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On-line liquid chromatography–electrospray ionisation mass spectrometry for κ-carrageenan oligosaccharides with a porous graphitic carbon column
Journal of Chromatography A, 1147 (2007) 37–41
On-line liquid chromatography–electrospray ionisation mass spectrometry for -carrageenan oligosaccharides with a porous graphitic carbon column Aristotelis Antonopoulos a , Patrick Favetta b , William Helbert c , Michel Lafosse a,∗ a
Institut de Chimie Organique et Analytique (ICOA), UMR CNRS 6005, Universit´e d’Orl´eans, B.P. 6759, 45067 Orl´eans Cedex 2, France b Institut de Chimie Organique et Analytique (ICOA), UMR CNRS 6005, ASUC, 21 rue de Loigny la Bataille, 28000 Chartres, France c UMR 7139 CNRS-UPMC-Laboratoires Go¨ emar, Station Biologique, Pl. G. Teissier, B.P. 74, 29682 Roscoff Cedex, France Received 18 October 2006; received in revised form 31 January 2007; accepted 7 February 2007 Available online 11 February 2007
Abstract Enzymatically digested oligosaccharides of -carrageenans were separated on a porous graphitic carbon (PGC) column and characterised on-line by electrospray ionisation mass spectrometry (ESI–MS). Two different developing ions were applied. Among them ammonium hydrogencarbonate showed more eluting power as it should on normal anion-exchange stationary phases. The oligosaccharides were detected by ESI–MS as fully deprotonated oligosaccharides. © 2007 Elsevier B.V. All rights reserved. Keywords: Carrageenan; LC–MS; Electrospary ionisation; Porous graphitic carbon
1. Introduction Carrageenans are a class of sulphated polysaccharides extracted from red seaweeds (Rhodophyta) [1,2]. Their primary backbone structure consists of repeating units of a 3-linked d-galactose unit and a 4-linked ␣-d-galactose unit, referred to as G and D units, respectively, in accordance with established nomenclature [3]. Carrageenans are distinguished by the occurrence of 3,6-anhydro bridges (unit A) in the ␣-linked galactose residues and by the substitution of ester sulphate groups in various hydroxyl groups on the primary backbone structure. These biopolymers rarely exist with their ideal form. In real, they are mixtures of different carrageenan families [3] yielding polysaccharide structures where their complexity depends, among other factors, on their life phase, their environmental conditions and their extraction procedures [2]. Even though these sulphated polysaccharides are known for their biological activities [4–6], their relation with their substitution pattern (position of sulphate group) is still ambiguous because of lack of analytical methods for the determination of the fine structure of carrageenans on the polysaccharide level.
∗
Corresponding author. Tel.: +33 238494575; fax: +33 238417281. E-mail address: [email protected] (M. Lafosse).
0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.02.023
The application of specific enzymes (carrageenases) [7] partially resolves the problem, and we now should be able to study certain structural differences. However, the final enzymatic product is a mixture of oligosaccharides which vary significantly in the length of the primary backbone structure and the number/position of sulphate groups. Therefore, analytical tools are needed in order to characterise such a complex mixture. Nuclear magnetic resonance (NMR) spectroscopy is mainly employed [8]; but prior to analysis, purification of the oligosaccharides is needed and this procedure is time consuming. On the other hand, electrospray ionisation mass spectrometry (ESI–MS) has been proven to be a valuable tool for the characterisation of sulphated oligosaccharides [9]. To this end, we presented an on-line LC/ESI–MS method based on ion-pair liquid chromatography [10]. Even though the method can indirectly determine and verify the exact number of sulphate groups as well as the molecular weight of the primary backbone structure of each oligosaccharide in the mixture, determination of sulphate position is hindered. In order to be able to determine the position of the sulphate groups with on-line LC/ESI–MS techniques, direct detection methods should be employed (detection of the deprotonated sulphated ions which are stable), using volatile salts in order to be compatible with the ionisation principle [11]. The above fact limits down the possibilities, besides size exclusion
38
A. Antonopoulos et al. / J. Chromatogr. A 1147 (2007) 37–41
chromatography which has no adequate resolution, only to anion-exchange chromatography techniques. An interesting stationary phase is the porous graphitic carbon (PGC) column [12] which presents simultaneous hydrophobic and electronic interactions that permit better elution control. PGC columns have been used for the off-line LC/ESI–MS profiling and structure elucidation of mucin-type oligosaccharides [13] and for the on-line LC/ESI–MS separation of sialylated [14] and sulphated N-linked oligosaccharides [15]. In all cases, a gradient of MeCN was used, while the concentration of the developing ion (trifluoroacetic acid, ammonium bicarbonate and ammonium acetate, respectively) was constant. Recently, we have reported on the chromatographic separation of - and -carrageenan oligosaccharides on a PGC column using a gradient of developing ion (ammonium acetate or ammonium hydrogencarbonate) coupled to an evaporative light scattering detector [16,17]. In this study, we present a method for the direct detection of the enzymatically digested -carrageenan oligosaccharides using an on-line LC/ESI–MS system in the negative-ion mode. 2. Materials and methods 2.1. Reagents Deionised water was obtained by an Elgastat UHQ II system (18 M) from Elga (Antony, France). HPLC grade acetonitrile (MeCN) was from J.T. Baker (Noisy le Sec, France). Analytical grade ammonium acetate, ammonium hydrogencarbonate and ammonia were from Fluka (St. Quentin Fallavier, France).
set at 30 eV, and the collision energy was set at 0 eV. 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 g/L, RbI 50 ng/L) in 2-propanol/H2 O (1:1, v/v). 2.4. Liquid chromatography on porous graphitic carbon column A PGC column, Hypercarb 150 × 4.6 mm I.D., particle size 5 m (Hypersil, France) used for the chromatographic analysis of the enzymatically digested oligosaccharides of carrageenan. Solvent A was water, solvent B was MeCN and solvent C was 1 M of ammonium acetate (pH 7.2) or 300 mM of ammonium hydrogencarbonate, (pH 8). The flow rate was 1.0 mL/min. For the ammonium acetate, the linear gradient program was as follows: 0–45 min, 85–15% of A, 15% of B, 0–70% of C. For the ammonium hydrogencarbonate, the linear gradient program was as follows: 0–30 min, 85–20% of A, 15–20% of B, 0–60% of C, 30–40 min, 10–5% of A, 20–25% of B, 70% of C. For the ammonium hydrogencarbonate developing ion the PGC column was thermostated under controlled temperature at 60 ◦ C. The heater oven was a block-type heater Gecko-Cil oven (Cil Cluzeau, Saint-Foy-La-Grande, France). The system was left 2 h to equilibrate before injecting the oligosaccharides of -carrageenans. All samples of -carrageenan were injected in a concentration of 2000 ppm in water.
2.2. Preparation of κ-carrageenan oligosaccharides 3. Results and discussion -Carrageenan (E. cottonii X-6913, CP-Kelco) was extracted from K. alvarezii. Oligosaccharides of -carrageenans were produced according to Rochas and Heyraud [18] using recombinant Alteromonas carrageenovora -carrageenase [7]. Prior to freeze-drying, degradation products were filtered through Amicon Centriprep (YM-30, Millipore). 2.3. Instrumentation Liquid chromatography was performed on a Hitachi L7100 (Merck, Nogent sur Marne, France) quaternary gradient pump model and a Rheodyne (Berkeley, CA, USA) model 7125 injector with a 20 L sample loop. The chromatographic system was coupled with passive split (1:10 ratio) to Quattro Ultima triple–quadrupole mass spectrometer (Micromass, Manchester, UK) with a Z-Spray electrospray ionisation source. Mass spectrometry was performed in the negative-ion mode. The desolvatation gas (N2 ) flow was 500 L/h, and the nebuliser gas (N2 ) flow rate was 50 L/h. The RF lens 1 was set at 0.0 V, the aperture was set at 0.0 V, and the RF lens 2 was set at 1.0 V. The probe temperature was held at 250 ◦ C, and the ion source temperature was 130 ◦ C. The electrospray capillary voltage was 2.75 kV, and the cone voltage was 32 V. 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
Fig. 1 depicts the total ion current (TIC) LC/ESI–MS chromatogram of the enzymatically digested -carrageenan oligosaccharides on a PGC column with ammonium acetate (Fig. 1a) and ammonium hydrogencarbonate (Fig. 1b) as developing ions. In both cases, the smallest oligosaccharide detected in the chromatogram corresponded to a disaccharide of -carrageenan (peak 1), verifying that the smallest oligosaccharide produced from the -carrageenase corresponded to the neocarrabiose-sulphate (DA-G4S) [19,20]. On the contrary, using ion-pair LC/ESI–MS [10] the neocarrabiose-sulphate unit could not be detected in the TIC chromatogram because the sulphate group was neutralised by the ion-pair agent. Using ammonium acetate as a developing ion, it was possible to detect up to a decasaccharide (DA-G4S)5 (Fig. 1a), while with the ammonium hydrogencarbonate, the largest oligosaccharide detected corresponded to a didecasaccharide (DA-G4S)10 (Fig. 1b). This difference is possible because of greater volatility of the latter than the ammonium acetate. Another difference is the separation of anomers (double peaks, Fig. 1a). This phenomenon has already been stated for carbohydrates [21] and carrageenan oligosaccharides [16]. In Fig. 2b, the PGC column was thermostated at 60 ◦ C and therefore the anomers were not separated because of the acceleration of the rate of interconversion between ␣ and  anomers.
A. Antonopoulos et al. / J. Chromatogr. A 1147 (2007) 37–41
39
Table 1 Main ions found for the -carrageenan oligosaccharides with negative-ion LC/ESI–MS Oligosaccharide
Disaccharide * *
Tetrasaccharide
*
Hexasaccharide
Octasaccharide
Decasaccharide
Dodecasaccharide Fig. 1. Total ion current chromatogram (LC/ESI–MS) of the enzymatically digested -carrageenans on a PGC column. Developing ions: (a) ammonium acetate, (b) ammonium hydrogencarbonate. The numbers above the peaks designate the repeating units of -neocarrabiose-sulphate (DA-G4S)n . For experimental conditions, see Section 2.
The influence of the developing ion to the detection of the -carrageenan oligosaccharides can be noted in Fig. 2. The negative-ion MS spectrum of disaccharide of -carrageenan with ammonium acetate (DA-G4S, peak 1 of Fig. 1a) is shown in Fig. 2a-1, while with ammonium hydrogencarbonate (DA-G4S, peak 1 of Fig. 1b) is shown in Fig. 2a-2. In both cases, the most abundant ion at m/z 403.2 corresponded to the molecular ion [DA-G4S]− . However, in the case of ammonium hydrogencarbonate, two additional ions were detected at m/z 807.4 and 824.3 ([(DA-G4S)2 + H]− and [(DA-G4S)2 + NH4 + ]− respectively, Table 1) which did not correspond to the tetrasaccharide of -carrageenan, since before detection, all oligosaccharides were separated in the PGC column. In addition, if this was the case, then we should have detected the molecular ion of the tetrasaccharide ([(DA-G4S)2 ]2− ) at m/z 394.4 (Fig. 2a-2). The m/z values (807.4 and 824.3) corresponded to clusters formed by ion/neutral reactions, a phenomenon already reported for the oligosaccharides of -carrageenan in the negative-ion mode [22]. Fig. 2b-1 depicts the MS spectrum of tetrasaccharide of -carrageenan (DA-G4S)2 (peak 2 of Fig. 1a) with ammonium acetate. The most abundant ion did not correspond to the molecular ion [(DA-G4S)2 ]2− (m/z 394.2) like the case
*
Ions
[DA-G4S]− [(DA-G4S)2 + H]− [(DA-G4S)2 + NH4 + ]− [(DA-G4S)2 ]2− [(DA-G4S)2 -SO3 + H]− [(DA-G4S)2 + H]− [(DA-G4S)2 + NH4 + ]− [(DA-G4S)4 + 3NH4 + ]− [(DA-G4S)3 ]3− [(DA-G4S)3 -SO3 + H]2− [(DA-G4S)3 + Na+ ]2− [(DA-G4S)3 -2SO3 + 2H]− [(DA-G4S)3 -SO3 + 2H]− [(DA-G4S)3 + H + NH4 + ]2− [(DA-G4S)3 + 2NH4 + ]− [(DA-G4S)3 + Ca2+ ]− [(DA-G4S)4 ]4− [(DA-G4S)4 -SO3 + H]3− [(DA-G4S)4 -2SO3 + 2H]2− [(DA-G4S)4 + 2NH4 + ]2− [(DA-G4S)4 + 3NH4 + ]− [(DA-G4S)4 + Ca2+ ]− [(DA-G4S)5 ]5− [(DA-G4S)5 -SO3 + H]4− [(DA-G4S)5 -3SO3 + 3H]2− [(DA-G4S)5 + 3NH4 + ]2− [(DA-G4S)6 ]6− [(DA-G4S)6 -SO3 + H]5− [(DA-G4S)6 -2SO3 + 2H]4− [(DA-G4S)6 + 4NH4 + ]2−
m/z Found
Calculated
403.2 807.4 824.3 394.4 709.4 789.4 806.3 1630.9 391.3 547.4 598.6 1015.4 1095.4 1192.4 1209.6 1212.9 389.9 493.3 700.3 797.6 1612.5 799.3 389.1 466.6 853.4 998.9 388.5 450.2 543.4 1200.5
403.1 807.1 824.2 394.1 709.1 789.1 806.2 1630.3 391.0 547.1 598.1 1015.2 1095.2 1192.1 1209.2 1213.1 389.6 493.1 700.2 797.2 1612.3 799.1 388.6 466.1 853.2 998.7 388.1 449.9 542.6 1200.2
Designates clusters formed by ion/neutral reactions.
of the ammonium hydrogencarbonate (Fig. 2b-2, peak 2 of Fig. 1b), but to the desulphated ion [(DA-G4S)2 -SO3 + H]− (m/z 709.2, 100% of relative intensity). The additional ions at m/z 789.4 and 806.3 (Fig. 2b-2) corresponded to the monocharged ions [(DA-G4S)2 + H]− and [(DA-G4S)2 + NH4 + ]− respectively (Table 1), while the ion at m/z 1630.9 corresponded to the [(DAG4S)4 + 3NH4 + ]− . Concerning the hexasaccharide (DA-G4S)3 (peak 3 of Fig. 1a and b) in contrast to ammonium acetate (Fig. 2c-1), ammonium hydrogencarbonate (Fig. 2c-2) gave an ion at m/z 391.3, which corresponds to the molecular ion [(DA-G4S)3 ]3− . With ammonium acetate, we could observe only the desulphated ions [(DA-G4S)3 -SO3 + H]2− , [(DA-G4S)3 -2SO3 + 2H]− and [(DA-G4S)3 -SO3 + 2H]− at m/z 547.4, 1015.4 and 1095.4, respectively. The ions at m/z 1095 and 1212.9 (Table 1) were not detected with the ammonium hydrogencarbonate. On the contrary, with this salt, we can observe the ([(DAG4S)3 + H + NH4 + ]2− and [(DA-G4S)3 + 2NH4 + ]− ; m/z 1192.4 and 1209.6, respectively). Fig. 2d-1 and d-2 depict the negative-ion MS spectra of the octasaccharide of -carrageenan (DA-G4S)4 , with ammonium acetate and ammonium hydrogencarbonate respectively (peak 4 of Fig. 1a and b). Using the former developing ion, the
40
A. Antonopoulos et al. / J. Chromatogr. A 1147 (2007) 37–41
Fig. 2. Negative ion electrospray mass spectra in LC/MS of the enzymatically digested -carrageenans of (a) disaccharide, (b) tetrasaccharide, (c) hexasaccharide, (d) octasaccharide and (e) decasaccharide using different developing ions. (1) Ammonium acetate. (2) Ammonium hydrogencarbonate.
oligosaccharide was detected practically only as a discharged ion [(DA-G4S)4 -2SO3 + H]2− (m/z 700.3) and to the less abundant ion at m/z 799.3 corresponding to the [(DA-G4S)4 + Ca2+ ]− . Using the latter developing ion, the most abundant ion corresponded to the molecular ion [(DA-G4S)4 ]4− (m/z 389.6). The ion at m/z 797.6 corresponded to the [(DA-G4S)4 + 2NH4 + ]2− , while the m/z 493.3 corresponded the desulphated ion [(DAG4S)4 -SO3 + H]3− . The same behaviour was detected for the decasaccharide (DA-G4S)5 of -carrageenan (peak 5 of Fig. 1a and b). With ammonium acetate, the most abundant ion corresponded to the discharged (Fig. 2e-1) with all other SO3 groups were cleaved off the molecule (m/z 853.4, [(DA-G4S)5 -3SO3 + 3H]2− ), while with ammonium hydrogencarbonate (Fig. 2e-2), the most abundant molecular ions corresponded to the molecular ions (m/z 389.1) and the ammonium adducts (m/z 998.9, [(DAG4S)5 + 3NH4 + ]2− ).
On the contrary, with ammonium hydrogencarbonate it was not observed the molecular ion of the oligosaccharide as the most abundant ion only after the dodecasaccharide. The ion at m/z 1200.5 corresponded at the [(DA-G4S)6 + 4NH4 + ]2− . The larger oligosaccharides of -carrageenan were detected either as desulphated ions corresponding to the formula [(DA-G4S)x SO3 + H](x−1) or as ions ammonium adducts corresponding to the formula [(DA-G4S)x + (x-2)NH4 + ]2− , where x corresponds to the number of repeating units of -carrageenan. To conclude, the advantage of this method is that the mobile phase (ammonium hydrogencarbonate, MeCN) used for the separation of the -carrageenan oligosaccharides on a PGC column, permits at the same time their MS detection as fully deprotonated ions and that this is valid for oligosaccharides with more than six sulphate groups. Future studies should be able to demonstrate that when these fully depronotated ions are fragmented, LC/ESI–MS–MS will
A. Antonopoulos et al. / J. Chromatogr. A 1147 (2007) 37–41
give information concerning the position of the sulphate group on a selected oligosaccharide. This is a clear advantage compared with ion-pair LC/ESI–MS where the sulphate position is based on assumptions that cannot always be verified. In general, this setup (LC–PGC/ESI–MS) should allow in one chromatographic run to separate and determine the fine structure (position of sulphate group) of the sulphated oligosaccharides in a mixture. Acknowledgement Funds from the State Scholarship Foundation of Greece are gratefully acknowledged. References [1] J.S. Craigie, in: K.M. Cole, R.G. Dheath (Eds.), Biology of the Red Seaweeds, Cambridge University Press, Cambridge, 1990, p. 221. [2] B. Kloareg, R.S. Quatrano, Oceanogr. Marine Biol. Annu. Rev. 26 (1988) 259. [3] S.H. Knutsen, D.E. Myslabodski, B. Larsen, A.I. Usov, Bot. Mar. 37 (1994) 163. [4] M.E. Perotti, A. Pirovano, D.M. Phillips, Biol. Reprod. 69 (2003) 933. [5] T. Yamada, A. Ogamo, T. Saito, J. Watanabe, H. Uchiyama, Y. Nakagawa, Carbohydr. Polym. 32 (1997) 51. [6] H. Desaire, T.L. Sirich, J.A. Leary, Anal. Chem. 73 (2001) 3513.
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[7] G. Michel, T. Barbeyron, D. Flament, T. Vernat, B. Kloareg, O. Dideberg, Acta Cryst. D55 (1999) 918. [8] F. van de Velde, S.H. Knutsen, A.I. Usov, H.S. Rollemay, A.S. Cerezo, Trends Food Sci. Technol. 13 (2002) 73. [9] J. Zaia, Mass Spectrom. Rev. 23 (2004) 161. [10] A. Antonopoulos, P. Favetta, W. Helbert, M. Lafosse, Anal. Chem. 77 (2005) 4125. [11] P. Kebarle, Y. Ho, in: R.B. Cole (Ed.), Electrospray Ionization Mass Spectrometry—Fundamentals Instrumentation and Applications, WileyInterscience, New York, 1997, p. 3. [12] P. Ross, LC-GC 13 (2000) 310. [13] J. Zhang, L.L. Lindsay, J.L. Hedrick, C.B. Lebrilla, Anal. Chem. 76 (2004) 5990. [14] N.G. Karlsson, N.L. Wilson, H.-J. Wirth, P. Dawes, H. Joshi, N.H. Packer, Rapid Commun. Mass Spectrom. 18 (2004) 2282. [15] N. Kawasaki, Y. Haishima, M. Ohta, S. Itoh, M. Hyuga, S. Hyuga, T. Hayakawa, Glycobiology 11 (2001) 1043. [16] A. Antonopoulos, B. Herbreteau, M. Lafosse, W. Helbert, J. Chromatogr. A 1023 (2004) 231. [17] A. Antonopoulos, P. Favetta, W. Helbert, M. Lafosse, J. Chromatogr. A 1059 (2004) 83. [18] C. Rochas, A. Heyraud, Polym. Bull. 5 (1981) 81. [19] P. Potin, C. Richard, T. Barbeyron, B. Henrissat, C. Gey, Y. Petillot, E. Forest, O. Dideberg, C. Rochas, B. Kloareg, Eur. J. Biochem. 228 (1995) 971. [20] S.H. Knutsen, M. Sletmoen, T. Kristensen, T. Barbeyron, B. Kloareg, P. Potin, Carbohydr. Res. 331 (2001) 101. [21] K. Koizumi, Y. Okada, M. Fukuda, Carbohydr. Res. 215 (1991) 67. [22] D. Ekeberg, S.H. Knutsen, M. Sletmoen, Carbohydr. Res. 334 (2001) 49.
On-line liquid chromatography–electrospray ionisation mass spectrometry for -carrageenan oligosaccharides with a porous graphitic carbon column Aristotelis Antonopoulos a , Patrick Favetta b , William Helbert c , Michel Lafosse a,∗ a
Institut de Chimie Organique et Analytique (ICOA), UMR CNRS 6005, Universit´e d’Orl´eans, B.P. 6759, 45067 Orl´eans Cedex 2, France b Institut de Chimie Organique et Analytique (ICOA), UMR CNRS 6005, ASUC, 21 rue de Loigny la Bataille, 28000 Chartres, France c UMR 7139 CNRS-UPMC-Laboratoires Go¨ emar, Station Biologique, Pl. G. Teissier, B.P. 74, 29682 Roscoff Cedex, France Received 18 October 2006; received in revised form 31 January 2007; accepted 7 February 2007 Available online 11 February 2007
Abstract Enzymatically digested oligosaccharides of -carrageenans were separated on a porous graphitic carbon (PGC) column and characterised on-line by electrospray ionisation mass spectrometry (ESI–MS). Two different developing ions were applied. Among them ammonium hydrogencarbonate showed more eluting power as it should on normal anion-exchange stationary phases. The oligosaccharides were detected by ESI–MS as fully deprotonated oligosaccharides. © 2007 Elsevier B.V. All rights reserved. Keywords: Carrageenan; LC–MS; Electrospary ionisation; Porous graphitic carbon
1. Introduction Carrageenans are a class of sulphated polysaccharides extracted from red seaweeds (Rhodophyta) [1,2]. Their primary backbone structure consists of repeating units of a 3-linked d-galactose unit and a 4-linked ␣-d-galactose unit, referred to as G and D units, respectively, in accordance with established nomenclature [3]. Carrageenans are distinguished by the occurrence of 3,6-anhydro bridges (unit A) in the ␣-linked galactose residues and by the substitution of ester sulphate groups in various hydroxyl groups on the primary backbone structure. These biopolymers rarely exist with their ideal form. In real, they are mixtures of different carrageenan families [3] yielding polysaccharide structures where their complexity depends, among other factors, on their life phase, their environmental conditions and their extraction procedures [2]. Even though these sulphated polysaccharides are known for their biological activities [4–6], their relation with their substitution pattern (position of sulphate group) is still ambiguous because of lack of analytical methods for the determination of the fine structure of carrageenans on the polysaccharide level.
∗
Corresponding author. Tel.: +33 238494575; fax: +33 238417281. E-mail address: [email protected] (M. Lafosse).
0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.02.023
The application of specific enzymes (carrageenases) [7] partially resolves the problem, and we now should be able to study certain structural differences. However, the final enzymatic product is a mixture of oligosaccharides which vary significantly in the length of the primary backbone structure and the number/position of sulphate groups. Therefore, analytical tools are needed in order to characterise such a complex mixture. Nuclear magnetic resonance (NMR) spectroscopy is mainly employed [8]; but prior to analysis, purification of the oligosaccharides is needed and this procedure is time consuming. On the other hand, electrospray ionisation mass spectrometry (ESI–MS) has been proven to be a valuable tool for the characterisation of sulphated oligosaccharides [9]. To this end, we presented an on-line LC/ESI–MS method based on ion-pair liquid chromatography [10]. Even though the method can indirectly determine and verify the exact number of sulphate groups as well as the molecular weight of the primary backbone structure of each oligosaccharide in the mixture, determination of sulphate position is hindered. In order to be able to determine the position of the sulphate groups with on-line LC/ESI–MS techniques, direct detection methods should be employed (detection of the deprotonated sulphated ions which are stable), using volatile salts in order to be compatible with the ionisation principle [11]. The above fact limits down the possibilities, besides size exclusion
38
A. Antonopoulos et al. / J. Chromatogr. A 1147 (2007) 37–41
chromatography which has no adequate resolution, only to anion-exchange chromatography techniques. An interesting stationary phase is the porous graphitic carbon (PGC) column [12] which presents simultaneous hydrophobic and electronic interactions that permit better elution control. PGC columns have been used for the off-line LC/ESI–MS profiling and structure elucidation of mucin-type oligosaccharides [13] and for the on-line LC/ESI–MS separation of sialylated [14] and sulphated N-linked oligosaccharides [15]. In all cases, a gradient of MeCN was used, while the concentration of the developing ion (trifluoroacetic acid, ammonium bicarbonate and ammonium acetate, respectively) was constant. Recently, we have reported on the chromatographic separation of - and -carrageenan oligosaccharides on a PGC column using a gradient of developing ion (ammonium acetate or ammonium hydrogencarbonate) coupled to an evaporative light scattering detector [16,17]. In this study, we present a method for the direct detection of the enzymatically digested -carrageenan oligosaccharides using an on-line LC/ESI–MS system in the negative-ion mode. 2. Materials and methods 2.1. Reagents Deionised water was obtained by an Elgastat UHQ II system (18 M) from Elga (Antony, France). HPLC grade acetonitrile (MeCN) was from J.T. Baker (Noisy le Sec, France). Analytical grade ammonium acetate, ammonium hydrogencarbonate and ammonia were from Fluka (St. Quentin Fallavier, France).
set at 30 eV, and the collision energy was set at 0 eV. 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 g/L, RbI 50 ng/L) in 2-propanol/H2 O (1:1, v/v). 2.4. Liquid chromatography on porous graphitic carbon column A PGC column, Hypercarb 150 × 4.6 mm I.D., particle size 5 m (Hypersil, France) used for the chromatographic analysis of the enzymatically digested oligosaccharides of carrageenan. Solvent A was water, solvent B was MeCN and solvent C was 1 M of ammonium acetate (pH 7.2) or 300 mM of ammonium hydrogencarbonate, (pH 8). The flow rate was 1.0 mL/min. For the ammonium acetate, the linear gradient program was as follows: 0–45 min, 85–15% of A, 15% of B, 0–70% of C. For the ammonium hydrogencarbonate, the linear gradient program was as follows: 0–30 min, 85–20% of A, 15–20% of B, 0–60% of C, 30–40 min, 10–5% of A, 20–25% of B, 70% of C. For the ammonium hydrogencarbonate developing ion the PGC column was thermostated under controlled temperature at 60 ◦ C. The heater oven was a block-type heater Gecko-Cil oven (Cil Cluzeau, Saint-Foy-La-Grande, France). The system was left 2 h to equilibrate before injecting the oligosaccharides of -carrageenans. All samples of -carrageenan were injected in a concentration of 2000 ppm in water.
2.2. Preparation of κ-carrageenan oligosaccharides 3. Results and discussion -Carrageenan (E. cottonii X-6913, CP-Kelco) was extracted from K. alvarezii. Oligosaccharides of -carrageenans were produced according to Rochas and Heyraud [18] using recombinant Alteromonas carrageenovora -carrageenase [7]. Prior to freeze-drying, degradation products were filtered through Amicon Centriprep (YM-30, Millipore). 2.3. Instrumentation Liquid chromatography was performed on a Hitachi L7100 (Merck, Nogent sur Marne, France) quaternary gradient pump model and a Rheodyne (Berkeley, CA, USA) model 7125 injector with a 20 L sample loop. The chromatographic system was coupled with passive split (1:10 ratio) to Quattro Ultima triple–quadrupole mass spectrometer (Micromass, Manchester, UK) with a Z-Spray electrospray ionisation source. Mass spectrometry was performed in the negative-ion mode. The desolvatation gas (N2 ) flow was 500 L/h, and the nebuliser gas (N2 ) flow rate was 50 L/h. The RF lens 1 was set at 0.0 V, the aperture was set at 0.0 V, and the RF lens 2 was set at 1.0 V. The probe temperature was held at 250 ◦ C, and the ion source temperature was 130 ◦ C. The electrospray capillary voltage was 2.75 kV, and the cone voltage was 32 V. 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
Fig. 1 depicts the total ion current (TIC) LC/ESI–MS chromatogram of the enzymatically digested -carrageenan oligosaccharides on a PGC column with ammonium acetate (Fig. 1a) and ammonium hydrogencarbonate (Fig. 1b) as developing ions. In both cases, the smallest oligosaccharide detected in the chromatogram corresponded to a disaccharide of -carrageenan (peak 1), verifying that the smallest oligosaccharide produced from the -carrageenase corresponded to the neocarrabiose-sulphate (DA-G4S) [19,20]. On the contrary, using ion-pair LC/ESI–MS [10] the neocarrabiose-sulphate unit could not be detected in the TIC chromatogram because the sulphate group was neutralised by the ion-pair agent. Using ammonium acetate as a developing ion, it was possible to detect up to a decasaccharide (DA-G4S)5 (Fig. 1a), while with the ammonium hydrogencarbonate, the largest oligosaccharide detected corresponded to a didecasaccharide (DA-G4S)10 (Fig. 1b). This difference is possible because of greater volatility of the latter than the ammonium acetate. Another difference is the separation of anomers (double peaks, Fig. 1a). This phenomenon has already been stated for carbohydrates [21] and carrageenan oligosaccharides [16]. In Fig. 2b, the PGC column was thermostated at 60 ◦ C and therefore the anomers were not separated because of the acceleration of the rate of interconversion between ␣ and  anomers.
A. Antonopoulos et al. / J. Chromatogr. A 1147 (2007) 37–41
39
Table 1 Main ions found for the -carrageenan oligosaccharides with negative-ion LC/ESI–MS Oligosaccharide
Disaccharide * *
Tetrasaccharide
*
Hexasaccharide
Octasaccharide
Decasaccharide
Dodecasaccharide Fig. 1. Total ion current chromatogram (LC/ESI–MS) of the enzymatically digested -carrageenans on a PGC column. Developing ions: (a) ammonium acetate, (b) ammonium hydrogencarbonate. The numbers above the peaks designate the repeating units of -neocarrabiose-sulphate (DA-G4S)n . For experimental conditions, see Section 2.
The influence of the developing ion to the detection of the -carrageenan oligosaccharides can be noted in Fig. 2. The negative-ion MS spectrum of disaccharide of -carrageenan with ammonium acetate (DA-G4S, peak 1 of Fig. 1a) is shown in Fig. 2a-1, while with ammonium hydrogencarbonate (DA-G4S, peak 1 of Fig. 1b) is shown in Fig. 2a-2. In both cases, the most abundant ion at m/z 403.2 corresponded to the molecular ion [DA-G4S]− . However, in the case of ammonium hydrogencarbonate, two additional ions were detected at m/z 807.4 and 824.3 ([(DA-G4S)2 + H]− and [(DA-G4S)2 + NH4 + ]− respectively, Table 1) which did not correspond to the tetrasaccharide of -carrageenan, since before detection, all oligosaccharides were separated in the PGC column. In addition, if this was the case, then we should have detected the molecular ion of the tetrasaccharide ([(DA-G4S)2 ]2− ) at m/z 394.4 (Fig. 2a-2). The m/z values (807.4 and 824.3) corresponded to clusters formed by ion/neutral reactions, a phenomenon already reported for the oligosaccharides of -carrageenan in the negative-ion mode [22]. Fig. 2b-1 depicts the MS spectrum of tetrasaccharide of -carrageenan (DA-G4S)2 (peak 2 of Fig. 1a) with ammonium acetate. The most abundant ion did not correspond to the molecular ion [(DA-G4S)2 ]2− (m/z 394.2) like the case
*
Ions
[DA-G4S]− [(DA-G4S)2 + H]− [(DA-G4S)2 + NH4 + ]− [(DA-G4S)2 ]2− [(DA-G4S)2 -SO3 + H]− [(DA-G4S)2 + H]− [(DA-G4S)2 + NH4 + ]− [(DA-G4S)4 + 3NH4 + ]− [(DA-G4S)3 ]3− [(DA-G4S)3 -SO3 + H]2− [(DA-G4S)3 + Na+ ]2− [(DA-G4S)3 -2SO3 + 2H]− [(DA-G4S)3 -SO3 + 2H]− [(DA-G4S)3 + H + NH4 + ]2− [(DA-G4S)3 + 2NH4 + ]− [(DA-G4S)3 + Ca2+ ]− [(DA-G4S)4 ]4− [(DA-G4S)4 -SO3 + H]3− [(DA-G4S)4 -2SO3 + 2H]2− [(DA-G4S)4 + 2NH4 + ]2− [(DA-G4S)4 + 3NH4 + ]− [(DA-G4S)4 + Ca2+ ]− [(DA-G4S)5 ]5− [(DA-G4S)5 -SO3 + H]4− [(DA-G4S)5 -3SO3 + 3H]2− [(DA-G4S)5 + 3NH4 + ]2− [(DA-G4S)6 ]6− [(DA-G4S)6 -SO3 + H]5− [(DA-G4S)6 -2SO3 + 2H]4− [(DA-G4S)6 + 4NH4 + ]2−
m/z Found
Calculated
403.2 807.4 824.3 394.4 709.4 789.4 806.3 1630.9 391.3 547.4 598.6 1015.4 1095.4 1192.4 1209.6 1212.9 389.9 493.3 700.3 797.6 1612.5 799.3 389.1 466.6 853.4 998.9 388.5 450.2 543.4 1200.5
403.1 807.1 824.2 394.1 709.1 789.1 806.2 1630.3 391.0 547.1 598.1 1015.2 1095.2 1192.1 1209.2 1213.1 389.6 493.1 700.2 797.2 1612.3 799.1 388.6 466.1 853.2 998.7 388.1 449.9 542.6 1200.2
Designates clusters formed by ion/neutral reactions.
of the ammonium hydrogencarbonate (Fig. 2b-2, peak 2 of Fig. 1b), but to the desulphated ion [(DA-G4S)2 -SO3 + H]− (m/z 709.2, 100% of relative intensity). The additional ions at m/z 789.4 and 806.3 (Fig. 2b-2) corresponded to the monocharged ions [(DA-G4S)2 + H]− and [(DA-G4S)2 + NH4 + ]− respectively (Table 1), while the ion at m/z 1630.9 corresponded to the [(DAG4S)4 + 3NH4 + ]− . Concerning the hexasaccharide (DA-G4S)3 (peak 3 of Fig. 1a and b) in contrast to ammonium acetate (Fig. 2c-1), ammonium hydrogencarbonate (Fig. 2c-2) gave an ion at m/z 391.3, which corresponds to the molecular ion [(DA-G4S)3 ]3− . With ammonium acetate, we could observe only the desulphated ions [(DA-G4S)3 -SO3 + H]2− , [(DA-G4S)3 -2SO3 + 2H]− and [(DA-G4S)3 -SO3 + 2H]− at m/z 547.4, 1015.4 and 1095.4, respectively. The ions at m/z 1095 and 1212.9 (Table 1) were not detected with the ammonium hydrogencarbonate. On the contrary, with this salt, we can observe the ([(DAG4S)3 + H + NH4 + ]2− and [(DA-G4S)3 + 2NH4 + ]− ; m/z 1192.4 and 1209.6, respectively). Fig. 2d-1 and d-2 depict the negative-ion MS spectra of the octasaccharide of -carrageenan (DA-G4S)4 , with ammonium acetate and ammonium hydrogencarbonate respectively (peak 4 of Fig. 1a and b). Using the former developing ion, the
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A. Antonopoulos et al. / J. Chromatogr. A 1147 (2007) 37–41
Fig. 2. Negative ion electrospray mass spectra in LC/MS of the enzymatically digested -carrageenans of (a) disaccharide, (b) tetrasaccharide, (c) hexasaccharide, (d) octasaccharide and (e) decasaccharide using different developing ions. (1) Ammonium acetate. (2) Ammonium hydrogencarbonate.
oligosaccharide was detected practically only as a discharged ion [(DA-G4S)4 -2SO3 + H]2− (m/z 700.3) and to the less abundant ion at m/z 799.3 corresponding to the [(DA-G4S)4 + Ca2+ ]− . Using the latter developing ion, the most abundant ion corresponded to the molecular ion [(DA-G4S)4 ]4− (m/z 389.6). The ion at m/z 797.6 corresponded to the [(DA-G4S)4 + 2NH4 + ]2− , while the m/z 493.3 corresponded the desulphated ion [(DAG4S)4 -SO3 + H]3− . The same behaviour was detected for the decasaccharide (DA-G4S)5 of -carrageenan (peak 5 of Fig. 1a and b). With ammonium acetate, the most abundant ion corresponded to the discharged (Fig. 2e-1) with all other SO3 groups were cleaved off the molecule (m/z 853.4, [(DA-G4S)5 -3SO3 + 3H]2− ), while with ammonium hydrogencarbonate (Fig. 2e-2), the most abundant molecular ions corresponded to the molecular ions (m/z 389.1) and the ammonium adducts (m/z 998.9, [(DAG4S)5 + 3NH4 + ]2− ).
On the contrary, with ammonium hydrogencarbonate it was not observed the molecular ion of the oligosaccharide as the most abundant ion only after the dodecasaccharide. The ion at m/z 1200.5 corresponded at the [(DA-G4S)6 + 4NH4 + ]2− . The larger oligosaccharides of -carrageenan were detected either as desulphated ions corresponding to the formula [(DA-G4S)x SO3 + H](x−1) or as ions ammonium adducts corresponding to the formula [(DA-G4S)x + (x-2)NH4 + ]2− , where x corresponds to the number of repeating units of -carrageenan. To conclude, the advantage of this method is that the mobile phase (ammonium hydrogencarbonate, MeCN) used for the separation of the -carrageenan oligosaccharides on a PGC column, permits at the same time their MS detection as fully deprotonated ions and that this is valid for oligosaccharides with more than six sulphate groups. Future studies should be able to demonstrate that when these fully depronotated ions are fragmented, LC/ESI–MS–MS will
A. Antonopoulos et al. / J. Chromatogr. A 1147 (2007) 37–41
give information concerning the position of the sulphate group on a selected oligosaccharide. This is a clear advantage compared with ion-pair LC/ESI–MS where the sulphate position is based on assumptions that cannot always be verified. In general, this setup (LC–PGC/ESI–MS) should allow in one chromatographic run to separate and determine the fine structure (position of sulphate group) of the sulphated oligosaccharides in a mixture. Acknowledgement Funds from the State Scholarship Foundation of Greece are gratefully acknowledged. References [1] J.S. Craigie, in: K.M. Cole, R.G. Dheath (Eds.), Biology of the Red Seaweeds, Cambridge University Press, Cambridge, 1990, p. 221. [2] B. Kloareg, R.S. Quatrano, Oceanogr. Marine Biol. Annu. Rev. 26 (1988) 259. [3] S.H. Knutsen, D.E. Myslabodski, B. Larsen, A.I. Usov, Bot. Mar. 37 (1994) 163. [4] M.E. Perotti, A. Pirovano, D.M. Phillips, Biol. Reprod. 69 (2003) 933. [5] T. Yamada, A. Ogamo, T. Saito, J. Watanabe, H. Uchiyama, Y. Nakagawa, Carbohydr. Polym. 32 (1997) 51. [6] H. Desaire, T.L. Sirich, J.A. Leary, Anal. Chem. 73 (2001) 3513.
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