Sequencing of partially methyl-esterified oligogalacturonates by tandem mass spectrometry and its use to determine pectinase specificities

Complex mixtures of acidic oligosaccharides were produced by enzymatic-dige stion of partially methyl-esterified pectin with Aspergillus niger pectin l yase, endopolygalacturonase 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 esterific ation patterns of selected oligomers in unseparated pectin digests. Collisi on-induced dissociation in a nanoelectrospray ionization ion trap mass spec trometer was used to locate methyl-esterified galacturonic acid residues in oligomers up to a degree of polymerization of 10, Analysis of the methyl e sterification patterns gave insight into the substrate specificities of the se pectolytic enzymes. Isomeric fragment ions containing the reducing and n onreducing ends were differentiated by O-18-labeling of the reducing end.