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 pro- duced by enzymatic digestion of partially methyl-esterified pectin with Aspergillus niger pectin lyase, endopoly- galacturonase II, and exopolygalacturonase. To determine the specificities of these pectolytic enzymes toward non- esterified and methyl-esterified galacturonic acid residues, we have studied the methyl esterification patterns of selected oligomers in unseparated pectin digests. Colli- sion-induced dissociation in a nanoelectrospray ionization ion trap mass spectrometer was used to locate methyl- esterified galacturonic acid residues in oligomers up to a degree of polymerization of 1 0 . Analysis of the methyl esterification patterns gave insight into the substrate specificities of these pectolytic enzymes. Isomeric frag- ment ions containing the reducing and nonreducing ends were differentiated by 18 O-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). Galac- turonic 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 proteins 3 and are under investigation for medical applications such as lowering blood cholesterol levels. 4 Furthermore, pectic frag- ments have been shown to possess regulatory activity for plant defense mechanisms 5,6 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 studies 8,9 have shown that pectolytic digests can be analyzed by matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry to determine the degree of polymerization * Corresponding author: (phone) ( +45) 65572404; ( fax) ( +45) 65932781; ( e- mail) roe@ pr-group.ou.dk. † 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. (5) Co ˆ te ´, 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. 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. Anal. Chem. 1999, 71, 1421-1427 10.1021/ac981240o CCC: $18.00 © 1999 American Chemical Society Analytical Chemistry, Vol. 71, No. 7, April 1, 1999 1421 Published on Web 02/25/1999