This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).
The overall goal is to characterize the reducing end and internal region glycosal sequences of heteroxylans by tagging the original reducing end sugar residue of the water-soluble heravenaxylans and treating with an endoxylanase to generate a mixture of label dolara saccharides. This method can help answer key questions in glycomic field, such as alterations of fine structure of oligosaccharides or polysaccharides, in response to planned growth development, as well as abiotic and biotic stressors. The main advantage of this method is it describes the integrated approach to sequencing of reducing n sequence as well as internal regensequence of any glycan or polysaccharide.
Prepare a one molar sodium cyanoborohydride solution in one millilitre of water. Then, dissolve 27.2 milligrams of 2AB reagent in the sodium cyanoborohydride solution by heating at 65 degrees Celcius and adjusting the pH of the reaction mixture to pH 5.5 with 10 percent acetic acid. Add 200 microlitres of the reaction mixture to one milligram of water-soluble arabinoxylans in a glass tube with a cap and mix using a vortex mixer.
Incubate the sample for two hours at 65 degrees Celcius in a fume hood. Following incubation, cool the suspension to room temperature and add four volumes of absolute ethanol. Place the suspension in a cold storage overnight to precipitate polysaccharides.
Then, centrifuge the sample and remove the supernatant. Wash the pellet extensively with absolute ethanol, acetone, and methanol, centrifuging between each wash. Vacuum dry the pellet at 40 degrees Celcius overnight.
Dissolve one milligram of the prepared 2AB labeled water-soluble arabinoxylans in 500 microlitres of sodium acetate buffer in a microcentrifuge tube. Add four units of endoxylanase and incubate at 37 degrees Celcius for 16 hours. Destroy the enzyme activity by heating the reaction mixture for 10 minutes in a boiling water bath.
Cool the suspension to room temperature and transfer to a glass tube with a cap. After adding four volumes of absolute ethanol, place the suspension in a cold storage overnight to precipitate any undigested polysaccharides. Centrifuge to pellet the undigested polysaccharides from the endoxylanase generated oligosaccharides.
Then, decant the supernatant into a clean plastic tube and place it into a warm water bath. Evaporate the ethanol under a stream of nitrogen gas to an endpoint volume. Freeze the supernatant at minus 80 degrees Celcius for four hours before drying the frozen supernatant in a freeze dryer to recover xylooligosaccharides.
To generate the xylooligosaccharides from KO8 soluble arabinoxylans and 2AB labelling, see the text protocol. To prepare the maldi matrix solution, add a small scoop of DHB to 500 millilitres of 50 percent acetonitrile containing 0.1 percent formic acid in a tube. Mix, using a vortex.
If it dissolves quickly, add another small scoop of DHB. Deposit the native aqueous oligosaccharide samples onto a maldi target plate. Add 0.3 microlitres of the maldi matrix solution using a separate tip and mix by pipetting up and down.
Allow the mixture to dry at room temperature. Introduce the target plate into the MS source and operate in positive ion mode. Adjust the accelerator voltage to 19 kilovolts at ion source one, and 16.3 kilovolts at ion source two.
Adjust the laser power to greater than 70 percent. Select the sample spot on the maldi target plate and click start to begin the laser shots. Analyze native endoxylanse generated oligosaccharides using nano HPLC, coupled with an electro spray ionization quadrupole time-of-flight MS instrument with online chromatographic separation using a reversed-based C18 column.
Transfer the endoxylanase generated aqueous oligosaccharide mixture into a vial and place into the HCLC autosampler. Program the elution gradient of five to 80 percent with the mobile phases 0.1 percent formic acid in water and 0.1 percent formic acid in acetonitrile, respectively, over 60 minutes. Run the liquid chromatographic program and allute the oligosaccharides.
Press add sample, type the sample name, and press the okay button. Select mass spectroscopy method file and press the submit button. And then start sample.
The resultant total ion chromatogram, or TIC, is saved automatically by the software. Open the saved TIC and select extract chromatogram. Type the expected masses at the command line.
Scan the chromatogram by clicking enter before processing the selected ion scans using software according to the manufacturer's instructions. Insert one to two microlitres of per O methylated oligosaccharide sample in 50 percent acetonitrile into a nanospray tip using a syringe. Trim the nanospray using a glass cutter to fit into the discrete nanospray holder attached to the MS.Set the mass according to the expected mass range, the curtain gas to 10, the ion spray voltage to 1900 volts, and the polarity to positive.
Press the acquire button to open the relevant window. Then, enter the data file name to obtain a total ion scan before pressing the stop button. Change the scan type from product ion to fragment a piece of interest.
Enter the mass of interest to fragment and adjust the mass range. Press the acquire button and adjust the collision energy to achieve entire fragmentation of the parent ion and acquire a fragment ion scan. Dissolve the endoxylanase generated mixture of oligosaccharides in one millilitre of deuterium oxide in a ten millilitre plastic test tube.
Freeze the suspension at minus 80 degrees Celcius for four hours before drying the frozen suspension in a freeze dryer to recover xylooligosaccharides. Next, dissolve the dried oligosaccharides in 0.6 millilitres of deuterium oxide and add 0.5 microlitres of five percent acetone in deuterium oxide as an internal standard. Transfer the deuderated oligosaccharides into the NMR sample tube.
Hold the NMR tube containing sample by the top and insert the sample tube in a plastic spinner. Place the spinner in the sample depth gauge. Push or pull the sample tube to adjust the depth of the sample to ensure that the center line of the sample top and bottom depth gauges are equal.
Remove the depth gauge and insert the sample into the autosampler attached to a 600 megaHertz NMR spectrometer equipped with a cryoprobe. Proceed to run the NMR experiment and analyze the resulting spectra as described in the text protocol. The signals in the maldi tof MS spectrum oligosaccharides derived from 2AB labelled native water soluble arabinoxylans include a series of unlabelled internal region neutral and acidic oligosaccharides as well as oligosaccharides originally labelled with 2AB at their reducing end.
Shown here are selected ion scans of the elected ion scans of the ESI cutoff MS chromatograms of native oligosaccharides derived from internal region oligosaccharides generated by endoxylanase treatment of the 2AB tagged water-soluble arabinoxylans. Also shown are scans of native oligosaccharides derived from reducing endoligosaccharides with 2AB tag and region acidic oligosaccharides generated in the same way. Unmethylated hydroxyl groups generated during gas phase fragmentation of per-O methylated oligosaccharides in MS provide a 14 Dalton mass difference scar used to identify the branching pattern and the glycosal sequences.
Each scar generated by the fragmentation event is marked as a solid line, as several isomeric structures are possible for a defined mass. Y-ions are labelled in red, and B-ions are labelled in black. Analysis of the electrospray ionization tandem MS spectra of per-o-methylated reducing and neutral glycosal alditol consisting of four pentose sugar residues and xylitol at mass to charge ratio 885 indicates that the reducing end glycosal sequence consists of the arabinose side chain attached at the reducing end xylitol residue and/or both the reducing end xylitol and the penultimate xylosil residue.
Following this procedure, other methods like chromprioansomichro poly profiling and electropulses can be performed in order to answer the questions like antibody epitop specificity and rapid screening of changes of fine structure. After its development, this technique paved the way for the researchers in the field of glycomics to explore the structure features of glycan, including the type and pattern of substitutions with glycan or non-glycan residues in different type of tissues, developmental stages and species. This approach can be applied to other classes of polysaccharides by using appropriate endrohydrilasis.
Don't forget that working with metal alide and sodium cyanoborohydride can be extremely dangerous, and precautions such as wearing proper gloves, working in fume hood, should always be taken while performing this procedure.
