Anal. Chem. 2007, 79, 6390-6397
On-Line HPLC/ESI-MS Separation and Characterization of Hyaluronan Oligosaccharides from 2-mers to 40-mers Nicola Volpi*
Department of Biologia Animale, University of Modena and Reggio Emilia, Modena, Italy
A new method for the separation and identification of oligosaccharides obtained by enzymatic digestion of hyaluronic acid (HA) with hyaluronidase (EC 3.2.1.35) using on-line high-performance liquid chromatography/ electrospray mass spectrometry (HPLC/ESI-MS) is presented. Reversed-phase ion pairing-HPLC, based on tributylamine salts and a volatile mobile phase, provided excellent chromatographic resolution and separation was achieved for HA oligosaccharides containing 2-40 monomers (from 2- to 40-mers). Using the on-line ion trap mass analyzer, complete identification and structural information for each HA oligomer species was obtained. In particular, a series of negatively charged species of different m/z ratios are seen for each oligosaccharide. Smaller HA species, from 2- to 4-mers, exhibit mainly [M - H]-1 anions, whereas the 6-10-mers exist predominantly as the charge state of -2. The HA oligomers from 12- to 18-mers are mainly represented by [M - 3H]-3 anions while species from 20- to 28/30-mers are characterized by a charge state of -4. HA oligosaccharides from 32- to 40-mers exist as [M - 5H]-5 anions. Furthermore, for smaller HA species, from 4/6- to 18/20mers, ESI-MS revealed, generally in low relative abundance, anions related to the loss of one/two monosaccharide unit(s) from the oligomers, and no odd-numbered anions were produced for HA species greater than 20-mers. Hyaluronic acid (HA) is a linear, polydisperse, natural polysaccharide with the repeating disaccharide units consisting of D-glucuronic acid and N-acetylglucosamine linked by a β-1,4 linkage.1 Currently, HA is used as a drug with (possible) applications and benefits in wound healing,2 inflammatory processes,3 osteoarthritis,3 and cancer,4 as well as a pharmaceutical excipient in hydrogel formation5 and drug attachment.6 A key finding in this field is that HA oligosaccharides or small polymer fragments may have several important biological and pharmacological functions, such * E-mail:
[email protected]. Fax: 0039 (0)59 2055548. Tel: 0039 (0)59 2055543. (1) Fraser, J. R. E.; Laurent, T. C.; Laurent, U. B. G. J. Intern. Med. 1997, 242, 27-33. (2) Gao, F.; Cao, M.; Yang, C.; He, Y.; Liu, Y. J. Biomed. Mater. Res. B: Appl. Biomater. 2006, 78, 385-92. (3) Ghosh, P.; Guidolin, D. Semin. Arthritis Rheum. 2002, 32, 10-37. (4) Rooney, P.; Kumar, S.; Ponting, J.; Wang, M. Int. J. Cancer 1995, 60, 6326. (5) Barbucci, R.; Lamponi, S.; Borzacchiello, A.; Ambrosio, L.; Fini, M.; Torricelli, P.; Giardino, R. Biomaterials 2002, 23, 4503-13. (6) Pouyani, T.; Prestwich, G. D. Bioconjugate Chem. 1994, 5, 339-47.
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as the stimulation of angiogenesis2 and in various drug delivery systems.7 Various enzymes, named hyaluronidases, are able to catalyze the HA depolymerization. They are generally classified into three main families according to their catalytic mechanism: hyaluronate 4-glycanohydrolase (EC 3.2.1.35), hyaluronate 3-glycanohydrolase (EC 3.2.1.36), and hyaluronate lyase (EC 4.2.2.1).8-10 Electrospray ion mass spectrometry (ESI-MS) characterization of saturated HA oligomers derived from digestion with testicular hyaluronidase11-13 and of unsaturated oligosaccharides resulting from enzymatic degradation with hyaluronate lyase has been reported.7 The highly complex nature of the HA oligomer mixtures obtained by enzymatic digestion makes specific analysis a challenge. For a rapid determination of complex mixtures of HA oligosaccharides from digests, without the further derivatization step, analytical separation is usually required with specific evaluation by means of MS. In fact, UV detection does not provide specific structural information, and there are no standard oligomers available to enable a proper assignment according to the observed electrophoretic/chromatographic migration times. At the moment, off-line liquid chromatography (LC)14 or direct coupled with ESI-MS15 and on-line capillary electrophoresis with ESI-MS7 have been used for the separation and identification of HA oligosaccharides. However, the HA oligomers have been obtained by hyaluronate lyase treatment producing unsaturated derivatives, and the separation has been obtained for HA species up to 16mers.7,14 In fact, no on-line HPLC direct coupled with ESI-MS has been investigated for the separation and characterization of saturated HA oligomers derived by the treatment with hyaluronate glycanohydrolase(s) but just previous chromatographic preparative approaches to isolate HA fractions for subsequential off-line MS analysis.11-13,16 (7) Ku ¨ hn, A. V.; Ru ¨ ttinger, H. H.; Neubert, R. H.; Raith, K. Rapid Commun. Mass Spectrom. 2003, 17, 576-82. (8) Linhardt, R. J.; Galliher, P. M.; Cooney, C. L. Appl. Biochem. Biotechnol. 1986, 12, 135-76. (9) Mio, K.; Stern, R. Matrix Biol. 2002, 21, 31-7. (10) Blundell, C. D.; Almond, A. Anal. Biochem. 2006, 353, 236-47. (11) Prebyl, B. S.; Kaczmarek, C.; Tuinman, A. A.; Baker, D. C. Carbohydr. Res. 2003, 338, 1381-7. (12) Tawada, A.; Masa, T.; Oonuki, Y.; Watanabe, A.; Matsuzaki, Y.; Asari, A. Glycobiology 2002, 12, 421-6. (13) Mahoney, D. J.; Aplin, R. T.; Calabro, A.; Hascall, V. C.; Day, A. J. Glycobiology 2001, 11, 1025-33. (14) Price, K. N.; Tuinman, A.; Baker, D. C.; Chisena, C.; Cysyk, R. L. Carbohydr. Res. 1997, 303, 303-11. (15) Kuhn, A. V.; Raith, K.; Sauerland, V.; Neubert, R. H. J. Pharm. Biomed. Anal. 2003, 30, 1531-7. 10.1021/ac070837d CCC: $37.00
© 2007 American Chemical Society Published on Web 07/04/2007
MALDI time-of-flight (TOF) MS11,17 and ESI-MS11-13 have been the most favored approaches for the analysis of HA-derived oligosaccharides. Both ESI-MS and MALDI-TOF-MS offer particular advantages in the analysis of large ionic macromolecules. In MALDI, the major ion formed is typically the singly charged species, making MALDI-TOF-MS well suited to the analysis of mixtures. However, the ion yield depends on the chemical nature and size of the analyte, and MALDI is sufficiently energetic to damage the analyte. By contrast, ESI-MS utilizes a flowing stream containing the analyte, ensures very gentle ionization, and can be easily combined with on-line liquid-phase separation techniques, such as high performance liquid chromatography (HPLC) and capillary electrophoresis. Strong anion-exchange (SAX)-HPLC conventionally used to fractionate glycosaminoglycan- and HAderived oligosaccharide mixtures18 is difficult to interface with MS due to the high ionic strength mobile phases required to elute multiply charged oligosaccharides. On the contrary, reversedphase ion pairing (RPIP)-HPLC, based on tributylamine salts and a volatile mobile phase, provides excellent chromatographic resolution and MS compatibility.19 In fact, this analytical approach has been applied to the separation and characterization of oligosaccharides derived from heparosan20 and of highly sulfated species from heparin.19 However, to our knowledge, HA fully saturated oligosaccharides obtained by enzymatic treatment have not yet been investigated by on-line HPLC/ESI-MS. Thus, the aim of this work is to demonstrate the feasibility of this approach applied to both small and large HA-derived oligosaccharides. EXPERIMENTAL SECTION Materials. HA sodium salt from rooster comb and hyaluronidase from bovine testes (EC 3.2.1.35, 300 units/mg of solid) were purchased from Sigma-Aldrich. Acetonitrile, MS-grade, and all other reagents, of the purest grade available, were from SigmaAldrich. Preparation of HA Oligosaccharides. HA was partially degraded by bovine testicular hyaluronidase. To a solution containing 20 mg of polysaccharides in 2 mL of 100 mM sodium acetate buffer adjusted to pH 5.2 with acetic acid containing 150 mM NaCl, 5000 units of hyaluronidase was added, and enzymatic digestion was performed at 37 °C for 1-8 h. The incubation time of the hyaluronidase varied according to the size of HA oligosaccharides to be obtained, assessed at various time points by running 5 µL of the reaction mixture on the SAX-HPLC. The reactions were stopped by boiling for 20 min. The sample was centrifuged at 10 000 rpm for 30 min at 5 °C, and the supernatant was run in HPLC/ESI-MS. Analytical SAX-HPLC. High-performance liquid chromatography equipment was from Jasco (pump model PU-1580, UV detector model UV-1570, Rheodyne injector equipped with a 20µL loop, software Jasco-Borwin rel. 1.5). The HA oligosaccharides (16) Takagaki, K.; Kojima, K.; Majima, M.; Nakamura, T.; Kato, I.; Endo, M. Glycoconjugate J. 1992, 9, 174-9. (17) Yeung, B.; Marecak, D. J. Chromatogr., A 1999, 852, 573-81. (18) Imanari, T.; Toida, T.; Koshiishi, I.; Toyoda, H. J. Chromatogr., A 1996, 720, 275-93. (19) Thanawiroon, C.; Rice, K. G.; Toida, T.; Linhardt, R. J. J. Biol. Chem. 2004, 279, 2608-15. (20) Kuberan, B.; Lech, M.; Zhang, L.; Wu, Z. L.; Beeler, D. L.; Rosenberg, R. D. J. Am. Chem. Soc. 2002, 124, 8707-18.
Figure 1. Strong anion-exchange HPLC of HA oligosaccharide mixtures produced by testicular hyaluronidase digestion for 8 h and detected at 214 nm.
were analyzed by SAX-HPLC separation using a 150 × 4.6-mm stainless steel column Spherisorb 5-SAX (5 µm, trimethylammoniopropyl groups SiCH2CH2CH2N+(CH3)3 in Cl- form, from Phase Separations Ltd., Deeside Industrial Park, Deeside Clwyd, U.K.) and detection at 214 nm. Isocratic separation was performed using 50 mM NaCl pH 4.00 for 5 min followed by a 5-60 min linear gradient from 100% 50 mM NaCl pH 4.00 to 100% 1.2 M NaCl pH 4.00, at a flow rate of 1.2 mL/min. HPLC/ESI-MS. HPLC separation was performed on a 3-µm Gemini C18 110 Å column (4.6 × 150 mm) from Phenomenex (Torrance, CA). According to Thanawiroon et al.,19 eluent A was water/acetonitrite (80:20) and eluent B was water/acetonitrı`le (35:65). Tributylamine (15 mM) and ammonium acetate (50 min) were added to both eluents. The mobile phase pH was adjusted to 7.0 with acetic acid. The sample (20 µL) was injected, and a linear gradient (from 0 to 100% eluent B in 90 min) at a flow rate of 0.3 mL/min was used for elution. ESI mass spectra were obtained using an Agilent 1100 VL series (Agilent Technologies, Inc.). The electrospray interface was set in negative ionization mode with the capillary voltage at 3,500 V and a heat source of 325 °C in full scan spectra (200-2200 Da, 10 full scans/s). Nitrogen was used as a drying (9 L/min) and nebulizing gas (40 psi). Software versions were 4.0 LC/MSD trap control 4.2 and Data Analysis 2.2 (Agilent Technologies, Inc.). RESULTS AND DISCUSSION Hyaluronidase is used in reducing the molecular weight of HA to prepare low molecular weight derivatives and to facilitate the isolation and purification of specific HA oligosaccharide components.11-13 HA was depolymerized by partial controlled digestion with testicular hyaluronidase and separated into oligosaccharides from 2-mers to over 40-mers by analytical anion-exchange chromatography (Figure 1). Extensive experiments were undertaken to optimize the HPLC/ESI-MS on-line separation of both small and large HAderived oligosaccharides. According to Thanawiroon et al.,19 RPIPHPLC with an MS-friendly mobile phase including the volatile ionpairing reagent tributylamine, the volatile inorganic salt ammonium Analytical Chemistry, Vol. 79, No. 16, August 15, 2007
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Figure 2. TIC of HA oligosaccharides up to 40-mers in negative ion mode separated by means of RPIP-HPLC. The inlet panel shows the chromatogram expanded in the region from 6 to 42 min.
acetate, and the organic modifier acetonitrile was used. Furthermore, the choice of a small analytical RP-C18 column having particle size of 3 µm and pore size of 110 Å enabled the separation and analysis of discrete amounts of HA digest mixture, ∼200 µg, useful for ESI-MS detection, by maintaining the capacity to resolve each single oligosaccharide species in a relatively short time. The total ion chromatogram (TIC) of the HA oligosaccharides in negative ion mode is illustrated in Figure 2. As can be clearly seen, HA oligosaccharides up to 40-mers are separated in ∼35 min. Calculated m/z values for HA oligomers are shown in Table 1. The negative ESI-MS spectrum of the peak at the retention time of 6.5 min is illustrated in Figure 3A, showing the main ion at m/z 396.1 with a relative abundance of 100%. The peak with the retention time of 7.5 min was identified as the HA 4-mer oligosaccharide having the main ion at m/z 775.3 with a relative abundance of 85% (Figure 3B and Table 2). The presence of the ion at m/z 572.2 corresponding to the 3-mer species and having a relative abundance of 15% probably deriving from the loss of one monosaccharide unit7,11,13,14 was also detected (Figure 3B). The ESI-MS spectrum of the peak at the retention time of 9.0 min is reported in Figure 3C, showing various ions, at m/z 1154.4 belonging to the HA 6-mer oligomer and at m/z 576.7 corresponding to the 6-mer having a charge state of -2. The 5-mer ion with a charge state of -2 at m/z 475.2 and the 4-mer ion at m/z 775.2 were also identified as being derived from the cleaved HA 6-mer species.7,11,13,14 The peak at 11.2 min was easily identified as the 8-mer oligosaccharide by the presence of two main ions with a charge state of -2 at m/z 766.3 corresponding to the 8-mer species and at m/z 664.7 corresponding to the 7-mer species (Figure 3D). Minor ions were the 6-mer species with a charge state of -2 at m/z 576.7 and the 5-mer species with a charge state of -2 at m/z 475.1 (Table 2). The ESI-MS spectrum of the peak at the retention time of 13.8 min is shown in Figure 3E, having various ions, at m/z 956.0 belonging to the HA 10-mer oligomer with a charge state of -2 and at m/z 854.4 corresponding to the 9-mer having a charge state of -2. The 10-mer ion with a charge state of -3 at 6392
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m/z 637.0 and the 9-mer ion at m/z 569.4 were also identified as being derived from the cleaved HA 6-mer species.7,11,13,14 The peak at 17.1 min was identified as the 12-mer oligomer by the presence of two main ions at m/z 1154.4 corresponding to the 12-mer species with a charge state of -2 and at m/z 763.5 species with a charge state of -3 (Figure 3F). Other ions were the 11-mer species with a charge state of -3 at m/z 695.7 and the 10-mer species with a charge state of -2 at m/z 955.9 (Table 2). The 14-mer HA species at the retention time of 20.9 min was identified by the presence of the ions with the charge state of -2 at m/z 1334.9 and of -3 at m/z 889.9 (Figure 3G). Two other ions having low abundance (Table 2) were the 12-mer species at m/z 695.7 and the 13-mer species at m/z 955.9 both with a charge state of -3. The 16-mer HA oligomer at the retention time of 23.7 min showed a main ion with the charge state of -3 at m/z 1016.4 (Figure 3H) while the 18-mer species (retention time of 25.9 min) was identified by the presence of two main ions at m/z 1142.9 corresponding to the 18-mer oligosaccharide with a charge state of -3 and at m/z 856.9 with a charge state of -4 (Figure 3I). The ESI-MS spectra of the HA oligomers from 20- to 28-mers showed a main corresponding ion with a charge state of -4 of high relative abundance with a progressive decrease in the ions having a charge state of -3 (Figure 3 from L to P, Table 2). The ESI-MS spectra of the HA oligomers from 30- to 40-mers showed a progressive increase in the main ions with a charge state of -5 with a parallel decrease in the ions having a charge state of -4 and the appearance of ion species of a charge state of -6, -7, -8, -9, and -10 (Figure 3 from Q to V, Table 2). Several studies have been published on the ESI-MS characterization of HA oligosaccharides of defined length produced by partial depolymerization with hyaluronidase.7,11-14 However, these researches adopt time-consuming and repetitive preparative chromatographic separations to isolate HA fractions having a suitable grade of purity. Furthermore, these purified HA oligomers are submitted to extensive removal of salts to obtain species compatible with ESI-MS. To our knowledge, this is the first paper
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6393
mol mass
397.1 573.1 776.2 952.3 1155.3 1331.4 1534.4 1710.5 1913.6 2089.6 2292.7 2468.7 2671.7 2847.8 3050.9 3226.9 3430.1 3606.6 3809.3 3985.7 4188.4 4364.8 4567.7 4744.2 4947.4 5124.0 5327.2 5503.8 5707.2 5883.8 6087.2 6263.8 6467.2 6643.8 6847.2 7023.8 7227.2 7403.8 7607.2
oligomer
2-mer 3-mer 4-mer 5-mer 6-mer 7-mer 8-mer 9-mer 10-mer 11-mer 12-mer 13-mer 14-mer 15-mer 16-mer 17-mer 18-mer 19-mer 20-mer 21-mer 22-mer 23-mer 24-mer 25-mer 26-mer 27-mer 28-mer 29-mer 30-mer 31-mer 32-mer 33-mer 34-mer 35-mer 36-mer 37-mer 38-mer 39-mer 40-mer
396.1 572.1 775.3 951.3 1154.3 1330.4 1533.3 1709.5 1912.6 2088.6 2291.7 2467.7 2670.7 2846.8 3049.9 3225.9 3429.1 3605.6 3808.3 3984.7 4187.4 4363.8 4566.7 4743.2 4946.4 5123.0 5326.2 5502.8 5706.2 5882.8 6086.2 6262.8 6466.2 6642.8 6846.2 7022.8 7226.2 7402.8 7606.2
(-1)
285.5 387.1 475.2 576.7 664.7 766.2 854.2 955.9 1044.3 1145.8 1233.3 1334.8 1422.9 1524.5 1612.5 1714.1 1802.3 1903.7 1991.9 2093.2 2181.4 2282.9 2371.1 2472.7 2561.0 2662.6 2750.9 2852.6 2940.9 3042.6 3130.9 3232.6 3320.9 3422.6 3510.9 3612.6 3700.9 3802.6
(-2)
316.4 384.1 442.8 510.5 569.2 636.9 695.5 763.4 821.9 889.6 948.3 1016.0 1074.6 1142.4 1201.2 1268.8 1327.6 1395.1 1453.9 1521.6 1580.4 1648.1 1707.0 1774.7 1833.6 1901.4 1960.3 2028.1 2086.9 2154.7 2213.6 2281.4 2340.3 2408.1 2466.9 2534.7
(-3)
331.8 382.6 426.6 477.4 521.4 572.2 616.2 666.9 711.0 761.7 805.7 856.5 900.7 951.3 995.4 1046.1 1090.2 1140.9 1185.1 1235.9 1280.0 1330.8 1375.0 1425.8 1470.0 1520.8 1565.0 1615.8 1660.0 1710.8 1755.0 1805.8 1850.0 1900.8
(-4)
341.1 381.7 416.9 457.5 492.7 533.3 568.6 609.2 644.4 685.0 720.3 760.9 796.1 836.7 872.0 912.5 947.8 988.5 1023.8 1064.4 1099.8 1140.4 1175.8 1216.4 1251.8 1292.4 1327.8 1368.4 1403.8 1444.4 1479.8 1520.4
(-5)
347.3 381.1 410.4 444.3 473.6 507.5 536.8 570.7 600.1 633.9 663.3 697.1 726.5 760.3 789.7 823.6 853.0 886.9 916.3 950.2 979.6 1013.5 1043.0 1076.9 1106.3 1140.2 1169.6 1203.5 1233.0 1266.9
(-6)
351.7 380.7 380.7 434.8 460.0 489.0 514.2 543.2 568.4 597.3 622.5 651.5 676.7 705.8 731.0 760.0 785.3 814.3 839.5 868.6 893.8 922.9 948.1 977.2 1002.4 1031.5 1056.7 1085.7
(-7)
333.0 380.4 402.4 427.8 449.8 475.2 497.2 522.6 544.6 570.0 592.0 617.4 639.5 664.9 687.0 712.4 734.5 759.9 782.0 807.4 829.5 854.9 877.0 902.4 924.5 949.9
(-8)
357.5 380.1 399.7 422.3 441.9 464.4 484.0 506.5 526.1 548.7 568.3 590.9 610.5 633.1 652.8 675.4 695.0 717.6 737.2 759.8 779.4 802.0 821.6 844.2
(-9)
359.7 379.9 397.6 417.8 435.5 455.8 473.4 493.7 511.4 531.7 549.4 569.7 587.4 607.7 625.4 645.7 663.4 683.7 701.4 721.7 739.4 759.7
(-10)
361.3 379.8 395.8 414.2 430.3 448.8 464.8 483.3 499.3 517.8 533.9 552.4 568.4 586.9 603.0 621.5 637.5 656.0 672.1 690.6
(-11)
362.7 379.6 394.4 411.3 426.0 442.9 457.7 474.6 489.3 506.3 521.0 537.9 552.7 569.6 584.3 601.3 616.0 632.9
(-12)
363.9 379.6 393.2 408.8 422.4 438.0 451.6 467.2 480.8 496.5 510.1 525.7 539.3 554.9 568.5 584.2
(-13)
365.0 379.5 392.1 406.7 419.3 433.8 446.4 460.9 473.6 488.1 500.7 515.2 527.8 542.4
(-14)
Table 1. Calculated Monoisotopic m/z Values for Fully Saturated HA Oligomers Separated by Means of RPIP-HPLC
365.9 379.5 391.3 404.8 416.6 430.1 441.9 455.5 467.3 480.8 492.6 506.1
(-15)
366.7 379.5 390.5 403.2 414.2 427.0 438.0 450.7 461.7 474.5
(-16)
367.5 379.4 389.8 401.8 412.2 424.1 434.5 446.5
(-17)
368.1 379.4 389.2 400.5 410.3 421.6
(-18)
368.7 379.4 388.7 399.4
(-19)
369.2 379.4
(-20)
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Figure 3. ESI-MS spectrum in the negative mode of each single HA oligosaccharide species separated by means of RPIP-HPLC from 2- to 40-mers.
showing the separation and characterization of HA oligomers using on-line HPLC/ESI-MS with no further preparative or derivative approach. Thanawiroon et al.19 reported the use of online HPLC/ESI-MS for the analysis of complex mixtures of heparin oligosaccharides. In this paper, the suitability of this analytical 6396
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approach for the separation and characterization of HA oligosaccharides up to ∼40-mers is evaluated. Up to 40-mers fully saturated HA oligomers produced by partial depolymerization with hyaluronidase were separated by RPIP-HPLC and characterized by negative ESI-MS. For each HA oligosaccharide, a series of
Table 2. Retention Times (RT) of the HA Oligomers Separated by Means of RPIP-HPLC and the Main Ion Species Related to Each Oligosaccharide with Their Relative Abundance oligomer
RT, min
mol mass
ions (charge) m/z
rel abund, %
2-mer 4-mer
6.5 7.5
397.1 776.2
6-mer
9.0
1155.3
396.1 775.3 572.1 1154.3 775.3 576.7 475.2 766.2 664.7 576.7 475.2 955.9 854.2 636.9 569.2 1145.8 955.9 763.4 695.5 1334.8 889.9 822.1 763.5 1524.5 1016.0 948.3 1142.9 1074.6 856.5 805.7 1268.8 1201.2 951.3 900.7 760.9 1395.1 1046.1 836.7 1521.6 1140.9 912.5 760.3
100 85 15 4 10 33 53 55 35 5 5 55 4 15 26 11 3 58 28 3 85 10 2 3 5 92 55 1 34 10 27
