Guillaumie, Fanny2; Justesen, Sune F. L.2; Mutenda, Kudzai E.3; Roepstorff, Peter3; Jensen, Knud J.5; Thomas, Owen R. T.2
1 Department of Basic Sciences and Environment, Faculty of Life Sciences, Københavns Universitet2 BioCentrum-DTU3 Syddansk Universitet4 Administration, Department of Chemistry, Faculty of Science, Københavns Universitet5 Administration, Department of Chemistry, Faculty of Science, Københavns Universitet
initial steps towards sequencing of oligosaccharides
This work presents the optimized separation of pectin oligomers, their analysis by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), their subsequent immobilization to supports, and our initial steps towards solid-support assisted sequencing. The ambient pressure strong anion-exchange resin Source 15Q combined with ammonium formate buffer (AF) was used for the separation of unsaturated and saturated pectic oligogalacturonides (OGAs) derived from enzymatic digestion of pectin. Routinely, multi-milligram quantities of defined sizes OGAs with DPs from 5 to 19 were produced in excellent purity (>95%). Elution of OGAs followed by direct analysis of the peak fractions by MALDI-TOF MS. Purified OGAs (DP 5-7) were chemoselectively immobilized onto aminooxy-terminated polyethylene glycol polyacrylamide (PEGA) supports. Solid-phase anchoring took place at the reducing end of the oligosaccharide and resulted in the formation of an oxime linkage. The very high coupling yields confirmed the general suitability of aminooxy-PEGA resins for the immobilization of OGAs of different lengths. The OGA-functionalized PEGA supports were subsequently treated with aq TFA at 40 or 60 degrees C, and the chemical degradation products released from the support were analyzed by ESIMS. In all cases, the original OGA was degraded into smaller oligomers of various sizes down to the monomer. This work illustrates some of the basic principles underlying a strategy ultimately aimed at solid-support assisted sequencing of oligosaccharides.
Carbohydrate Research, 2006, Vol 341, Issue 1, p. 118-129
Carbohydrate Sequence; Chemical Fractionation; Chromatography, Ion Exchange; Hexuronic Acids; Oligosaccharides; Pectins; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization