Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H⁺

This method can help answer questions in the field of plant Glycoscience such as how N-glycans of plant origin can be quickly analyzed and characterized. The main advantage of this technique is that it doesn't require any buffer changes between the enzymatic release of N-glycans and the derivatization reactions with 2-aminobenzamide. Demonstrating the procedure will be miss Ya-Min Du, and miss Shen-Li Zhen;two graduate students from the Glycomics and Glycan Bioengineering Research Center at Nanjing Agricultural University.

To begin isolating the proteins, use a kitchen blender to homogenize approximately 100 grams of fresh radish for ten minutes. Transfer the resulting slurry to a 50 milliliter centrifuge tube. Centrifuge at 14, 000 times gravity at four degrees Celsius for 20 minutes to remove the insoluble material.

After this, carefully transfer the supernatant into a new 50 milliliter centrifuge tube. Add an equal amount of a two molar trichloroacetic acid solution. Centrifuge at 14, 000 times gravity at four degrees Celsius for 30 minutes.

And then remove the supernatant. Wash the pellet with 20 milliliters of deionized water. Centrifuge again at 14, 000 times gravity at four degrees Celsius for five minutes to remove soluble oligosaccharides and polysaccharides.

Then, re-suspend the pellet with one milliliter of deionized water. And transfer the protein solution to a fresh 1.5 milliliter centrifuge tube. To begin preparing the N-glycans, mix 50 microliters of the protein solution with 0.2 milliunits of recombinant PNGase H+in ten millimolar acetic acid.

Incubate at 37 degrees Celsius for 12 hours. Then, centrifuge at 14, 000 times gravity for five minutes to remove the extra protein and enzyme. Transfer the supernatant to a fresh 1.5 milliliter centrifuge tube until ready to purify the N-glycans.

To begin purifying the N-glycans, wash the solid phase extraction column with three milliliters of water. Activate the column by adding three milliliters of an 80 percent acetonitrile solution containing trifluoroacetic acid. Then, three milliliters of deionized water to equilibrate the column.

Transfer the sample onto the column and discard the flow through. Wash the column by adding 1.5 milliliters of deionized water. Failure to remove salts and add impurities in the sample mixture may negatively impact the efficiency of the fluorescence derivatization reaction.

Then, add 1.5 milliliters of an aqueous 20 percent acetonitrile solution containing 0.1 percent TFA to elute the N-glycans into a two milliliter tube. Remove the solvent by centrifugal evaporation at room temperature, evaporating until the sample is completely dry. To begin fluorescence derivatization of N-glycans, prepare the 2-AB solution as outlined in the text protocol.

Add five microliters of the prepared 2-AB solution to the dried samples. Vortex until each sample is completely dissolved. Incubate at 65 degrees Celsius for two hours.

After this, let the sample cool to room temperature for five minutes. Add five microliters of deionized water and 40 microliters of acetonitrile to the cooled sample. Centrifuge at 14, 000 times gravity for three minutes.

Then, transfer 48 microliters of the resulting supernatant into a 300 microliter high recovery HPLC vial. Store at negative 20 degrees Celsius for up to one month or when ready to use. To begin profiling, use a standard UPLC system equipped with a hydrophobic interaction liquid chromatography UPLC glycan column and connect it to an online fluorescence detector to analyze the samples.

Set the excitation and emission wavelengths to 330 nanometers and 420 nanometers, respectively. Set the column temperature to 60 degrees Celsius. Next, inject 45 microliters of the sample into the UPLC system.

Elute the N-glycans by UPLC using retention times of 15 and 40 minutes. Using two milliliters centrifuge tubes collect each observed UPLC peak fraction. Then, use centrifugal evaporation to dry the samples.

Inject approximately one picomole of 2-AB labeled dextran standard to calibrate the plant N-glycan profiling and to assign their elution times and to standardize glucose units. To begin MALDI-TOF analysis, dissolve the dried samples in five microliters of LCMS grade water. Mix one microliter of this solution with one microliter of ATT solution.

Then, pipet this mixture into the MALDI-TOF sample carrier. After this, use a MALDI-TOF mass spectrometer instrument to analyze the samples as outlined in the text protocol. In this study, N-glycan profiles are compared from various cultivars of radish.

Representative UPLC chromatograms show that derivatized N-glycans and the relative abundance of each and different radishes can be successfully analyzed through this process. After this, MALDI-TOF mass spectroscopy is used to analyze the structures of 2-AB derivatized N-glycans. Though this method can provide insight into the composition of N-glycans from different Raphanus sativus cultivars, it can also be applied to other systems such as peanuts or other allergenic plant species.

Once mastered, samples can be prepared within two working days which already includes the UPLC analysis procedure. When attempting this procedure, it's important to remember that the solid phase extraction columns used to isolate the N-glycans are sufficiently moistened with distilled water prior to sample application. Otherwise, binding of N-glycans to the column may be hampered.

For additional verification of N-glycan structures, other methods can also be used. This could be, for example, sequential exoglycosidase treatments or MALDI-TOF MS N based fragmentation techniques. After its development, this technique paved the way for researchers in the field of Glycoscience to explore the composition and function of plant N-glycans more rapidly.

After watching this video, you should have a good understanding of how to prepare N-glycans from different cultivars of radish using PNGase H+and how to analyze the 2-aminobenzamide derivatives by UPLC. Don't forget that working with trifluoroacetic acid and trichloroacetic acid can be extremely hazardous and precautions such as wearing protective gear should always be taken while performing this procedure.