CE-MSn of complex pectin-derived oligomers
Coenen, G.J. ; Kabel, M.A. ; Schols, H.A. ; Voragen, A.G.J. - \ 2008
Electrophoresis 29 (2008)10. - ISSN 0173-0835 - p. 2101 - 2111.
anion-exchange chromatography - capillary zone electrophoresis - trap mass-spectrometry - hairy ramified regions - maldi-tof ms - rhamnogalacturonan-i - 8-aminonaphthalene-1,3,6-trisulfonic acid - galacturonic acid - apple pectin - oligosaccharides
As pectin molecules are too large and heterogeneous to analyze as a whole, the polymer is usually degraded to smaller oligomers, which are often analyzed by high-performance anion exchange chromatography (HPAEC). However, the high salt concentration necessary to elute pectin oligomers by HPAEC is incompatible with online mass detection. To overcome such a disadvantage, a CE-IT-MS system was set up to further elucidate the fine structure of charged oligosaccharides. An effective separation of differently substituted galacturonic acid containing oligomers was obtained by low-pH CE-LIF analysis. By adapting the buffer and capillary online MS detection was enabled. Moreover, with MS/MS it was possible to localize sugar residues' substitutions. With this combined CE-MS approach LIF electropherograms of xylogalacturonan and rhamnogalacturonan I digests could be annotated. The method was further exemplified by a complex oligomer mixture of acid hydrolyzed apple pectin, which was separated and characterized by CE-MSn. Oligomers present in low amounts could be localized by their corresponding m/z, as was demonstrated by selected mass range representation.
Partially esterified oligogalacturonides are the preferred substrates for pectin methylesterase of Aspergillus niger.
Alebeek, G.J.W.M. van; Scherpenzeel, K. van; Beldman, G. ; Schols, H.A. ; Voragen, A.G.J. - \ 2003
Biochemical Journal 372 (2003). - ISSN 0264-6021 - p. 211 - 218.
flight mass-spectrometry - maldi-tof ms - methyl esterase - pectinesterase - purification - cloning - plants - lyase - mode
Investigations on the mode of action of Aspergillus niger pectin methylesterase (PME) towards differently C-6(-) and C-1-substituted oligogalacturonides (oligoGalpA) are described. De-esterification of methyl-esterified (un)saturated oligoGalpA proceeds via a specific pattern, depending on the degree of polymerization. Initially, a first methyl ester of the oligomer is hydrolysed, resulting in one free carboxyl group. Subsequently, this first product is preferred as a substrate and is de-esterified for a second time. This product is then accumulated and hereafter deesterified further to the final product, i.e. oligoGalpA containing one methyl ester located at the non-reducing end residue for both saturated and unsaturated oligoGalpA, as found by post-source decay matrix-assisted laser-desorption/ionization-time-of-flight MS. The saturated hexamer is an exception to this: three methyl esters are removed very rapidly, instead of two methyl esters. When unsaturated oligoGalpA were used, the formation of the end product differed slightly, suggesting that the unsaturated bond at the non-reducing end influences the de-esterification process. In vivo, PME prefers methyl esters, but the enzyme appeared to be tolerant for other C-6- and C-1-substituents. Changing the type of ester (ethyl esterification) or addition of a methyl glycoside (C-1) only reduced the activity or had no effect respectively. The specific product pattern was identical for all methyl- and ethyl-esterified oligoGalpA and methyl-glycosidated oligoGalpA, which strongly indicates that one or perhaps two non-esterified oligoGalpA are preferred in the active-site cleft.