The goal of this research is to address the effects of drought on agriculture. As the climate continues to change, there is an increase in drought, and drought affects food security. Metabolomics is important to identify and quantify the impact drought has on our food.
A big challenge in metabolomics is attaining a high level of certainty of identified compounds. By using methoxyamine instead of hydroxylamine in the oximation step, we've improved the efficiency of the subsequent silylation step as the methoxy group won't react with MSTFA. Current methods to observe plant responses to environmental stress focus on specific processes or tissue.
While this method is more integrative by detecting chemical signals in the final grain product. In the future, we would like to use this method to understand how drought and intercropping affect barley. Barley is an important grain for food security, and we want to understand how a changing climate will affect it.
To begin, load 200 milligrams of barley flour into a solid phase extraction, or SPE, solvent reservoir. Soak the flour with 200 microliters of methanol for 20 minutes to macerate. Remove the methanol through evaporation under vacuum for 30 minutes.
Upon drying, attach the column to a vacuum manifold. Extract non-polar components fraction A with dichloromethane into a 10 milliliter vial, and run the vacuum manifold until the flour is dry. Next, add a mixture of methanol and deionized water to extract the polar components fraction B into a 10 milliliter vial.
Run the vacuum manifold until the flour is dry. Add standard 1 and standard 2 to fraction A and mix by repeated pipetting. Fully evaporate the solvent under reduced pressure using a rotary evaporator.
Then redissolve the sample in methyl tert-butyl ether, or MTBE, before adding methanol and sodium methoxide into the vial. Allow transesterification to proceed for 90 minutes at room temperature under magnetic stirring. Add aqueous hydrochloric acid to the solution.
Mix the acidified solution with dichloromethane. Collect the contents of the vial with a long Pasteur pipette, and allow the phases to separate in the pipette. Wash the organic phase by adding aqueous hydrochloric acid.
Again, collect the contents of the vial with a long Pasteur pipette and allow the phases to separate in the pipette. Transfer the lower organic phase to a new vial and evaporate to dryness using a rotary evaporator. Next, add anhydrous sodium sulfate to an SPE column.
Dissolve the evaporated sample in dichloromethane and load it onto the SPE column. Elute fraction 1 with three portions of n-Hexane and MTBE into a 10 milliliter vial. Evaporate the solution containing fraction 1 to dryness on a rotary evaporator.
Redissolve the sample in n-Hexane and transfer it to an autosampler vial for GC/MS. Next elute pre-fraction 2 from the SPE column with three subsequent portions of n-Hexane and MTBE into a 10 milliliter vial. After evaporating the pre-fraction 2 to dryness redissolve it in anhydrous pyridine and add MSTFA.
Using zip ties, secure a nitro glove over the end of a syringe with the plunger removed. Fill the balloon with anhydrous argon and attach a disposable needle to the syringe. Push the balloon syringe through the septum of the bottle containing the anhydrous reagent and insert a second syringe with a long needle to draw up the solvent.
Flush the sample vial with a gentle stream of anhydrous argon. After sealing the vial with parafilm, place it in a 70 degree Celsius oil bath for 15 minutes. Following silylation, cool fraction 2 at room temperature for five minutes and load it into an autosampler vial.
Add standards 3 and 4 to fraction B and mix by repeated pipetting. Transfer two and four milliliters of fraction B mixture to a new vial to process into fraction 3 and fraction 4 respectively. Evaporate pre-fraction 3 to dryness on a rotary evaporator, followed by coevaporation with anhydrous pyridine.
redissolve the pre-fraction 3 in anhydrous pyridine and add trimethylsilyimidazole. Flush the sample vial with a gentle stream of anhydrous argon. After sealing the sample vial with parafilm, heat for 20 minutes in a 70 degree Celsius oil bath.
After cooling the solution to room temperature, add n-Hexane and deionized water. Stir the mixture and transfer the upper organic phase to an autosampler vial using a long Pasteur pipette. Evaporate pre-fraction 4 to dryness on a rotary evaporator, followed by coevaporation with anhydrous pyridine.
Redissolve the sample in anhydrous pyridine and add methoxylamine hydrochloride. After flushing the vial with anhydrous argon, oxamate the parafilm sealed vial for 30 minutes in a 70 degree Celsius oil bath. Once the vial is cooled to room temperature, add MSTFA and silylate for 20 minutes in an oil bath.
Next, add n-Hexane and deionized water into the vial and collect the lower aqueous phase using a long Pasteur pipette. After the last wash, evaporate the aqueous phase to dryness using a rotary evaporator followed by coevaporating the remaining material with anhydrous acetonitrile. Redissolve the sample in anhydrous acetonitrile and MSTFA.
After flushing the vial with anhydrous argon, heat the sealed vial for 60 minutes in a 70 degree Celsius oil bath. Load samples onto the GC/MS using the specified injection volumes. Run the samples with a split flow of 14 milliliters per minute of helium at an injection temperature of 250 degrees Celsius.
For fractions 1 and 2, ramp the column temperature from 100 to 300 degrees Celsius at a rate of 4 degrees Celsius per minute. After reaching 300 degrees Celsius, hold the temperature for 10 minutes. For fraction 3, heat and hold the column at 300 degrees Celsius for the entirety of the run.
For fraction 4, ramp the column temperature from 70 to 142 degrees Celsius at 2.5 degrees Celsius per minute. Hold the temperature at 142 degrees Celsius for 10 minutes. Then increase the temperature from 142 to 235 degrees Celsius at 4 degrees Celsius per minute, holding for 10 minutes.
Finally, bake the column at 320 degrees Celsius for 8 minutes.
