Fractionation, solid-phase immobilization and chemical degradation of long pectin oligogalacturonides. 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.
Guillaumie
Fanny
F
Center for Microbial Biotechnology, BioCentrum-DTU, Technical University of Denmark, Building 223, Søltofts Plads, DK-2800 Kgs. Lyngby, Denmark.
Justesen
Sune F L
SF
Mutenda
Kudzai E
KE
Roepstorff
Peter
P
Jensen
Knud J
KJ
Thomas
Owen R T
OR
D002240:Carbohydrate Sequence; D005591:Chemical Fractionation; D002852:Chromatography, Ion Exchange / Q000379:methods; D006603:Hexuronic Acids / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry*; D010368:Pectins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006603:Hexuronic Acids; D009844:Oligosaccharides; C007819:galacturonic acid; D010368:Pectins