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 digestion of partially methyl-esterified pectin with Aspergillus niger pectin lyase, 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 esterification patterns of selected oligomers in unseparated pectin digests. Collision-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 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.
Körner
R
R
Department of Molecular Biology, Odense University, Denmark.
Limberg
G
G
Christensen
T M
TM
Mikkelsen
J D
JD
Roepstorff
P
P
D002240:Carbohydrate Sequence; D006603:Hexuronic Acids / Q000032:analysis*; D013058:Mass Spectrometry; D008745:Methylation; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis*; D011096:Polygalacturonase / Q000378:metabolism*; D013379:Substrate Specificity
D006603:Hexuronic Acids; D009844:Oligosaccharides; C007819:galacturonic acid; D011096:Polygalacturonase