Structure elucidation of arabinoxylan isomers by normal phase HPLC-MALDI-TOF/TOF-MS/MS.
Normal phase-high performance liquid chromatography (NP-HPLC) coupled to matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry is evaluated for the detailed structural characterization of various isomers of arabinoxylan (AX) oligosaccharides produced from endo-beta-(1-->4)-xylanase (endoxylanase) digestion of wheat AX. The fragmentation characteristics of these oligosaccharides upon MALDI-TOF/TOF high-energy collision induced dissociation (CID) were investigated using purified AX oligosaccharide standards labeled at the reducing end with 2-aminobenzoic acid (2-AA). A variety of cross-ring cleavages and 'elimination' ions in the fragment ion spectra provided extensive structural information, including Araf substitution patterns along the xylan backbone and comprehensive linkage assignment. The off-line coupling of this MALDI-CID technique to capillary normal phase HPLC enabled the separation and identification of isomeric oligosaccharides (DP 4-8) produced by endoxylanase digestion of AX. Furthermore, this technique was used to characterize structurally different isomeric AX oligosaccharides produced by endoxylanase enzymes with different substrate specificities.
Maslen
Sarah L
SL
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
Goubet
Florence
F
Adam
Alex
A
Dupree
Paul
P
Stephens
Elaine
E
D000586:Amination; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry; D007536:Isomerism; D009844:Oligosaccharides / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014990:Xylans / Q000737:chemistry*; D062367:ortho-Aminobenzoates / Q000737:chemistry
D009844:Oligosaccharides; D014990:Xylans; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; C085118:arabinoxylan; D043364:Endo-1,4-beta Xylanases