Anal. Chem. 2005, 77, 4125-4136
On-Line Liquid Chromatography Electrospray Ionization Mass Spectrometry for the Characterization of K- and ι-Carrageenans. Application to the Hybrid ι-/ν-Carrageenans Aristotelis Antonopoulos,† Patrick Favetta,† William Helbert,‡ and Michel Lafosse*,†
UMR CNRS 6005, Institut de Chimie Organique et Analytique (ICOA), Universite´ d’Orle´ ans, BP 6759, 45067 Orle´ ans Cedex 2, France, and UMR 7139 CNRS-UPMC-Laboratoires Goe¨mar, Station Biologique, Pl. G. Teissier, BP 74, 29682 Roscoff Cedex, France.
An on-line liquid chromatography electrospray ionization mass spectrometry (MS) method was developed for the characterization of polymers of K- (extracted from Kappaphycus alvarezii), ι-, and hybrid ι-/ν-carrageenans (both extracted from Eucheuma denticulatum) enzymatically digested with specific carrageenase enzymes. Applying either CID MS/MS or in-source fragmentation mechanisms, the results demonstrated that none of the polymers of K- or ι-carrageenans existed with their ideal repeating units. On the polymer of K-carrageenan, the nonideal structures identified consisted of ι-neocarrabiose sulfate units. On the polymer of ι-carrageenan, the nonideal structures identified consisted of the following: (i) K-neocarrabiose sulfate units, (ii) ι-neocarrabiose sulfate units with an additional sulfate group, and (iii) ι-neocarrabiose sulfate units with an additional sulfate and a pyruvate acetal group. For both K- and ι-carrageenans, the nonideal structures were randomly distributed on the polymers. The method was then applied for the characterization of a hybrid polymer of ι-/ν-carrageenans, enzymatically digested with ι-carrageenase. The results did not reveal an ideal oligosaccharide of ν-carrageenan, suggesting that the ι-carrageenase enzyme could cleave only reduced “densities” of ν-carrageenan repeating units. In addition, information about the sequence of hybrid ι-/νcarrageenans from E. denticulatum is deduced. Matrix polysaccharides of algae cell walls have been the subject of intense research during the past 30 years mostly because of their thickening and gelling properties.1-3 Cell walls of marine algae differ mainly from land plant cell walls by the prevalence of the polyanionic (e.g., sulfated) polysaccharides over the neutral * Corresponding author. Phone: +33(0)238494575. Fax: +33(0)238417281. E-mail:
[email protected]. † Universite´ d’Orle´ans. ‡ Station Biologique. (1) De Ruiter, G. A.; Rudolph, B. Trends Food Sci. Technol. 1997, 8, 389-395. (2) Stanley, N. F. In Food Gels; Harris, P., Ed.; Elsevier Applied Science: London, 1990; pp 79-119. (3) Therkelsen, G. H. In Industrial Gums, 3rd ed.; Whisthler, R. L., BeMiller, J. N., Eds.; Academic Press: San Diego, CA, 1993; pp 145-180. 10.1021/ac050091o CCC: $30.25 Published on Web 05/11/2005
© 2005 American Chemical Society
polysaccharides.4 This fact suggests that these polymers have specific functions in relation to the marine environment; i.e., they are involved in mechanical, osmotic, and ionic regulation.4 Carrageenans are a family of highly sulfated galactans that occur in the above marine matrixes, specifically in the red algae (Rhodophyta), with a primary backbone structure based on alternatively 3-linked, β-D-galactose (unit G) and 4-linked, R-Dlinked galactose (unit D). Carrageenans are distinguished by the occurrence of 3,6-anhydro bridges (unit A) in the R-linked galactose residues and by the substitution of ester sulfate groups.5,6 The main industrially exploited carrageenans are κ- (G4S-DA) and ι- (G4S-DA2S) carrageenans owing to their gelling properties,7 while λ- (G2S-D2S,6S) has no gelling properties (Figure 1). κ-Carrageenan is conventionally described as the repetition of the disaccharidic motif 4-sulfate O-β-D-galactopyranosyl-(1,4)-3,6-anhydro-R-D-galactose, also referred to as κ-neocarrabiose sulfate. ι-Carrageenan differs from κ-carrageenan by the presence of one additional sulfate substituent per repeating disaccharide, at C-2 on the R-linked D-galactose residues also referred to as ι-neocarrabiose sulfate. However, native polysaccharides are rarely in their ideal form. Their heterogeneity largely depends on the algal sources, the life stages, and the extraction procedures of the polymers.8,9 This structural complexity is attributed to the occurrence of a mixture of carrageenans in extracts as well as to a combination of ideal carrabiose repeating units in purified carrageenans, giving rise to a hybrid or copolymer chain.5,9,10 The most classical copolymers of carrageenan are those found in native or unprocessed κ- and ι-carrageenan chains that usually contain fractions of their biosynthetic precursors6,11,12 named µ- (G4S-D6S) and ν- (G4S-D2S,6S) carrageenans, respectively (Figure 1). Other carrabiose (4) Kloareg, B.; Quatrano, R. S. Oceanogr. Mar. Biol. Annu. Rev. 1988, 26, 259-315. (5) Knutsen, S. H.; Myslabodski, S. H.; Larsen, B.; Usov, A. I. Bot. Mar. 1994, 37, 163-169. (6) Craigie, J. S.; Wong, K. F. Proc. Int. Seaweeds Symp. 1978, 369-377. (7) Van de Velde, F.; Peppelman, H. A.; Rollema, H. S.; Tromp, R. H. Carbohydr. Res. 2001, 331, 271-283. (8) Amimi, A.; Mouradi, A.; Givernaud, T.; Chiadmi, N.; Lahayec, M. Carbohydr. Res. 2001, 333, 271-279. (9) Graigie, J. S. In Biology of the Red Seaweeds; Cole, K. M., Dheath, R. G., Eds.; Cambridge University Press: Cambridge, U.K., 1990; pp 221-257. (10) Usov, A. I. Food Hydrocolloids 1998, 12, 301-308. (11) Bellion, C.; Brigand, G. Carbohydr. Res. 1983, 119, 31-48.
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Figure 1. Proposed biosynthetic pathway of κ- and ι-carrageenans from Craigie and Wong.6 All carrageenan families shown in the figure are biosynthesized from the ideal backbone structure (G-D, carrageenan) via sulfotransferases and sulfohydrolases.
combinations have been also demonstrated such as the κ-/ιcarrageenan hybrids (or κ2-carrageenan) in several species of the Gigartinacae family7,13,14 and the κ-/β- (β, G-DA) carrageenan copolymer found in Furcellaria sp. and Euchema gelatinae.15,16 An additional layer of complexity is reached when the hydroxyl groups are substituted by methyl17 and pyruvate18,19 groups. (12) Van de Velde, F.; Rollema, H. S.; Grinberg, N. V.; Burova, T. V.; Grinberg, V. Y.; Tromp, R. H. Biopolymers 2002, 65, 299-312. (13) Greer, C. W.; Yaphe, W. Bot. Mar. 1984, 27, 479-484. (14) Bixler, H. J. Hydrobiologia 1996, 326/327, 35-57. (15) Greer, C. W.; Yaphe, W. Bot. Mar. 1984, 27, 473-478. (16) Knusten, S. H.; Grasdalen, H. Bot. Mar. 1987, 30, 497-505.
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The existence of nonideal structures, known as “kinks”,20 introduces modifications in their physicochemical properties.10,21,22 The term “kinks” refers to a conformational defect in the chain, (17) Chiovitti, A.; Bacic, A.; Craik, D. J.; Kraft, G. T.; Liao, M. L. Carbohydr. Res. 2004, 339, 1459-1466. (18) Chiovitti, A.; Bacic, A.; Craik, D. J.; Munro, S. L. A.; Kraft, G. T.; Liao, M. L. Carbohydr. Res. 1997, 299, 229-243. (19) Chiovitti, A.; Bacic, A.; Craik, D. J.; Kraft, G. T.; Liao, M. L.; Falshaw, R.; Furneaux, R. H. Carbohydr. Res. 1998, 310, 77-83. (20) Rees, D. A. Adv. Carbohydr. Chem. Biochem. 1969, 24, 267-332. (21) Pearce-Pratt, R.; Phillips, D. M. Biol. Reprod. 1996, 54, 173-182. (22) Potin, P.; Bouarab, K.; Salau ¨ n, J. P.; Pohnert, G.; Kloareg, B. Curr. Opin. Plant Biol. 2002, 5, 1-10.
where this defect is mandatory for the physicochemical properties. At present, there are no reports connecting this conformational defect with biological activity. The chemical and spectrometric methods that have been developed until now and applied to the structural analysis of carrageenans usually lead to the determination of linkages and averaged composition. However, the fine description of the organization of carrabiose units along the carrageenan chains is still an unsolved problem. Enzymes could be a very helpful tool for a better understanding of the carrabiose sequences in carrageenans. Carrageenase specificity permits the degradation of specific structures of carrageenans (i.e., κ-carrageenases degrade κ-carrageenan but are inactive on ι-carrageenan) without drastic chemical treatment that may interfere in the solving of the native structure. Strong analytical tools are therefore needed in order to gain insight into the complexity of different carrageenan structures. To address this issue, nuclear magnetic resonance (NMR) spectroscopy,7,23,24 matrix-assisted laser desorption/ionization (MALDI) mass spectrometry,25 electrospray ionization-mass spectrometry (ESI-MS), FT-IR,26,27 and FT-Raman spectroscopy27 have been employed. However, the absence of model compounds yields NMR spectra that are difficult to exploit,24 and highly sulfated oligosaccharides as such give a very poor mass spectrometric response, almost independent of the ionization technique, because the extremely polar (ionic) character of these compounds renders it difficult to produce intact gas-phase ions for mass spectrometric analysis.28 That is why the analysis of sulfated carbohydrates with basic peptides was recently reported for MALDI29,30 and ESI-MS31 instruments. However, in both cases, it was shown that the type of peptide to be used had to be carefully selected (or more often synthesized), since its complexation with the sulfates of the molecules to be examined did not always result in ionization of the molecule.31 On ESI-MS instruments, this strategy was applied, but for a particular molecule (sucrose octasulfate) and not online.31 This means that, in carrageenan oligosaccharides, this methodology could not be easily applied because (i) pure oligosaccharides are hard to find and challenging to purify and (ii) the oligosaccharides are expected to be fairly heterogeneous in the number of sulfate groups (see hybrid ι-/ν-carrageenans). Nevertheless, ESI-MS has been proved to be a powerful and reliable tool for the analysis of saccharides32,33 offering precise results, analytical versatility, and high sensitivity,33 and when (23) Tojoa, E.; Prado, J. Carbohydr. Polym. 2003, 53, 325-329. (24) Van de Velde, F.; Knutsen, S. H.; Usov, A. I.; Rollema, H. S.; Cerezo, A. S. Trends Food Sci. Technol. 2002, 13, 73-92. (25) Ackloo, S.; Terlouw, J. K.; Ruttink, P. J. A.; Burgers, P. C. Rapid Commun. Mass Spectrom. 2001, 15, 1152-1159. (26) C ˇ erna´, M.; Barros, A. S.; Nunes, A.; Rocha, S. M.; Delgadillo, I.; C ˇ opı´kova´, J.; Coimbra, M. A. Carbohydr. Polym. 2003, 51, 383-389. (27) Pereira, L.; Mesquita, J. F. J. Appl. Phycol. 2004, 16, 369-383. (28) Juhasz, P.; Biemann, K. Carbohydr. Res. 1995, 270, 131-147. (29) Juhasz, P.; Biemann, K. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4333-4337. (30) Venkataraman, G.; Shriver, Z.; Raman, R.; Sasisekharan, R. Science 1999, 286, 537-542. (31) Siegel, M. M.; Tabei, K.; Kagan, M. Z.; Vlahov, I. R.; Hileman, R. E.; Linhardt, R. J. J. Mass Spectrom. 1997, 32, 760-772. (32) Stahl, M.; Von Brocke, A.; Bayer, E. In Carbohydrate Analysis by Modern Chromatography and Electrophoresis; El Rassi, Z., Ed.; Elsevier Science: Amsterdam, 2002; pp 961-1042. (33) Zaia, J. Mass Spectrom. Rev. 2004, 23, 161-227.
instruments cannot offer adequate resolution, separation mechanisms are complementary tools that enhance its analytical power. In this respect, size exclusion chromatography was coupled to the ESI-MS34 to analyze chondroitin sulfate. Ion-pair liquid chromatography coupled to ESI-MS has been used for heparosan oligosaccharide characterization35 and for sequencing highly sulfated heparin-derived oligosaccharides.36 In our case, this method was used recently to analyze enzymatically digested oligomers of κ-carrageenans37 up to tetratriacontasaccharide (G4S-DA)17. The oligomers were then characterized off-line with ESI-MS in the positive-ion mode. Formerly, experiments to couple anion-exchange mechanisms to the mass spectrometer failed due to the high salt concentration needed to separate the oligosaccharides.38 In the present work, we demonstrate for the first time the characterization of κ- and ι-carrageenans, (extracted from Kappaphycus alvarezii and Eucheuma denticulatum, respectively) enzymatically digested with κ- and ι-carrageenases respectively, using ion-pair liquid chromatography coupled to ESI-MS in the negative-ion mode. The method was then applied to characterize a hybrid sample of ι-/ν-carrageenans (extracted from E. denticulatum) enzymatically digested with ι-carrageenase. EXPERIMENTAL SECTION Materials. Deionized water was obtained by an Elgastat UHQ II system (18 MΩ) from Elga (Antony, France). HPLC grade methanol (MeOH) was from J. T. Baker (Noisy le Sec, France) and analytical grade heptylamine (C7H15NH2) and formic acid were from Fluka (St. Quentin Fallavier, France). Preparation of Oligosaccharides of K- and ι-Carrageenans. κ-Carrageenan (E. cottonii X-6913, CP-Kelco) was extracted from K. alvarezii, and transformed ι-carrageenans (E. spinosum X-6908, CP-Kelco) and untransformed ι-/ν-carrageenan (E. spinosum X-6043, CP-Kelco) were from E. denticulatum. Oligomers of κ- and ι-carrageenans were produced according to Rochas and Heyraud39 using recombinant Alteromonas carrageenovora κ-carrageenase, and Alteromona fortis ι-carrageenase.40 Prior to freeze-drying, degradation products were filtered through Amicon Centriprep (YM-30, Millipore). Instrumentation and Mass Spectrometry. Liquid chromatography was performed on a Hitachi L7100 (Merck, Nogent sur Marne, France) quaternary gradient pump model and a Rheodyne (Berkeley, CA) model 7125 injector with a 20-µL sample loop. The chromatographic system was coupled to Quattro Ultima triplequadrupole mass spectrometer (Micromass Ltd., Manchester, U.K.) with a Z-Spray electrospray ionization source. Mass spectrometry was performed in the negative-ion mode. The desolvatation gas (N2) flow was 500 L/h, and the nebulizer gas (N2) flow rate was 50 L/h. The rf lens 1 was set at 0.0, the (34) Zaia, J.; Costello, C. E. Anal. Chem. 2001, 73, 233-239. (35) Kuberan, B.; Lech, M.; Zhang, L.; Wu, Z. L.; Beeler, D. L.; Rosenberg, R. D. J. Am. Chem. Soc. 2002, 124, 8707-8717. (36) Thanawiroon, C.; Rice, K. G.; Toida, T.; Linhardt, R. J. J. Biol. Chem. 2004, 279, 2608-2615. (37) Antonopoulos, A.; Favetta, P.; Helbert, W.; Lafosse, M. Carbohydr. Res. 2004, 339, 1301-1309. (38) Antonopoulos, A.; Herbreteau, B.; Lafosse, M.; Helbert, W. J. Chromatogr., A 2004, 1023, 231-238. (39) Rochas, C.; Heyraud, A. Polym. Bull. 2003, 5, 81-86. (40) Michel, G.; Barbeyron, T.; Flament, D.; Vernat, T.; Kloareg, B.; Dideberg, O. Acta Crystallogr. 1999, D55, 918-920.
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aperture was set at 0.0, and the rf lens 2 was set at 1.0. The probe temperature was held at 250 °C and the ion source temperature at 130 °C. The electrospray capillary voltage was 2.75 kV, and the cone voltage was 32 V unless stated otherwise. The LM resolution 1 and HM resolution 1 were both set at 12.5, while the ion energy 1 was at 1.1 eV. The entrance and exit of the collision cell were both set at 30 eV, and the collision energy was set at 0 eV unless stated otherwise (In CID MS/MS studies while infusion, the entrance and exit of the collision cell were set at -1 and 1, respectively). The LM resolution 2 and HM resolution 2 were both set at 15.0, and the ion energy 2 was set at 1.5 eV. The multiplier was set at 650. The pressure in the analyzer was 8.5 × 10-6 and 1.0 × 10-4 mbar in the gas cell. Full scan mode was used in negative-ion mode. The m/z range 90-3000 was scanned in 3 s. The instrument was calibrated using NaIRb (NaI 2 µg/µL, RbI 50 ng/µL) in 2-propanol/H2O (1:1, v/v). Ion-Pair Liquid Chromatography. An Alltima C18 5U column (150 × 4.6 mm i.d., particle size 5 µm) purchased from Alltech (Templemars, France) was coupled to the MS with a split ratio of 1:10. The mobile phase consisted of water (eluent A), MeOH (eluent B), and 20 mM heptylammonium formate (pH 4) (eluent C). All samples were injected to the system with a concentration of 2000 ppm in water. The chromatographic separation of oligomers of κ-carrageenans was performed with the following linear gradient program: 0-15 min, 40-20% A, 3555% B, 25% C; 15-40 min, 20-10% A, 55-65% B, 25% C, 40-50 min, 10% A, 65% B, 25% C. The chromatographic separation of oligomers of ι-carrageenans was performed with the following linear gradient program: 0-5 min, 30-17% A, 45-58% B, 25% C; 5-25 min, 17-10% A, 58-65% B, 25% C; 25-45 min, 10% A, 65% B, 25% C. The chromatographic separation of the hybrids ι-/νcarrageenans was performed with the following linear gradient program: 0-5 min, 45-32% A, 45-58% B, 10% C; 5-25 min, 3225% A, 58-65% B, 10% C; 25-45 min, 25% A, 65% B, 10% C. Dead volume was determined by injecting NaNO3 with MeOH/ water (30:70) as mobile phase. It corresponded to the very first peak of each chromatogram. Preparation of Ion-Pairing Agent. The ion-pairing agent was a mixture of heptylamine and formic acid. They were prepared by imposing the alkylammonium concentration value and a pH value of 4. The formate ion concentration and the ionic strength of each eluent were calculated by PHoEBus,41 an application program help for buffer studies. For the preparation of ion-pairing agent heptylammonium formate (20 mM heptylammonium ion, pH 4), 0.283 mL of formic acid and 0.745 mL of heptylamine were diluted into a volume of 250 mL. The pH value was checked with a Beckman pH meter (model F10, Gagny, France). Infusion of the Oligosaccharides Mixture. Oligosaccharides of κ-, ι-, and hybrid ι-/ν-carrageenans were injected into the ESI probe of the mass spectrometer via direct infusion with a Harvard Apparatus pump 11 (Harvard Apparatus) flowing at 5 µL min-1. The analyte, 200 ppm of mixture of oligosaccharides of κ-, ι-, or hybrid ι-/ν-carrageenans was injected with 5 mM heptylammonium formate (pH 4) in 75:25 MeOH/water mobile phase. Data Processing. Data acquisition and processing was performed using MASSLYNX 4.0 software (Micromass Ltd.). All (41) Morin, P.; Vangrevelinghe, E.; Mayer, S. PHoEBus, version 1.3, Analis, Namur, Belgium.
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chromatograms were smoothed twice using the Savitzky-Golay algorithm with three window-size scans. All mass spectra were smoothed twice using Savitzky-Golay algorithm with 0.75-Da peak width. The molecular weights of all carrageenan oligosaccharides with the counterions of heptylamine were calculated with Macisotopes software, version 1.2. RESULTS AND DISCUSSION Characterization of Ideal Oligosaccharides of K- and ι-Carrageenans. Previous reports have selected nonlinear alkylammonium ion-pairing agents in LC.35,36 In this work, we chose heptylamine as an ion-pairing agent37 because of its sufficiently large molecular weight (115.1) and its volatility; in addition, the low content (5 mM or less) required to retain oligosaccharides of κ- and ι-carrageenans is compatible with mass spectrometry. Enzymatically digested oligosaccharides of κ-carrageenan were detected up to hexatriacontasaccharide (G4S-DA)18 (Figure 2a) and oligosaccharides of ι-carrageenan up to decasaccharide (G4SDA2S)5 (Figure 2b). For the ι-carrageenan family, a disaccharide unit was not produced. This is consistent with the fact that the ι-neocarratetraose sulfate (G4S-DA2S)2 is the smallest oligosaccharide of ι-carrageenan that can be produced from the A. fortis ι-carrageenase.42 For the κ-carrageenan family however, a disaccharide unit was detected (Figure 2a, inset) with retention time (Rt) 3.23 min. This is consistent with a previous report,43 which demonstrated that recombinant κ-carrageenase was able to produce κ-neocarrabiose sulfate. Nevertheless, this disaccharide unit could not be detected directly from the total ion current chromatogram possibly because the κ-neocarrabiose sulfate (G4SDA) has only one sulfate group, which is mostly neutralized by the heptylamine molecule and therefore cannot easily carry a negative charge. However, when it carries a negative charge, the m/z 403.1 detected corresponded to the ion [G4S-DA]-. The κ-neocarratetraose sulfate (peak 2, Figure 2a) was mainly detected as a 1- (monocharged) ion (m/z 904.2, Figure 3a, Table 1) (100%, relative intensity, RI) attached with one counterion of heptylamine, while its 2- (twice charged) ion (m/z 394.0) was found in relatively low abundance (6.2% RI). As for the 1- ion with one sulfate group protonated (m/z 789.1) as well as the desulfated 1- ion (m/z 709.2), it will be shown that they derived from in-source fragmentation of the m/z 904.2 (see below). In contrast to the κ-neocarratetraose sulfate, the ι-neocarratetraose sulfate (peak 2, Figure 2b) was detected as 1- and 2- ions (Figure 3b, Table 1) in about the same relative abundance (82.1 and 99.3% RI, respectively). In both tetrasaccharides, the ion m/z 394.1 was observed which corresponded to the structure of (G4SDA)2. It should be noted that in Figure 3a this ion corresponds to the molecular ion of tetrasaccharide of κ-carrageenan with all sulfates in their deprotonated form, while in Figure 3b, it corresponds to a fragmentation product ion deriving from the ion m/z 589.2, and not a tetrasaccharide of κ-carrageenan that eluted at the same time with the tetrasaccharide of ι-carrageenan. It is significant that the relative instability of the ι-neocarratetraose sulfate compared with that of κ-neocarratetraose sulfate can (42) Knutsen, S. H.; Sletmoen, M.; Kristensen, T.; Barbeyron, T.; Kloareg, B.; Potin, P. Carbohydr. Res. 2001, 331, 101-106. (43) Potin, P.; Richard, C.; Barbeyron, T.; Henrissat, B.; Gey, C.; Petillot, Y.; Forest, E.; Dideberg, O.; Rochas, C.; Kloareg, B. Eur. J. Biochem. 1995, 228, 971-975.
Figure 2. Total ion current chromatograms of the enzymatically digested (a) κ- and (b) ι-carrageenans. The number above the peaks designates the repeating neocarrabiose sulfate units of κ- (G4S-DA)n and ι-carrageenans (G4S-DA2S)n. (a) Peaks designated K1-K3 correspond to nonideal κ-neocarrabiose sulfate structures in the κ-carrageenans, (b) and I1-I7 correspond to nonideal ι-neocarrabiose sulfate structures in the ι-carrageenans. Inset in (a) depicts the extracted ion current of the disaccharide of κ-carrageenan (m/z 403.1).
Figure 3. Negative ion electrospray mass spectra of the ideal κ- and ι-carrageenans in LC/MS. Neocarratetraose sulfate of (a) κ- and (b) ι-carrageenans, while (c) and (d) are the neocarrahexaose sulfate of κ- and ι-carrageenans, respectively.
be partially attributed to the fact that ι-neocarratetraose sulfate is twice as sulfated as the κ-neocarratetraose sulfate. Therefore, it should be expected that the κ- and ι-neocarrahexaose sulfate (Figure 3c and d, respectively, Table 1) would have a more extensive fragmentation. However, when they are compared with the tetrasaccharides, the former were shown to be more stable with less in-source fragmentation product ions. This phenomenon can be attributed to the distribution of energy to a larger molecule and therefore did not reach the quantities required for fragmentation. Overall, the number of heptylamine ions (C7H15NH3+) attached to each oligosaccharide is found to be dependent on the number of sulfate groups. The same principle was recently reported for highly sulfated heparin-derived oligosaccharides.36 It follows that
in our case the formula for the κ-carrageenans takes the form [(G4S-DA)n + (C7H15NH3+)x](n-x)-, while for the ι-carrageenans, it is [(G4S-DA2S)n + (C7H15NH3+)x](2n-x)-, where n is the number of neocarrabiose units and x is the number of C7H15NH3+ counterions. Figure 4 depicts in bubble plot the number and the percentage of heptylamine molecules attached to each κ- and ι-neocarrabiose sulfate. In most cases, there was only one charge state that principally dominated. The linear trend observed for the two carrageenan families confirms that indeed the number of heptylamine molecules attached to each oligosaccharide depends only on the number of sulfate groups. In addition, the fact that the slope of ι-carrageenans is almost twice the slope of κ-carrageenans is related to the fact that the repeating unit of the former is twice sulfated as the repeating unit of the latter. Analytical Chemistry, Vol. 77, No. 13, July 1, 2005
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Table 1. Ions Identified in the Ideal Tetrasaccharide (Peak 2 in Figure 2a and b) and Hexasaccharide (Peak 3 in Figure 2a and b) of the Enzymatically Digested K- and ι-Carrageenans, (G4S-DA)n and (G4S-DA2S)n, Respectivelya oligomer (G4S-DA)n
assignment
m/z found
m/z calcd
tetrasaccharide (n ) 2)
[(G4S-DA)2 + C7H15NH3+][(G4S-DA)2 + H][(G4S-DA)2 - SO3 + H][(G4S-DA)2]2[(G4S-DA)3 + (C7H15NH3+)2][(G4S-DA)3 + H + C7H15NH3+][(G4S-DA)3 - SO3 + H + C7H15NH3+][(G4S-DA)3 - 2SO3 + 2H][(G4S-DA)3 + C7H15NH3+]2[(G4S-DA)3 - SO3]2[(G4S-DA)3]3-
904.2 789.1 709.2 394.0 1405.3 1290.2 1210.3 1015.1 644.8 547.3 391.2
904.2 789.1 709.1 394.1 1405.4 1290.3 1210.3 1015.2 644.7 547.1 391.0
hexasaccharide (n ) 3)
oligomer (G4S-DA2S)n tetrasaccharide (n ) 2)
hexasaccharide (n ) 3)
a
assignment +)
]-
[(G4S-DA2S)2 + (C7H15NH3 3 [(G4S-DA2S)2 + H + (C7H15NH3+)2][(G4S-DA2S)2 - SO3 + 2H + C7H15NH3+][(G4S-DA2S)2 - 2SO3 + 2H + C7H15NH3+][(G4S-DA2S)2 - 2SO3 + 3H][(G4S-DA2S)2 - 3SO3][(G4S-DA2S)2 + (C7H15NH3+)2]2[(G4S-DA2S)2 + H + C7H15NH3+]2[(G4S-DA2S)2 - SO3 + H + (C7H15NH3+)2]2[(G4S-DA2S)2 - SO3 + 2H]2[(G4S-DA2S)2 - 2SO3 + 2H]2[(G4S-DA2S)3 + (C7H15NH3+)5][(G4S-DA2S)3 + (C7H15NH3+)4]2[(G4S-DA2S)3 + H + (C7H15NH3+)3]2-
m/z found
m/z calcd
1294.4 1179.2 984.0 904.1 789.0 709.1 589.2 531.7 492.1 434.2 394.1 1991.0 937.4 879.8
1294.4 1179.3 984.2 904.1 789.1 709.1 589.2 531.6 492.1 434.0 394.0 1990.7 937.3 879.7
These ions are observed in spectra of Figure 3 and obtained by LC/MS.
Figure 4. Bubble plot of the number of heptylamine molecules attached to the neocarrabiose sulfate unit of κ- and ι-carrageenans.
Characterization of Nonideal Oligosaccharides of K- and ι-Carrageenans. The minor peaks detected in Figure 2a (peaks K1 to K3) did not correspond to ideal oligosaccharides of κ-carrageenan. The quantity of these compounds in the sample was limited, and the resolution was too poor to isolate them for identification. Therefore, on-line LC/MS coupling with high efficiency was the only method for their characterization. Their m/z values revealed that these oligosaccharides consisted of ideal κ-neocarrabiose sulfates, each one with an additional sulfate in their structure (Table 2). The comparison of the retention time of peak K1 with that of ideal κ-neocarrahexaose sulfate (G4S-DA)3 demonstrated that retention was affected by 4130
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the number of sulfate groups (compare also K2-(G4S-DA)4 and K3-(G4S-DA)5). In addition, the selectivity between the nonideal structures K1-K3 and the (G4S-DA)n, n ) 3-5, respectively, is about the same. In our case, the more sulfate groups present, the greater the possibility of reacting with the counterion, resulting in increased hydrophobicity of the compound. The phenomenon that an additional sulfate group can significantly alter the retention time has already been described for SEC.44 However, while peak K1 is not masked by peak 5, peak K2 is close to peak 6 and peak K3 is almost masked by peak 7. Therefore, the nonideal structures were not just these three peaks, but the rest of the nonideal structures were masked by the ideal structures. However, their structure was of the type (G4S-DA)n + SO3, where n is the number of κ-neocarrabiose sulfate, n g 3. Likewise, peaks I1-I7 were not related to ideal oligosaccharides of ι-carrageenan (Figure 2b, Table 2) but consisted of a combination of ideal ι-neocarrabiose sulfates plus or minus one sulfate or pyruvate (P) group. In detail, I2 and I5 were found to correspond to (G4S-DA2S)4 and (G4S-DA2S)5, respectively, but with one sulfate less on their structure. On the contrary, I3 and I6 were found to correspond to the structures (G4S-DA2S)3 and (G4SDA2S)4, respectively, but each one found with an additional sulfate group. This explains why these structures were more retained than the ideal ι-neocarrahexaose and ι-neocarraoctaose sulfate, which had the same structure, respectively, but lacking the additional sulfate. (44) Ekeberg, D.; Knutsen, S. H.; Sletmoen, M. Carbohydr. Res. 2001, 334, 4959.
Table 2. Ions Identified in the Nonideal Enzymatically Digested K- and ι-Carrageenans (LC/MS) oligomer
a
no.Sa
K1
4
K2 K3 I1 I2 I3 I4 I5 I6 I7
5 6 5 7 7 7 9 9 9
assignment +)
[(G4S-DA)3 + SO3 + [(G4S-DA)3 + SO3 + [(G4S-DA)4 + SO3 + [(G4S-DA)5 + SO3 + [(G4S-DA2S)2 + (GP-DA2S) + (C7H15NH3+)3]2[(G4S-DA2S)4 - SO3 + (C7H15NH3+)5]2[(G4S-DA2S)3 + SO3 + (C7H15NH3+)5]2[(G4S-DA2S)3 + (GP-DA2S) + (C7H15NH3+)5]2[(G4S-DA2S)5 - SO3 + (C7H15NH3+)7]2[(G4S-DA2S)4 + SO3 + (C7H15NH3+)7]2[(G4S-DA2S)4 + (GP-DA2S) + (C7H15NH3+)7]2(C7H15NH3 2]2H + C7H15NH3+]2(C7H15NH3+)3]2(C7H15NH3+)4]2-
m/z found
m/z calcd
742.5 684.4 993.1 1243.6 874.8 1188.1 1035.1 1223.0 1536.3 1383.5 1571.6
742.2 684.6 992.8 1243.9 874.8 1188.4 1034.9 1223.4 1537.0 1384.0 1572.0
Number of sulfate groups.
Conversely, I1, I4, and I7 were found to correspond to the structures (G4S-DA2S)2, (G4S-DA2S)3, and (G4S-DA2S)4, respectively, each one with an additional (GP-DA2S) disaccharide unit. The latter structure has already been identified in the Australian red algae.18,19 Comparison of In-Source and CID MS/MS Fragmentations of Oligosaccharides of K- and ι-Carrageenans. Oligosaccharides of κ- and ι-carrageenans ionize poorly, due to the high salt concentrations needed for their isolation from the algae. Moreover, studies of highly sulfated saccharides with a simple quadrupole system are quite difficult.34 However, infusion of the mixture of oligosaccharides with heptylamine in the negative-ion mode (data not shown) produced ions of the expected mass (formerly identified by LC/ESI-MS) and therefore their study was possible. In the spectrum obtained by infusing the κ-carrageenan oligosaccharide mixture, the adduct of tetrasaccharide with a heptylamine molecule C7H15NH2 was the ion m/z 904.2 [(G4SDA)2 + C7H15NH3+]- and this ion was selected and fragmented by CID MS/MS under various collision energies. Figure 5a illustrates the breakdown curves: From the ion [(G4S-DA)2 + C7H15NH3+]- (m/z 904.2), a heptylamine molecule was cleaved leaving the sulfate group protonated (m/z 789.1). The neutralized sulfate was then cleaved by the molecule, giving the product ion [(G4S-DA)2 + H - SO3]- (m/z 709.2). The same fragmentation by CID MS/MS was observed for the ion m/z 1294.4 (ιneocarratetraose sulfate) isolated from the spectrum of ι-carrageenans (Figure 5b), meaning that heptylamine and sulfate groups were expelled consecutively until the oligosaccharide remained only with the charged sulfates, which are more stable than the protonated ones.45,46 Only when the ion reached that stage could we observe backbone cleavage. This phenomenon has already been described for chondroitin sulfate, in which sulfate groups that do not bear a net charge because of pairing with protons result in losses of neutral SO3 from the precursor ion being more likely than the formation of product ions via backbone cleavage.46 In CID MS/MS of the ι-carratetraose sulfate, the [(G4S-DA2S)2 + (C7H15NH3+)2]2- ion (m/z 589.2) (noted in Table 1 in LC/MS) was not observed, suggesting that this ion was not a product ion (45) Yagami, T.; Kitagawa, K.; Aida, C.; Fujiwara, H.; Futaki, S. J. Pept. Res. 2000, 56, 239-249. (46) McClellan, J. E.; Costello, C. E.; O′Connor, P. B.; Zaia, J. Anal. Chem. 2002, 74, 3760-3771.
Figure 5. Relative intensity in CID-MS/MS versus collision energy of the neocarratetraose sulfate of κ- and ι-carrageenans, respectively. (a) [(G4S-DA)2 + C7H15NH3+]- ion (m/z 904.2), (b) [(G4S-DA2S)2 + (C7H15NH3+)3]- ion (m/z 1179.2).
of in-source fragmentation but derived from the ionization process. In addition, this suggested that all product ions kept the same charge state. For both the neocarratetraose sulfate of κ- and ι-carrageenans in CID-MS/MS, the final product ion before backbone cleavage was the [(G4S-DA)2 - SO3 + H]- (m/z 709.1). Since the first step of CID MS/MS was always the cleavage of a heptylamine molecule (C7H15NH2, 115.1 u), we were able to identify each time the charge state of the selected ion, i.e., for an oligosaccharide of m/z M, if the charge state was x-, the first product ion should be detected at m/z M - (115.1/x). Since sulfates are the most acidic groups in the carrageenans, as in chondroitin sulfate,47,48 they carry the negative charge under nearly all the experimental conditions.49 This means that from the charge state of the selected ion we were (47) Zaia, J.; McClellan, J. E.; Costello, C. E. Anal. Chem. 2001, 73, 6030-6039. (48) Zaia, J.; Costello, C. E. Anal. Chem. 2003, 75, 2445-2455.
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Figure 6. CID MS/MS infusion of oligosaccharides of κ- and ι-carrageenans (a-c). In-source fragmentation (LC/MS) of oligosaccharides of κ- and ι-carrageenans (d-f). Extracted ion current of m/z 985.5 of ι-carrageenans (g). Negative ion CID MS/MS of a low (left panel) and a high (right panel) collision energy of (a) [(G4S-DA)3 + (C7H15NH3+)2]- ion (m/z 1405.3), (b) [(G4S-DA)3 + SO3 + (C7H15NH3+)2]2- ion (m/z 742.5), (c) [(G4S-DA2S)2 + (GP-DA2S) + (C7H15NH3+)3]2- ion (m/z 874.8). Negative-ion in-source fragmentation at low (left panel) and high (right panel) cone voltage of the (d) hexasaccharide of κ-carrageenan (G4S-DA)3, (e) K1 (G4S-DA)3 + SO3, (f) I1 (G4S-DA2S)2 + (GP-DA2S), and (h) I5 (G4S-DA2S)5 - SO3 + [(C7H15NH3+)7]2-.
able to figure out the number of deprotonated sulfate groups of the oligosaccharide. Since the last product ion before backbone cleavage was always the primary backbone structure left with only the deprotonated sulfates, for example, m/z 1015.1 (whose number is known from the charge state of the ion), multiplying the m/z value of this last product ion with its charge state (which is not altered during the fragmentation steps), we obtain the molecular weight of the primary backbone structure of the oligosaccharide attached with the deprotonated sulfates. However, since during CID MS/MS the only groups that are cleaved are the heptylamines and the sulfate groups attached to them, multiplying the difference between the last and the first product ion with its charge state, (49) Gunay, N. S.; Tadano-Aritomi, K.; Toida, T.; Ishizuka, I.; Linhardt, R. J. Anal. Chem. 2003, 75, 3226-3231.
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and then dividing by the sum of heptylamine and sulfate (115.1 + 80 ) 195.1 u), we obtain the number of sulfates that are cleaved during CID MS/MS. For example, Figure 6a presents the CID MS/MS of the hexasaccharide of κ-carrageenan after infusion and selection of the ion m/z 1405.3. From the left panel (10 eV), we observe that the first product ion is the m/z 1290.3, whose difference from the m/z 1405.3 is 115, meaning that it is monocharged. From the right panel (60 eV) of the same Figure 6a, we observe the final product ion m/z 1015.1, which in this case, since the ion is monocharged, represents the molecular weight of the primary backbone structure of the hexasaccharide (G-DA)3 plus the deprotonated sulfate (molecular weight calculated, 1015.2). Then, the difference m/z 1405.3 - 1015.1 () 390.2 u), multiplied by its charge state () 390.2 × 1 ) 390.2) and then divided by the 195.1 u, gives the number 2, which stands for the
Figure 7. Structures proposed for the nonideal oligosaccharides of (a) κ-carrageenans and (b-d) ι-carrageenans. (a) Kx (n ) x + 2), or K1 (n ) 3), K2 (n ) 4), and K3 (n ) 5), (b) I2 (n ) 4), I5 (n ) 5), (c) I3 (n ) 3), I6 (n ) 4), and (d) I1 (n ) 2), I4 (n ) 3), and I7 (n ) 4).
number of protonated sulfates that were cleaved from the hexasaccharide. Following the procedure described above, we verified that the structure K1 of κ-carrageenans (Figure 6b) and the I1 of ι-carrageenans (Figure 6c) corresponded to the structures proposed. For example, for the K1 (m/z 742.5) structure, the first product ion in fragmentation mechanisms, both CID MS/MS and in-source fragmentation (Figure 6b and f, respectively), was the m/z 684.9, indicating that its charge state was 2-. This means that the oligosaccharide K1 had two sulfate groups carrying the negative charge. The last product ion before backbone cleavage was the m/z 547.2 corresponding to a hexasaccharide with two deprotonated sulfates. The difference between m/z 742.5 and 547.2 when multiplied with the charge state of the ions () 742.5 - 547.2 ) 195.3, 195.3 × 2 ) 390.6), and then divided by the molecular weight of sulfate and heptylamine (390.6/195.2 ) 2), equaled 2, a number that corresponded to the sulfates that cleaved during the fragmentation process. When the above information was assembled we obtained a hexasaccharide with four sulfate groups in total. This information matches the structure proposed in Table 2. However, for the I1 oligosaccharide, whose primary structure is composed of (G-DA)2 + (GP-DA), CID MS/MS showed that all heptylamines and protonated sulfates were cleaved from the molecule. The m/z 560.2 corresponds to the cleavage of the carboxylate group50 from the pyruvate acetal group. However, this phenomenon was not observed with the other pyruvated structures (I4 and I7), suggesting that the energy was distributed to a (50) Que´me´ner, B.; De´sire´, C.; Debrauwer, L.; Rathahao, E. J. Chromatogr., A 2003, 984, 185-194.
larger molecule. Taking advantage of the lability of the protonated sulfates (nonstable) and increasing the cone voltage resulted in the same ions as CID MS/MS (Figure 6d-f). In the case of oligosaccharide I5, the only m/z that could be identified by infusion was the m/z 985.5, but this value corresponded also to the tetrasaccharide (G4S-DA2S)2 (Figure 6g). This means that infusion CID MS/MS cannot be used for the study of this oligosaccharide. To address this issue, in-source fragmentation can be used since prior to this the two different structures were separated in the column. Figure 6h depicts the in-source fragmentation of I5. The results showed that, for an unknown oligosaccharide, we were able to detect the molecular weight of its primary backbone structure, the number of sulfate groups that existed in the oligosaccharide, and its charge state. This information was valuable in order to verify the structure proposed above for the ideal oligosaccharides (Table 1) as well as for the nonideal oligosaccharides (Table 2). Applying the above procedures, we verified that the molecular weight of the primary backbone structure and the number of the sulfate groups of all structures corresponded to the proposed structures (Tables 1 and 2). The proposed structures for the nonideal carrageenan oligosaccharides are shown in Figure 7. Structure of K- (K. alvarezii) and ι-Carrageenans (E. denticulatum). The results confirmed that neither κ-carrageenan from K. alvarezii nor ι-carrageenan from E. denticulatum existed as their pure ideal repeating units. The nonideal κ-carrageenan oligosaccharides were found with an additional sulfate group whose position on the primary backbone structure was not defined (Figure 7a). Since the difference between κ- and ι-neocarrabiose sulfate is only one sulfate group, we suggest that these Analytical Chemistry, Vol. 77, No. 13, July 1, 2005
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nonideal structures were in fact hybrids of κ-/ι-carrageenans. This fact has already been suggested in a recent report.44 However, this hybridization was limited, and the ideal repeating unit of κ-carrageenan was the one that dominated, since the incorporation of ι-carrageenan repeating units to the κ-carrageenan oligosaccharides did not exceed, in any case, the one repeating unit for each κ-oligosaccharide. This observation indicates that the ι-carrageenan disaccharides were randomly distributed on the polymer of κ-carrageenan. On the other hand, ι-carrageenan polymer of E. denticulatum was a more complicated one. The nonideal oligosaccharides identified corresponded to the ideal repeating unit of ι-carrageenan, but with one less sulfate group (Figure 7b). This implies the reverse phenomenon from that described for the κ-carrageenans; i.e., the ι-carrageenan polymer existed as a copolymer with κ-carrageenan repeating units. However, as in κ-carrageenans, this type of hybridization was significantly limited. Moreover, since the incorporation of κ-carrageenan repeating units into the ι-carrageenan oligosaccharides did not exceed the one repeating unit for each ι-oligosaccharide, this suggests that the κ-carrageenan disaccharides were randomly distributed on the polymer of ι-carrageenan. Finally, they were identified oligosaccharides consisting of the ideal repeating unit of ι-carrageenan but with an additional sulfate (Figure 7c). This implies that their structure consisted of an anhydrogalactose group plus three sulfate groups for each repeating unit. As for the oligosaccharides of ι-carrageenan identified with a pyruvate group (Figure 7d), all studies reported that the pyruvic acetal residue was situated at the C-4 and C-6 positions of the 3-linked, β-D-galactose (unit G).18,19,51,52 It should be noted that the enzymatic degradation of κ- and ι-carrageenan polymers (from K. alvarezii and E. denticulatum, respectively) was almost quantitative and the enzyme-resistant fraction was not of carrageenan nature, meaning that the enzymatic products found here reflect the structure of the polymers. Application: Characterization of Hybrid ι-/ν-Carrageenans (E. denticulatum). Using the same gradient program as for ι-carrageenan analysis, the more charged oligosaccharides of hybrid ι-/ν-carrageenans were retained in the column. Therefore, to separate the oligosaccharides, it was necessary to modify the mobile phase. Increasing the organic modifier content while holding the heptylamine concentration constant resulted in coeluted peaks. The 2 mM heptylamine in the mobile phase was found to be adequate in order to retain the oligosaccharides. Figure 8 presents the separation of the enzymatically digested hybrid oligosaccharides of ι-/ν-carrageenans by LC/ESI-MS. The first two major peaks corresponded to the tetrasaccharide (G4SDA2S)2 and the hexasaccharide (G4S-DA2S)3 of ι-carrageenans, respectively. The mass spectra of these peaks had the same fingerprint as the tetrasaccharide and hexasaccharide of the enzymatically digested ι-carrageenans even with this heptylamine concentration (2-
