulfation Sequence in Chondroitin Sulfate Oligosaccharides Joseph Zaia,* Joseph E. McClellan, and Catherine E. Costello

Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine, 715 Albany Street, R-806, Boston, Massachusetts 02118

Chondroitin sulfate (CS) is a glycosaminoglycan consisting of repeating uronic acid, N-acetylgalactosamine sulfate disaccharide units [-UroA(β1,3)-GalNAcS(β1,4)]n. Chondroitin sulfate type A (CSA) contains glucuronic acid, and 90% of the GalNAc residues are sulfated at the 4-position with 10% at the 6-position. Chondroitin sulfate type C (CSC) contains glucuronic acid, and 90% of the GalNAc residues are sulfated at the 6-position with 10% sulfated at the 4-position. These molecules are fragile due to their high degree of sulfation and are challenging to analyze as a result. This work presents the first evidence that tandem mass spectrometry can be used for the determination of a CS oligosaccharide sequence with respect to the positions of GalNAc sulfation. Using this technique, it is possible to analyze individual components from mixtures, saving much purification effort. Oligosaccharides produced from CSA and CSC are used in this work to demonstrate that CID MS/MS can be used to distinguish positional sulfation isomers. For charge states where charge equals the number of sulfates, abundant oddnumbered Bn and Yn ions are observed. The percent total ion abundances of these ions indicate the position of sulfation. Glycosaminoglycans (GAGs) are linear oligosaccharides found in virtually every animal species and tissue1-3 attached to proteoglycan core proteins. A large number of proteins bind GAGs including those involved in blood coagulation, many growth factors, and several extracellular matrix proteins.4 Mutation in GAG biosynthetic genes leads to severe malformations, indicating the critical roles these carbohydrates play in development.5-8 The diverse nature of GAG structures renders them difficult to purify and hinders correlation of structural determinants and biological activities. To date, difficulties in obtaining sufficient quantities of * Corresponding author: (e-mail) [email protected]; (fax) 617-638-6760. (1) Kraemer, P. M. Biochemistry 1971, 10, 1445-51. (2) Kraemer, P. M. Biochemistry 1971, 10, 1437-45. (3) Nader, H. B.; Ferreira, T. M.; Toma, L.; Chavante, S. F.; Dietrich, C. P.; Casu, B.; Torri, G. Carbohydr. Res. 1988, 184, 292-300. (4) Conrad, H. E. Heparin Binding Proteins; Academic Press: New York, 1998. (5) Hacker, U.; Lin, X.; Perrimon, N. Development 1997, 124, 3565-73. (6) Bellaiche, Y.; The, I.; Perrimon, N. Nature 1998, 394, 85-8. (7) Sen, J.; Goltz, J. S.; Stevens, L.; Stein, D. Cell 1998, 95, 471-81. (8) Perrimon, N.; Bernfield, M. Nature 2000, 404, 725-8.

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pure samples have limited the correlation of GAG structure with function. Proteoglycans are major components of extracellular matrixes and cell surfaces. A wide variety of these molecules contain chondroitin sulfate (CS) linked to serine residues via a xylosecontaining linker. CS consists of repeating units of -GlcA(β1,3)GalNAc(β1,4)- polymerized in chains of 20-50-kDa size, depending on the core protein and tissue location. Examples of CScontaining proteoglycans include the hyalectins (aggrecan, versican, neurocan, brevican), small leucine-rich proteoglycans (decorin, biglycan), bamacan (a basement membrane proteoglycan), and syndecan (a cell surface proteoglycan also modified with heparin sulfate). CS type A (CSA) is sulfated primarily (90%) at the 4-position of GalNAc and CS type C (CSC) is sulfated primarily (90%) at the 6-position. There is evidence to suggest that CS isoforms differing in the position and degree of sulfation play distinct functional roles during development.9 Monoclonal antibodies have been used to map functionally distinct tissue domains10 and demonstrate patterned expression of CS epitopes. In addition, CS epitopes are distributed in a nonrandom manner within CS chains, possibly facilitating interactions with respondent proteins.11 For cartilage aggrecan, changes in CS chain length and sulfation pattern have been correlated with tissue remodeling during skeletal development,12,13 aging,14 and development of osteoarthritis.15 Changes have been demonstrated to the nonreducing terminal saccharides of CS chains,16 to the linker region,17,18 and to the CS core domain.19 (9) Mark, M. P.; Butler, W. T.; Ruch, J. V. Dev. Biol. 1989, 133, 475-88. (10) Sorrell, J. M.; Mahmoodian, F.; Schafer, I. A.; Davis, B.; Caterson, B. J. Histochem. Cytochem. 1990, 38, 393-402. (11) Sorrell, J. M.; Carrino, D. A.; Caplan, A. I. Matrix 1993, 13, 351-61. (12) Roughley, P. J.; White, R. J. J. Biol. Chem. 1980, 255, 217-24. (13) Thonar, E. J.; Buckwalter, J. A.; Kuettner, K. E. J. Biol. Chem. 1986, 261, 2467-74. (14) Bayliss, M. T.; Osborne, D.; Woodhouse, S.; Davidson, C. J. Biol. Chem. 1999, 274, 15892-900. (15) Shinmei, M.; Miyauchi, S.; Machida, A.; Miyazaki, K. Arthritis Rheum. 1992, 35, 1304-8. (16) Plaas, A. H.; Wong-Palms, S.; Roughley, P. J.; Midura, R. J.; Hascall, V. C. J. Biol. Chem. 1997, 272, 20603-10. (17) Shibata, S.; Midura, R. J.; Hascall, V. C. J. Biol. Chem. 1992, 267, 654855. (18) Lauder, R. M.; Huckerby, T. N.; Nieduszynski, I. A. Biochem. J. 2000, 347, 339-48. (19) Plaas, A. H.; West, L. A.; Wong-Palms, S.; Nelson, F. R. J. Biol. Chem. 1998, 273, 12642-9. 10.1021/ac015577t CCC: $20.00

© 2001 American Chemical Society Published on Web 11/15/2001

Quantitative measurement of disaccharides released from enzymatic depolymerization has been used to estimate the changes to CS fine structure in different samples. CS disaccharides are readily resolved using capillary electrophoresis.20 Fluorophoreassisted carbohydrate electrophoresis has been used to profile CS saccharides in conjunction with mercuric ion treatment as a means of determining CS fine structure.21,22 High-pH anionexchange chromatography has been used to fingerprint CS di-, tetra-, and hexasaccharides produced from different sources of CS.18,23 At the present time, direct structural determination of CS oligosaccharides using nuclear magnetic resonance spectroscopy24-27 requires the analysis of milligram quantities of pure oligosaccharide. Sulfated GAG oligosaccharides are easily detected using electrospray mass spectrometry.28-31 The molecular mass distribution can be extracted from these spectra, and results have been reported for CS up to tetradecasaccharide size.28 GAGs produce intense negative electrospray signals, but the spectra are often complex due to the inherent polydispersity of GAG samples, the large number of possible charge states, and the adduction with alkali ions. As a result, it is not presently possible to interpret the electrospray mass spectra of full-length (20-50 kDa) CS chains. The interpretation of mass spectra generated from larger GAG oligosaccharides will be facilitated by application of improved analyzer designs now available with electrospray ionization including orthogonal quadrupole time-of-flight (Q-oTOF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers. Matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrometry has been used to determine the sequences of purified GAG molecules.32 This technique involves pairing the oligosaccharide with a basic peptide and determining the mass of the peptide-oligosaccharide complex.33,34 By measuring the mass before and after partial enzymatic or chemical digestion, it is possible to determine the oligosaccharide residue sequence. Using this approach, it is necessary to purify the GAG oligosaccharide to homogeneity prior to the MALDI-TOF sequencing procedures. (20) Pervin, A.; al-Hakim, A.; Linhardt, R. J. Anal. Biochem. 1994, 221, 182-8. (21) Calabro, A.; Benavides, M.; Tammi, M.; Hascall, V. C.; Midura, R. J. Glycobiology 2000, 10, 273-81. (22) Calabro, A.; Hascall, V. C.; Midura, R. J. Glycobiology 2000, 10, 283-93. (23) Lauder, R. M.; Huckerby, T. N.; Nieduszynski, I. A. Glycobiology 2000, 10, 393-401. (24) Chai, W.; Kogelberg, H.; Lawson, A. M. Anal. Biochem. 1996, 237, 88102. (25) Kinoshita, A.; Yamada, S.; Haslam, S. M.; Morris, H. R.; Dell, A.; Sugahara, K. J. Biol. Chem. 1997, 272, 19656-65. (26) Kitagawa, H.; Tanaka, Y.; Yamada, S.; Seno, N.; Haslam, S. M.; Morris, H. R.; Dell, A.; Sugahara, K. Biochemistry 1997, 36, 3998-4008. (27) Chai, W.; Lawson, A. M.; Gradwell, M. J.; Kogelberg, H. Eur. J. Biochem. 1998, 251, 114-21. (28) Takagaki, K.; Kojima, K.; Majima, M.; Nakamura, T.; Kato, I.; Endo, M. Glycoconjugate J. 1992, 9, 174-9. (29) Chai, W.; Luo, J.; Lim, C. K.; Lawson, A. M. Anal. Chem. 1998, 70, 20606. (30) Kim, Y. S.; Ahn, M. Y.; Wu, S. J.; Kim, D. H.; Toida, T.; Teesch, L. M.; Park, Y.; Yu, G.; Lin, J.; Linhardt, R. J. Glycobiology 1998, 8, 869-77. (31) Beeson, J. G.; Chai, W.; Rogerson, S. J.; Lawson, A. M.; Brown, G. V. Infect. Immun. 1998, 66, 3397-402. (32) Venkataraman, G.; Shriver, Z.; Raman, R.; Sasisekharan, R. Science 1999, 286, 537-42. (33) Juhasz, P.; Biemann, K. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4333-7. (34) Juhasz, P.; Biemann, K. Carbohydr. Res. 1995, 270, 131-47.

Using multidimensional MS, however, it is possible to select an ion from a complex mixture in the first MS dimension, eliminating the need to produce pure oligosaccharides. Collisionalinduced dissociation (CID) of the selected ion produces a pattern of ions that is useful for structural analysis. Sequencing of linear and branched oligosaccharides has been demonstrated using tandem MS.35,36 Recent improvements in high-resolution mass analyzers such as the Q-oTOF and FTICR will facilitate direct sequencing of GAG oligosaccharides without the need for purification. Mass spectrometric differentiation of positional sulfation isomers has been demonstrated for CS-derived disaccharides 2-acetamido-2-deoxy-3-O-(β-D-gluco-4-enepyranosyluronic acid)-4-Osulfo-D-galactose (∆Di4S) and 2-acetamido-2-deoxy-3-O-(β-D-gluco4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (∆Di6S) using negative ion fast atom bombardment (FAB) CID mass analyzed ion kinetic energy spectra.37 This work has been extended to negative ionization electrospray using quadrupole ion trap38 and triple quadrupole39 instruments to show that the CID MS/MS spectra can be used to determine sulfation ratios in mixtures produced by chondroitin lyases without derivatization or purification. Negative ion FAB CID MS/MS has been used to structurally analyze CS tetrasaccharide tri- and tetrasulfate monoclonal antibody antigenic determinants as potassium salts.40 Biological CS samples presented for analysis will be heterogeneous with respect to both oligosaccharide length and sulfate composition. CS sulfation occurs principally on the 4- or the 6-position of GalNAc residues and less commonly at the 2-positon of UroA. The differentiation of 2-sulfation from 4- or 6-sulfation will be straightforward since the mass of ions produced by glycosidic bond cleavage during CID will determine the position.37,41 A similar argument applies for oversulfated CS sequences. The determination of 4- and 6-sulfation is more difficult since both positions lie on the same residue. The present work demonstrates the use of negative ion electrospray Q-oTOF mass spectrometry for the 4S/6S sequence analysis of CS oligosaccharides up to octamers. Oligosaccharides prepared from CSA and CSC produce distinct patterns of product ion abundances in CID MS/MS spectra that allow the determination of sulfate position for individual GalNAc residues. MATERIALS AND METHODS Preparation of Saturated Chondroitin Sulfate Saccharides. CSA and CSC (Seikagaku/Associates of Cape Cod, Falmouth, MA) were digested with testicular hyaluronidase and fractionated using gel filtration chromatography as described.39 Preparation of Reduced, ∆-Unsaturated Chondroitin Sulfate Saccharides. CSA or CSC (10 µL, 10 mg/mL) was mixed with water (10 µL), tris-HCl buffer (2 µL, 1 M, pH 7.4), ammonium acetate (1 µL, 1 M), and chondroitinase ACI (5 µL, 5 mU/µL, (35) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409. (36) Reinhold: V. N.; Reinhold: B. B.; Costello, C. E. Anal. Chem. 1995, 67, 1772-84. (37) Lamb, D. J.; H. M., W.; Mallis, L. M.; Linhardt, R. J. J. Am. Soc. Mass Spectrom. 1992, 3, 797-803. (38) Desaire, H.; Leary, J. J. Am. Soc. Mass Spectrom. 2000, 11, 916-20. (39) Zaia, J.; Costello, C. E. Anal. Chem. 2001, 73, 233-9. (40) Ii, T.; Kubota, M.; Hirano, T.; Ohashi, M.; Yoshida, K.; Suzuki, S. Glycoconjugate J. 1995, 12, 282-9. (41) Ii, T.; Okuda, S.; Hirano, T.; Ohashi, M. Glycoconjugate J. 1994, 11, 12332.

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Seikagaku), digested for 10 min at 37 °C, and boiled for 1 min. An aliquot (10 µL) was then mixed with sodium borodeuteride (20 µL, 0.5 M in 0.05 M sodium hydroxide, Aldrich Chemical Co., Milwaukee, WI) and allowed to react for 2 h at 37 °C. Acetic acid was added (1 µL), and the volume of the reaction mixture was reduced to ∼10 µL in vacuo. The reduced ∆-unsaturated oligosaccharides were fractionated using gel filtration chromatography as described.39 Disaccharides ∆Di4S and ∆Di6S (Seikagaku) were reduced with sodium borodeuteride and purified using gel filtration chromatography using similar procedures. Electrospray Mass Spectrometry. All mass spectra were acquired in the negative ion mode using an Applied Biosystems/ MDS-Sciex API QSTAR Pulsar quadrupole orthogonal time-offlight mass spectrometer. Dried samples were dissolved in water and diluted in sufficient volume of 30% methanol to achieve a 1 pmol/µL solution. Aliquots (3 µL) were infused into the mass spectrometer source using 2-µm orifice nanospray42 tips pulled in-house using a capillary puller (Sutter Instrument Co., Novato, CA). Steady ion signal was typically observed using a needle potential of -1000 V, and all spectra were calibrated externally. RESULTS AND DISCUSSION Figure 1 shows the CID product ion spectra of ions at m/z 461.10 (calculated value m/z 461.083) corresponding reduced ∆Di4S (∆Di-4S-OL, 1a) and reduced ∆Di-6S (∆Di-6S-OL, 1b). The CID product ion spectrum of ∆Di-4S is characterized by an abundant Y1 ion and a Z1 ion of low abundance.37-39 An abundant ion corresponding to Y1 is observed in the CID product ion spectrum of ∆Di-4S-OL (Figure 1a) with ions corresponding to Z1-SO3 and Y1-SO3 observed at increased relative abundance compared with the spectrum of ∆Di-4S. An abundant Z1 ion is observed in the CID product ion spectrum of ∆Di-6S, and the difference in Y1/Z1 ion abundances is used as the basis for mass spectrometric determination of mixtures of the two isomers.38,39 The CID product ion spectrum of ∆Di-6S-OL (Figure 1b), by contrast, is characterized by an abundant Y1 ion, a Z1 ion in low abundance, and abundant ions corresponding to Y1-SO3 and Z1-SO3. The pattern of product ions is quite similar to that observed for ∆Di-4S-OL (Figure 1a), with the primary distinguishing factor the relative abundances of the ions resulting from sulfate loss. Representative calculated and observed m/z values for all spectra are shown in Table 1. These spectra indicate that the sulfate position-dependent glycosidic bond fragmentation observed for ∆Di-4S and ∆Di-6S requires a closed reducing terminal disaccharide residue. This observation has bearing on the spectra of reduced CS oligosaccharides discussed in the following sections. CS oligosaccharides were generated by digestion with chondroitinase ACI or by testicular hyaluronidase and purified by gel filtration chromatography before acquiring mass spectra.39 In theory, ∆-unsaturated CS oligosaccharides (produced by chondroitinases) produce isobaric Cn and Zn ions where n ) even. Saturated CS oligosaccharides (produced by testicular hyaluronidase) produce isobaric Bn and Zn ions and isobaric Cn and Yn ions where n ) even. Reduced ∆-unsaturated CS oligosaccharides, however, produce unique calculated m/z values for Bn, Cn, Yn, and Zn ions. Therefore, the spectra of these derivatives were (42) Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 16780.

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Figure 1. Negative ion electrospray CID product ion spectra of (a) ∆Di4SOL and (b) ∆Di6SOL, acquired at -35 V collision energy. The disaccharide structure and cleavages resulting in product ions are shown in (c): ∆Di4SOL, R ) SO3-, R′ ) H; ∆Di6SOL, R ) H, R′ ) SO3-.

acquired to allow unambiguous assignment of ions based on observed m/z. Spectra of underivatized CS hyaluronidase oligosaccharides differ in structure at both the reducing and nonreducing termini and were acquired to facilitate identification of internal product ions. The composition of saturated CS oligosaccharide will be given as (X,Y,Z) z-, where X ) number of UroA residues, Y ) number of GalNAc residues, and Z ) number of sulfate groups. The composition of reduced ∆-unsaturated CS oligosaccharides will be abbreviated ∆(X,Y,Z)OL z-. Figure 2 shows CID product ion spectra for the ion corresponding to CSA ∆(2,2,2)OL 2- (m/z 459.59, calculated value m/z 459.572), acquired at collision energy -10 (a), -20 (b), and -30 V (c). Representative observed and calculated m/z values for product ions observed in the CID product ion spectra of CS oligosaccharides are included in Table 1. The precursor ion fragments to a minimal degree at -10 V collision energy. At -20 V, ions corresponding to Y11-, Y32-, B31-, and the precursor are observed at moderate relative abundance. The Y11- ion is the most abundant in the spectrum at -30 V collision energy, and the precursor ion is no longer detected. The ion at m/z 282.08 matches the calculated value for Z11- for an unreduced CS oligosaccharide (m/z 282.028) but differs significantly from the expected value for the Z11- ion for a deuterated reduced alditol (m/z 285.050). While the singly charged ion at m/z 458.08 is consistent with C21-

Table 1. Observed and Calculated m/z Values for CS Product and Precursor Ions reduced, ∆-unsaturated

iona B11B31B52B73Y11Y32Y53Y74Z1 precursor ionse dimer [M - H][M - H - SO3]tetramer [M - 2H]2[M - SO3 - 2H]2hexamer [M - 3H]3[M - SO3 - 3H]3octamer [M - 4H]4[M - SO3 - 4H]4-

saturated

obsdb

calcd

composition

obsdb

calcd

composition

157.07 616.10 537.08 510.75 303.06 380.57 406.40 419.32 ndc,d

157.014 616.082 537.071 510.734 303.061 380.561 406.394 419.311 285.050

C6H5O5 C20H26NO19S C34H46N2O33S2 C48H66N3O47S3 C8H15DNO9S C22H35DN2O23S2 C36H55 DN3O37S3 C50H75DN4O51S4 C8H13DNO8S

ndc 634.11 546.10 516.75 300.09 379.06 405.40 418.57 282.04

175.024 634.092 546.076 516.738 300.039 379.050 405.387 418.555 282.028

C6H7O6 C20H28NO20S C34H48N2O34S2 C48H68N3O48S3 C8H14NO9S C22H34N2O23S2 C36H54N3O37S3 C50H74N4O51S4 C8H12NO8S

461.10 381.16 459.59 419.62 459.08 432.46 458.83 438.84

461.083 381.126 459.572 419.594 459.068 432.416 458.816 438.827

C14H21DNO14S C14H21DNO11 C28H41DN2O28S2 C28H41DN2O25S C42H61DN3O42S3 C42H61DN3O39S2 C56H81DN4O56S4 C56H81DN4O53S3

467.09 427.10 464.06 437.41 462.58 442.59

467.067 427.088 464.064 437.412 462.564 442.574

C28H42N2O29S2 C28H42N2O26S C42H62N3O43S3 C42H62N3O40S2 C56H82N4O57S4 C56H82N4O541S3

a Observed m/z values are given for B and Y ions from CSA ∆(4,4,4)OL 4- and CSA (4,4,4)4-; the mass accuracies typify those obtained for n n dimer, tetramer, and hexamer samples. b External calibration was used for all samples. c nd, not detected d This ion is not detected in CID MS/ MS spectra of CS tetramers, hexamers, and octamers. It is detected in spectra of reduced, ∆-unsaturated dimers (Figure 1) at m/z 285.10. e The values were taken from the CID MS/MS spectra of CSA oligosaccharides. Similar values were obtained for CSC oligosaccharides.

for a ∆-unsaturated oligosaccharide (expected m/z 458.060), this assignment is incorrect as will be shown below. The CID product ion spectrum of the ion corresponding to CSA (2,2,2) 2- (Figure 3) shows increasing complexity of fragmentation as collision energy is decreased. Ions corresponding to Y11-, Y32-, and B31- are observed at -20 V collision energy (Figure 3b). The precursor is not detected at -30 V collision energy, and ions at m/z 282 and 458 are observed (Figure 3c). Since CSA (2,2,2) 2- differs in structure from ∆(2,2,2)OL 2- at both the reducing and nonreducing termini, the ions observed at m/z 282 and 458 contain neither terminus and result from multiple bond cleavage. These ions have low abundances between -10 and -20 V collision energy. Figure 4 shows plots of percent total ion abundance for ions produced from CSA ∆(2,2,2)OL 2- (a-c) and CSA (2,2,2) 2(d-f) ions. The ion observed at m/z 458 reaches a maximum percent total ion abundance at -25 to -30 V collision energy for both tetrasaccharides (Figure 4a and d) and diminishes between -30 and -40 V. The ion observed at m/z 282, by contrast, steadily increases in abundance, reaching a maximum at -40 V collision energy. Loss of sulfate from the precursor ion is observed at greater percent total ion abundance for the ion corresponding to ∆(2,2,2)OL 2- (Figure 4a) relative to (2,2,2) 2- (Figure 4d), indicating that the sulfate on the reduced GalNAc is lost. The Y11ion for both tetrasaccharides (Figure 4b and e) reaches a broad maximum between -25 and -35 V collision energy, a pattern distinct from that observed for Y32- for which a maximum is reached between -20 and -25 V. This observation is consistent with the production of ions isobaric with Y11- by multiple bond cleavage between -25 and -40 V collision energy. An ion corresponding to the Y2 ion is not observed at any collision energy for either tetrasaccharide. The B31- ion is observed for both (Figure 4c and f) at a maximum at -20 V collision energy, below which the percent total ion abundance diminishes rapidly. The

ion labeled B11- (m/z 157.07) for CSA ∆(2,2,2)OL 2- (Figure 4c) is observed to increase, reaching a broad plateau between -30 and -40 V collision energy, consistent with its formation from other product ions. An ion is observed at the same m/z value in Figure 4f, consistent with its production by internal fragmentation. The B21- ion has a low percent ion abundance for ∆(2,2,2)OL 2(Figure 4c) and is isobaric with the m/z 458 ion for (2,2,2)2-. From these data it can be concluded that that the ions observed at m/z 282 and 458 in the CID product ion spectra of CSA tetrasaccharides correspond to internal product ions. On the basis of their diminished percent total ion abundance between -30 and -40 V collision energy, several ions appear to undergo further fragmentation. The concomitant increases in percent total ion abundance observed for the ions corresponding to 2821-, Y11-, and B11- are consistent with their formation from multiple bond fragmentation. Figure 5 shows CID product ion spectra for the ion corresponding to CSA ∆(3,3,3)OL 3- acquired at -10 (a), -17.5 (b), and -27.5 V (c) collision energy. At -10 V collision energy, the product ions have very low abundances relative to the precursor ion. The precursor ion remains abundant at -17.5 V collision energy, and the ions corresponding to Y11-, Y32-, Y53-, B31-, and B52- are observed. At -27.5 V collision energy, the precursor ion has very low abundance and those at m/z 282 and 458 have increased abundances. The trend is similar for the CID product ion spectra of CSA (3,3,3) 3- (Figure 6). At -17.5 V collision energy, Y11-, Y32-, Y53-, B31-, and B52- are observed and other ions including m/z 282 and 458 appear at -27.5 V. The presence of ions at m/z 282 and 458 in both sets of spectra (Figures 5 and 6) indicates that these ions contain neither the reducing nor the nonreducing terminus. Odd-numbered glycosidic bond cleavages predominate for these hexamers. Figure 7 compares plots of percent total ion abundance as a function of collision energy for the ions corresponding to CSA Analytical Chemistry, Vol. 73, No. 24, December 15, 2001

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Figure 2. Negative ion electrospray CID product ion spectra of CSA ∆(2,2,2)OL 2- acquired at collision energy (a) -10, (b) -20, and (c) -30 V. The structure and cleavages resulting in product ions are shown in (d).

∆(3,3,3)OL 3- and (3,3,3)3-. As observed in the plots for the tetramers (Figure 4), the percent total ion abundance for m/z 282 (Figure 7a and d) steadily increases between -20 and -40 V collision energy for both hexamers. The ion at m/z 458 is at maximum percent ion abundance at approximately -25 V collision energy and diminishes between -25 and -40 V, an observation consistent with the further fragmentation of this ion. Fragmentation of the ∆(3,3,3)OL 3- ion produces abundant ions corresponding toY11- and Y32- ions with a low-abundance Y53- ion (Figure 7b). The Y32- ion is of lower percent total ion abundance for CSA (3,3,3) 3- (Figure 7e), therefore indicating that its formation is more favored for the reduced hexasaccharide with an open ring reducing terminal GalNAc. The B31- and B52- ions are observed in spectra of both hexasaccharides (Figure 7c and f) to reach maximum percent total ion abundance between -15 and -20 V collision energy and to diminish between -25 and -40 V. An ion observed at m/z 157 (B11- in Figure 7c) in both cases increases 6034 Analytical Chemistry, Vol. 73, No. 24, December 15, 2001

Figure 3. Negative ion electrospray CID product ion spectra of CSA (2,2,2)2- acquired at collision energy (a) -10, (b) -20, and (c) -30 V. The structure and cleavages resulting in product ions are shown in (d).

steadily in abundance between -25 and -40 V collision energy, consistent with its formation from other ions by subsequent fragmentation. Note that ions corresponding to B21- and B42- for CSA (3,3,3)3- are isobaric with m/z 458 and are therefore not shown in Figure 7f. At -10 V collision energy, CID product ion spectra of ions corresponding to CS ∆(4,4,4)OL 4- (Figure 8) and (4,4,4)4- (Figure 9) show distinct Bn and Yn fragmentation at odd-numbered glycosidic bonds. The comparison CID product ion spectra of the reduced, ∆-unsaturated oligosaccharides (Figure 8) with those of the saturated oligosaccharides (Figure 9) allow unambiguous assignment of the ions based on their m/z values despite the redundancy of the repeating disaccharide structure of CS. The pattern of odd-numbered glycosidic cleavages, clear from the octamer spectra, indicates preferential scission of the GlcA-GlcNAc glycosidic bond for these molecules. It is important to note that data were collected for CS oligosaccharides with one negative

Figure 4. Percent total ion abundance versus collision energy for product ions of CSA ∆(2,2,2)OL 2- (a-c) and CSA (2,2,2)2- (d-f). See Figures 2d and 3d for structures and cleavages resulting in product ions.

charge per sulfate group. Under these conditions, all sulfates are present as free acids and sulfate losses are minimized. The relative abundances of product ions differ depending on whether the oligosaccharides are composed primarily of 4- or 6-sulfated GalNAc, as shown in Figures 8 and 9. Figure 10 compares percent total ion abundances for the ion corresponding to ∆(4,4,4)OL 4- derived from CSA and CSC. The Bn and Yn ions have greater abundances for CS octamers at collision energy -10 V than for tetramers (Figure 4) or hexamers (Figure 7). It is therefore clear that the extent of fragmentation at a given collision energy increases with increasing CS oligosaccharide size. The ions observed at m/z 282 and 458 have greater abundances for CSA ∆(4,4,4)OL 4- (Figure 10a) than for CSC ∆(4,4,4)OL 4(Figure 10d), indicating a difference in the formation of these ions resulting from the position of sulfation. The Yn ions have greater abundances for CSA ∆(4,4,4)OL 4- relative to CSC ∆(4,4,4)OL 4but follow similar trends for both types of ∆-unsaturated octamers (Figure 10b and e). The Bn ions are also lower in percent total ion abundance for CSC ∆(4,4,4)OL 4- relative to CSA ∆(4,4,4)OL 4-. Note that the ion labeled B11- in Figure 10c and f has an m/z value of 157. Figure 11 compares plots of percent total ion abundance for CSA (4,4,4) 4- (a-c) and CSC (4,4,4) 4- (d-f). The abundances of m/z 282 and 458 are again different for CSA and CSC (4,4,4) 4(Figure 11a and d). CSC (4,4,4) 4- fragments to produce m/z 458 in lower percent total ion abundance and an ion corresponding to [M-SO3-4H]4- in higher abundance relative to CSA (4,4,4) 4-. While the Y11- ion is higher in percent total ion abundance for CSA (4,4,4) 4-, the other Yn ions are of similar values for both octamers (Figure 11b and e). The CSA oligosaccharides (Figures 10c and 11c) produce Bn ions with greater percent total ion abundance that the CSC oligosaccharides (Figures 10f and 11f), indicating that the formation of these ions may be influenced by the position of the neighboring GalNAc sulfate group. In addition, the Y32- ion is significantly more abundant for the CSA oligosac-

charides than for those derived from CSC as shown in Figure 10. Odd-numbered Bn ions are observed for both the reduced ∆-unsaturated octamers (Figure 8) and the saturated octamers (Figure 9), corresponding to cleavage at the reducing side of GlcA residues. Cleavage to the reducing side of GalNAcS residues is not observed. Cleavage to the reducing side of GlcA also results in odd-numbered Yn ions as shown in Figures 12b and 13b. This trend is consistent among CS tetramers (Figures 2 and 3), hexamers (Figures 5 and 6), and octamers derived from both CSA and CSC. To further investigate these results, mixtures of purified CSA and CSC octamers were prepared and CID product ion spectra were acquired at -17.5 V collision energy. Figure 12 shows the percent total ion abundances of Bn ions for mixtures of CS octamers ranging from 1:0 CSA/CSC to 0:1 in graphical form. For the saturated octamers (Figure 12a), the B31-, B52-, and B73ions show a clear decrease in percent total ion abundance with decreasing CSA/CSC ratio. The trend is reversed for the [M 4H]4- ion, indicating that the CSC ion fragments to a lesser degree than the CSA ions at -17.5 V collision energy. The percent total ion abundance for the reduced ∆-unsaturated octamers show a similar trend for the ions corresponding to B31- and B52- as shown in Figure 12b. The B73- ion is of comparatively low abundance in the spectra and increases slightly in percent total ion abundance as the CSA/CSC ratio decreases. This difference between the abundance of the B7 ion in Figure 12a versus b is likely due to the open ring structure of the reducing GalNAc residue for the oligosaccharide alditols. Since the extent of internal bond fragmentation is low at -17.5 V collision energy, the ion at m/z 157 is labeled B11- in Figure 12. Figure 13 shows the trend in Yn ion percent total ion abundance with decreasing CSA/CSC ratio. For the saturated octasaccharides (Figure 13a), the Y11- ion shows a clear decrease in percent total ion abundance as the CSA/CSC ratio decreases. This trend is similar to the expected abundances based on the appearance of Analytical Chemistry, Vol. 73, No. 24, December 15, 2001

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Figure 5. Negative ion electrospray CID product ion spectra of CSA ∆(3,3,3)OL 3- acquired at collision energy (a) -10, (b) -17.5,-

the CID product ion spectra of ∆Di4S (abundant Y1 ion) and ∆Di6S (abundant Z1 ion).38,39 The abundances of the ions corresponding to Y32-, Y53-, and Y74-, however, show no significant change as the CSA/CSC ratio decreases. The Y11- ion of ∆(4,4,4)OL 4- shows no change with CSA/CSC ratio, as expected based on the open ring structure of the reducing end GalNAc residue (Figure 13b). The ion corresponding to Y32-, on the other hand, decreases in percent total ion abundance with decreasing CSA/ CSC ratio with a trend similar to that observed for Y11- for (4,4,4) 4-. The ion corresponding to Y53- shows a small decrease in percent total ion abundance as the CSA/CSC ratio decreases and that corresponding to Y74- does not change. CID product ion spectra were acquired on CS oligosaccharides for which charge equaled the number of sulfate groups, observed to be the most abundant charge state. Under these conditions, the loss of sulfate from the precursor ion was kept to a minimum. The need for precise control over the CID process is demonstrated by the observation of internal product ions at m/z 282 and 458 in spectra of both reduced, ∆-unsaturated and saturated CS oligosac6036 Analytical Chemistry, Vol. 73, No. 24, December 15, 2001

Figure 6. Negative ion electrospray CID product ion spectra of CSA (3,3,3) 3- acquired at collision energy (a) -10, (b) -17.5, and (c) -27.5 V. The structure and product ion cleavages are shown in (d).

charides. The internal fragment ions abundances are minimized when the collision energy is kept in the -10 to -20 V range, with the optimal voltage increasing as the size of the oligosaccharides increases. Several product ions (B11-, Y11-, and m/z 282) were observed to increase in abundance between -25 and -40 V collision energy, consistent with their production by multiple bond cleavage. Again, these processes are minimized by keeping the collision energy between -10 and -20 V. Of considerable interest is the observation that abundant Bn (other than B1) and Yn ions are observed for odd-numbered glycosidic bonds for CS oligosaccharides where charge equals number of sulfate groups. While these ions result from cleavage to the reducing side of GlcA residues, cleavage to the reducing side of GalNAcS was not observed. Thus, cleavage to the reducing side of a given GalA residue produces complementary Bn/Yn pairs since all sulfate groups are charged. Results for mixtures (Figures 12 and 13) show that while the percent total ion abundance for Bn ions decreases with the CSA/

Figure 7. Plots of percent total ion abundance versus collision energy for product ions of CSA ∆(3,3,3)OL 3- (a-c) and CSA (3,3,3)3- ion (d-f). See Figures 5d and 6d for structures and cleavages resulting in product ions.

Figure 8. Negative ion electrospray CID product ion spectra acquired at collision energy -17.5 V, (a) CSA ∆(4,4,4)OL 4- and (b) CSC ∆(4,4,4)OL 4-. The symbolic structure and product ion cleavages are shown in (c).

CSC ratio, the percent total ion abundance for the precursor ion increases. This observation is consistent with a greater degree of fragmentation for 4-sulfated (CSA) than for 6-sulfated (CSC) octasaccharides. For ∆(4,4,4)OL 4- (Figure 12b), the dependence of the ion corresponding to B73- on the CSA/CSC ratio is not

Figure 9. Negative ion electrospray CID product ion spectra acquired at collision energy -17.5 V, (a) CSA (4,4,4) 4- and (b) CSC (4,4,4) 4-. The symbolic structure (see Figure 8 for definitions) and product ion cleavages are shown in (c).

observed. The abundance of this ion appears to depend on the position of sulfation of the GalNAc to the reducing side in CID product ion spectra of (4,4,4)4- (Figure 12a). The B1 ion is observed with very low abundance, presumably due to its lack of a sulfate group, and its appearance provides no information of the position of sulfation of GalNAc-2. Significantly, both the CSA Analytical Chemistry, Vol. 73, No. 24, December 15, 2001

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Figure 10. Percent total ion abundance versus collision energy for product ions of CSA ∆(4,4,4)OL 4- (a-c) and CSC ∆(4,4,4)OL 4- (d-f). See Figure 8 for the structure and product ion cleavages.

Figure 11. Percent total ion abundance versus collision energy for product ions of CSA (4,4,4) 4- (a-c) and CSC (4,4,4) 4- (d-f). See Figure 9 for the structure and product ion cleavages.

and CSC ∆(4,4,4)OL 4- precursor ions have percent total ion abundances equal to that of CSC (4,4,4)4-. This observation indicates that a substantial fraction of the molecules of the CSA (4,4,4)4- fragment to the reducing side of GlcA-7. The fact that the B73- percent total ion abundance in Figure 12a is significantly less than that observed for its complement, Y11-, in Figure 13a, indicates the possibility of subsequent fragmentation. For (4,4,4)4-, the percent total ion abundance for Y11- decreases directly with the CSA/CSC ratio (Figure 13a). This trend is not observed for ∆(4,4,4)OL 4-, indicating that the abundance of this ion depends on the position of sulfation of the reducing GalNAc. The ion corresponding to Y32- for ∆(4,4,4)OL 4- (Figure 13a) decreases in percent total ion abundance with the CSA/CSC ratio in contrast to the flat response for the same ion for (4,4,4)4-. This 6038

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ion in Figure 13b produces a similar response to the Y11- ion in Figure 13a, indicating that its abundance depends on the position of sulfation at GalNAc-6. CONCLUSIONS These results support the conclusion that CID product ion abundances indicate the position of sulfation for three of the four GalNAc residues of unsaturated CS octamers. The pattern of abundances observed in Figures 12 and 13 is consistent with sequential GlcA-GalNAc glycosidic bond fragmentation beginning with the reducing end of the oligosaccharide. This conclusion is supported by the comparatively abundant ion corresponding to B73- and the low-abundance ion corresponding to Y74-, both of the same composition with respect to number of monosaccharide

Figure 12. Bar graph representations of percent total ion abundances for Bn ions produced from CSA/CSC octasaccharides mixtures, ratios as given: (a) (4,4,4)4-; (b) ∆(4,4,4)OL 4-. See Figure 8c for symbol definitions.

CSC dimers,37-39 tetramers (Figure 4), and hexamers (Figure 7) indicate that the pattern of ion abundances is general to CS oligosaccharides. The maximum CS oligosaccharide size that can be analyzed by tandem MS has yet to be defined. The information produced is clearly useful for defining the position of sulfation on CS oligosaccharides. In principle, the determination of the position of sulfation of GalNAc residues in an unknown CS oligosaccharide entails acquisition of a standard series of oligosaccharide mixtures such as presented in Figures 12 and 13. The position of sulfation at each GalNAc residue would be determined by superimposing the percent total ion abundances for Bn ions produced from the unknown oligosaccharide on the bar graph. Owing to the lack of a B1 ion for charge states where z equals the number of sulfates, the 4S/6S positional information cannot be produced on the GalNAc residue adjacent to the nonreducing terminus. In continuing work, the fragmentation patterns produced by charge states other than those studied here will be investigated in order to produce information on the position of sulfation of this residue. It is also not possible to determine the position of sulfation of the reducing terminal GalNAc for reduced oligosaccharides. The use of reducing terminal derivatives will be investigated to facilitate the analysis of CS oligosaccharides without losing the information regarding the position of sulfation of the reducing terminal GalNAc residue. Further development of tandem mass spectrometry for GAG sequencing will involve careful study of the CID process for these molecules. It will be important to assess the influence of charge state and the adduction of alkali ions on the information content of CID product ion spectra and to analyze larger CS oligosaccharides. While the Q-oTOF instruments are extremely sensitive, quadrupole ion trap and FTICR mass spectrometers allow greater control over the CID process. Using the latter two instruments, fragmentation is specific for the isolated ion, excitation of ions formed during CID is kept to a minimum, and the abundances of internal product ions are expected to be low. Investigation into the use of these techniques for sequencing GAGs is presently underway in our research group. Abbreviations used: CID, collision-induced dissociation; CS, chondroitin sulfate; CSA, chondroitin sulfate type A; CSC, chondroitin sulfate type C; ∆Di4S, 2-acetamido-2-deoxy-3-O-(β-D-gluco4-enepyranosyluronic acid)-4-O-sulfo-D-galactose; ∆Di6S, 2-acetamido2-deoxy-3-O-(β-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-Dgalactose; FAB, fast atom bombardment; FTICR, Fourier transform ion cyclotron resonance; GAG, glycosaminoglycan; MALDI-TOF, matrix-assisted laser desorption/ionization; MS, mass spectrometry.

Figure 13. Bar graph representations of percent total ion abundances for Yn ions produced from CSA/CSC octasaccharides mixtures, ratios as given, (a) (4,4,4)4-; (b) ∆(4,4,4)OL 4-. See Figure 8c for symbol definitions.

ACKNOWLEDGMENT This work was funded by NIH/NCRR grant P41 RR10888. The Applied Biosystems/MDS-Sciex API QSTAR Pulsar quadrupole orthogonal time-of-flight mass spectrometer was provided on loan from Applied Biosystems. Mark McComb (Boston University School of Medicine) provided expert advice on operation of the QSTAR instrument.

residues. Figures 10 and 11 show that the abundance of the latter ion does not increase at lower collision energies and that multiple fragmentation takes place between -25 and -40 V collision energy. The differences in Bn and Yn ion abundances for CSA and

Received for review July 27, 2001. Accepted October 12, 2001. AC015577T Analytical Chemistry, Vol. 73, No. 24, December 15, 2001

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