Anal. Chem. 1999, 71, 1421-1427
Sequencing of Partially Methyl-Esterified Oligogalacturonates by Tandem Mass Spectrometry and Its Use To Determine Pectinase Specificities Roman Ko 1 rner,† Gerrit Limberg,‡ Tove M. I. E. Christensen,‡ Jørn Dalgaard Mikkelsen,‡ and Peter Roepstorff*,†
Department of Molecular Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark, and Danisco Biotechnology, Langebrogade 1, DK-1001 Copenhagen K, Denmark
Complex mixtures of acidic oligosaccharides were produced by enzymatic digestion of partially methyl-esterified pectin with Aspergillus niger pectin lyase, endopolygalacturonase II, and exopolygalacturonase. To determine the specificities of these pectolytic enzymes toward nonesterified and methyl-esterified galacturonic acid residues, we have studied the methyl esterification patterns of selected oligomers in unseparated pectin digests. Collision-induced dissociation in a nanoelectrospray ionization ion trap mass spectrometer was used to locate methylesterified galacturonic acid residues in oligomers up to a degree of polymerization of 10. Analysis of the methyl esterification patterns gave insight into the substrate specificities of these pectolytic enzymes. Isomeric fragment ions containing the reducing and nonreducing ends were differentiated by 18O-labeling of the reducing end. Scientific interest in the structural analysis of polysaccharides is increasing rapidly due to their importance in industry and in biological functions such as structural elements in plants. Pectins are a family of complex, anionic polysaccharides found in the primary cell wall and intercellular regions of higher plants.1,2 They consist of an R-(1f4)-linked galacturonic acid homopolymer (smooth region) and L-rhamnose D-galacturonic acid repeating units carrying neutral sugar side chains (hairy regions). Galacturonic acid units in both regions are partially methyl-esterified and acetylated. A schematic presentation of the pectin structure is given in Figure 1. Pectins are widely used as functional ingredients in the food industry due to their ability to form aqueous gels and stabilize proteins3 and are under investigation for medical applications such as lowering blood cholesterol levels.4 Furthermore, pectic frag* Corresponding author: (phone) (+45) 65572404; (fax) (+45) 65932781; (email)
[email protected]. † Odense University. ‡ Danisco Biotechnology. (1) Visser, J.; Voragen, A. G. J. Pectins and Pectinases; Elsevier: Amsterdam, 1996. (2) Francis, B. J.; Bell, J.-M. K. Trop. Sci. 1975, 17, 25-44. (3) May, C. D. Carbohydr. Polym. 1990, 12, 79-99. (4) Endress, H.-U. In The Chemistry and Technology of Pectin; Walter, R. G., Ed.; Academic: London, 1991; pp 251-268. 10.1021/ac981240o CCC: $18.00 Published on Web 02/25/1999
© 1999 American Chemical Society
Figure 1. Schematic overview of the pectin structure. Pectin consists of a homogalacturonan backbone (smooth region) and L-rhamnose D-galacturonic acid repeating units carrying branched neutral sugar side chains (hairy region). The smooth region is composed of partially methyl-esterified R-(1f4)-linked D-galacturonic acids as shown in the inset.
ments have been shown to possess regulatory activity for plant defense mechanisms5,6 and plant development.5,7 The degree and distribution of methyl ester groups are critical for the functioning of pectins and are therefore subject to ongoing research. Since pectin is highly heterogeneous, enzymatic digestion plays an important role for identification of structural motifs of pectin. A large number of pectinases has been isolated from bacteria, fungi, and plants,1 but for most of these, the specificity toward natural, partially methyl-esterified pectin has not been determined in detail. Recent studies8,9 have shown that pectolytic digests can be analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to determine the degree of polymerization (5) Coˆte´, F.; Hahn, M. G. Plant Mol. Biol. 1994, 26, 1379-1411. (6) De Lorenzo, G.; Ranucci, A.; Bellincampi, A.; Salvi, G.; Cervone, F. Plant Sci. 1987, 51, 147-150. (7) Marfa`, V.; Gollin, D. J.; Eberhard, S.; Mohnen, D.; Darvill, A.; Albersheim, P. Plant J. 1991, 1, 217-225. (8) Daas, P. J. H.; Arisz, P. W.; Schols, H. A.; De Ruiter, G. A.; Voragen, A. G. J. Anal. Biochem. 1998, 257, 195-202. (9) Ko ¨rner, R.; Limberg, G.; Dalgaard Mikkelsen, J.; Roepstorff, P. J. Mass Spectrom. 1998, 33, 836-842.
Analytical Chemistry, Vol. 71, No. 7, April 1, 1999 1421
(DP) and the degree of methyl esterification (DE). However, not only the number but also the location of methyl-esterified residues is important to understand the structure-function relationship of pectins and the specificity of pectolytic enzymes. Since its introduction in 1989, electrospray ionization mass spectrometry (ESI-MS)10 has had a major impact in the structural analysis of biopolymers such as proteins, nucleic acids, and carbohydrates. The nanoelectrospray ionization (nanoESI)11,12 technique has further considerably increased the sensitivity of electrospray ionization by reducing the flow rate to 10-50 nL/ min. Quadrupole ion traps13,14 have the attractive feature that multiple steps of ion isolation and fragmentation (MSn) can be performed in a single experiment. Recently, several groups have used ion trap tandem mass spectrometry for the structural analysis of neutral carbohydrates,15-20 and Xie et al.21 have studied linear galacturonic acid oligomers up to DP 7 by quadrupole ESI-MS. Here we investigate the fragmentation pathways of oligogalacturonates, discuss the 18O-labeling concept16,18,22 to resolve isomers, and report on the use of tandem mass spectrometry for the determination of methyl-esterified sites in oligomers derived from crude pectolytic digests. Partially methyl-esterified oligogalacturonates up to a DP of 10 were fragmented in a quadrupole ion trap, and the results are discussed with respect to the substrate specificities of the pectolytic enzymes. EXPERIMENTAL SECTION Materials. Grindsted URS lime pectin with DE 81% was from Danisco (Danisco Ingredients, Brabrand, Denmark). Purified pectin lyase (PL), exopolygalacturonase (exoPG), and pectin methyl esterase (PME) were from Danisco Biotechnology whereas endopolygalacturonase II (PG II) was obtained from Dr. Jacques Benen and Dr. Jaap Visser (Wageningen Agricultural University, Wageningen, The Netherlands). All enzymes were from Aspergillus niger. Cation-exchange resins (50W-X8, 200-400 mesh, hydrogen form) were purchased from Bio-Rad (Richmont, CA) and 18O-water (97 atom %) was from Isotec (Miamisburg, OH). Nanoelectrospray needles were obtained from Protana (Odense, Denmark). Preparation of Pectins with DE of 58% and 31%. An aqueous solution of Grindsted URS pectin (10 mg/mL) was adjusted to pH 4.5 by addition of diluted sodium hydroxide and pectin methyl esterase from A. niger was added. The pH decrease (10) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (11) Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 167180. (12) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (13) Bier, M. E.; Schwartz, J. C. In Electrospray Ionization Mass Spectrometry; Cole, R. B., Ed.; John Wiley & Sons: New York, 1997; pp 236-289. (14) Cooks, R. G.; Glish, G. L.; McLuckey, S. A.; Kaiser, R. E. Chem. Eng. News 1991, (March 25), 26-41. (15) Reinhold: V. N.; Sheeley, D. M. Anal. Biochem. 1998, 259, 28-33. (16) Viseux, N.; de Hoffmann, E.; Domon, B. Anal. Chem. 1997, 69, 31933198. (17) Weiskopf, A. S.; Vouros, P.; Harvey, D. J. Rapid Commun. Mass Spectrom. 1997, 11, 1493-1504. (18) Asam, M. R.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1997, 8, 987-995. (19) Bahr, U.; Pfenninger, A.; Karas, M. Anal. Chem. 1997, 69, 4530-4535. (20) Gaucher, S. P.; Leary, J. A. Anal. Chem. 1998, 70, 3009-3014. (21) Xie, M.; Giraud, D.; Bertheau, Y.; Casetta, B.; Arpino, P. Rapid Commun. Mass Spectrom. 1995, 1572-1575. (22) Hofmeister, G. E.; Zhou, Z.; Leary, J. A. J. Am. Chem. Soc. 1991, 113, 59645970.
1422 Analytical Chemistry, Vol. 71, No. 7, April 1, 1999
due to the action of the methyl esterase was compensated by automatic titration with diluted sodium hydroxide until the desired change in DE was reached. The enzyme was inactivated by heating to 100 °C for 5 min. The pectin was precipitated by addition of 2-propanol and dried. Enzymatic Digestions. A 5-mg sample of pectin was dissolved in 1 mL of 50 mM sodium acetate buffer adjusted to pH 4.5 (PG II, exoPG) or pH 5.0 (PL). The enzymes were dissolved in 50 mM sodium acetate buffers (pH 4.5 for PG II and exoPG, pH 5.0 for PL) to a concentration of 5 units/mL. For digestion with PL and exoPG, 0.1 unit of the enzymes was added to 1 mL of the pectin solutions whereas PG II required 0.2 unit/mL pectin solution to reach a complete digestion within the incubation time. Crude pectin solutions were digested with PL and PG II in separate experiments. In one experiment, a PG II digest was further digested with exoPG. Incubations were carried out at 23 °C for 24 h, after which the enzyme was inactivated by heating the solution to 95 °C for 5 min. Samples were not further fractionated except for the preparation of the unsaturated pentamer of galacturonic acid, which was used as a standard to investigate fragmentation pathways (see below). Preparation of the Pentamer of Galacturonic Acid. A pectin lyase digest of Grindsted URS pectin was prepared as described above. Hydrolysis of the methyl ester groups was achieved by treatment of 1 mL of crude digestion mixture with 0.1 mL of 0.5 M NaOH (resulting pH ∼12) at 3 °C for 16 h. The sample was neutralized by addition of 0.1 mL of 0.5 M HCl. Aliquots of 100 µL were loaded onto a MonoQ HR 5/5 column (Pharmacia, Uppsala, Sweden) using the A¨ kta system (Pharmacia). Separation could be achieved by running a gradient using 0.05 M NH4HCO3 (eluent A) and 0.75 M NH4HCO3 (eluent B) in Milli-Q water. Gradient program: 8 column volumes (cv) of 100% A, 10 cv of 0-40% B, 30 cv of 40-100% B. Oligomers were detected by UV absorption at 235 nm (double bond at the nonreducing end), and fractions were collected with the A¨ kta system. 18O-Labeling. Oligosaccharides were 18O-labeled at the reducing end16,18,22 by dissolving ∼10 µg of desalted (as described below), freeze-dried sample in 10 µL of 18O-water containing 0.5% formic acid. After incubation for 48 h at room temperature, the acid-catalyzed exchange was more than 90% complete. About 0.5 µL of the sample was diluted 10 times with 16O-water and analyzed by ESI-MS without further purification. No significant backexchange of 16O was observed during the analysis time (
