This procedure begins with the homogenization of plant material using ceramic beads and subsequent di chloral methane extraction to obtain the organic phase. Following centrifugation samples are dried under air and prepared for vapor phase extraction. After ellucian from vapor phase extraction filters, one can proceed to plant hormone analysis using mass spectrometry and gas chromatography.
Hi, I'm Marie Engelberg from the JU Engelberg Lab in the Department of Biology at the University of Texas here in San Antonio, And I'm Ju Engelberg. Today we'll show you a simple procedure for the simultaneous quantifications of important plant hormones. We use this method routinely to monitor changes in these hormones and plans on the insect reward tech.
We can also monitor changes in the fatty acid composition. So let's get started. For the extraction, you first need to prepare two milliliters, screw cap vials.
To do this, add 400 microliters extraction solution and 10 microliters of the respective internal standard at 10 nanograms per microliter to a vial. Next, freeze the vials in liquid nitrogen. After freezing, add the ceramic beads to the vials.
While the vials are still positioned in liquid nitrogen, add the plant material between 50 and 200 milligrams. Keep in mind that the weight of the added plant material has to be estimated before further processing to allow for exact quantification later, which is based on the fresh weight. Make sure that all the liquid nitrogen is evaporated before tightly closing the vial with a cap.
Be sure that the cap has a rubber seal to prevent spills of solvents and extracted compounds during the homogenization. Homogenize the tissue in a fast prep or PSUs bead grinder. Homogenizer at a speed of 6, 000 for PSUs for 30 seconds.
Once homogenized, remove the vials from the homogenizer individually and carefully open the cap to add one milliliter of di chloral methane to each sample, close the vials again tightly and homogenize for 10 to 15 seconds at 6, 000 after homogenization, transfer the vials to a tabletop centrifuge and separate the organic phase from the aqueous phase at 10, 000 times. G for 60 seconds. After the phase separation, transfer the lower green layer from the individual vials to a clean four milliliter glass vial.
This is the organic phase containing the hormones and the internal standards. Avoid the transfer of water or the upper phase, blow off the solvent with air through a manifold for about 10 to 25 minutes. The length of time will depend on the sample.
Check with a single airflow tip to carefully see if the samples are dry. Be sure not to over dry the samples because this might cause loss of compounds. Once the samples are dried, we can start the methylation of the carboxylic acid containing compounds in our samples.
First, add 100 microliters of a D ethyl ether methanol mixture nine to one volume to volume to each vial. Next, add four microliters of a two molar trimethyl Cyl Diaz methane solution in hexane. Immediately close the vials with an open top screw cap fitted with a Teflon lined silicone septum em shake the vials gently and incubate room temperature for 25 to 30 minutes.
Turn on a heating block and set it at the desired collection temperature. The volatility of the methylated compounds will vary depending on their size and number of polar groups. Temperatures can range between 80 and 200 degrees after a 30 minute incubation stop the methylation reaction by adding four microliters of a two molar acetic acid solution in hexane to each vial.
This is done to avoid unwanted secondary reaction. The filters used for the vapor phase extraction are handmade. They're made out of Teflon tubes.
From one side, a well-fitting glass tube of about four centimeters length is inserted. You can see the two discs of stainless steel mesh, which hold about 20 to 30 milligrams of absorbent. Wash the filters that will be used for the collection of the methylated and volatile plant hormones using 200 microliters of methanol.
Next, wash the filters twice with 200 microliters di chloral methane. Each air dry the remaining solvent from the filters. The filters can now be used for the trapping of volatile hormones through vapor phase extraction.
After 30 minutes of incubation of the samples, the rubber septa of the four milliliter vial caps are cut with a scalpel. Now the extracted material is ready to be collected to collect the plant hormones by vapor phase extraction. The cleaned filters are connected to individual vacuum lines at a flow rate of about 800 milliliters per minute.
The filters are then inserted into the vial through the cut septa. A small pipette tip is also inserted to act as an air inlet. During this initial part of the procedure, the vials with the inserted filters have to be held in an upright position to avoid any of the liquid inside the vial to come in contact with the filter tip, which would contaminate the filter.
And subsequently the GCMS hold the vials by hand until all the liquid is evaporated through the filter. Then the vials with the attached filters are placed on the heating block and heat evaporated compounds are collected for three minutes. After this final vapor phase extraction step, place the vials with the filters still attached on a rack and allow them to cool down.
Prepare 1.5 milliliter GC vials fitted with inserts when the filters have reached room temperature. Again, a loop with 150 microliters of di chloral methane into the vials. After blowing out the remaining solvent from the filter, the vials are capped and can now be analyzed by gas chromatography mass spectrometry.
For analysis, use a gas chromatograph equipped with a split split list injector interfaced to a mass spectrometer operated in chemical ionization mode after injection compounds are separated on an HP one MS column held at 40 degrees Celsius for one minute. The temperature is programmed at 15 degrees Celsius per minute to two 50 Celsius for 10 minutes with helium as the carrier gas at a constant flow of 0.7 milliliters per minute. For the MS based analysis, both a quadruple and an ion trap based mass spectrometer can be as a CI gas.
Either isobutane or methanol can be used. However, in each case, the fragments of the ionization reaction should be checked before the selected ion count programs are created. We use a variant GC 3, 900 gas chromatograph system coupled with a Saturn MS 2200 mass spectrometer and methanol as the CI reagent.
Please see our written protocol for a description of our program. Be sure that all compounds are identified by comparison with authentic commercially available standards. Quantification of JA SA and A BA is done by correlating the peak area extracted ions with the peak area extracted ions of the respective internal standard and is also based on the fresh weight of the plant material used.
After extracting the data from the GCM S files and some calculations, we found that the ethin induced considerable accumulations of salicylic acid, monic acid, and also abic acid when compared to untreated controls. The data for each individual hormone was taken from a single run and demonstrates the capacity of this method for multiplex hormone analysis. We have just shown you an easy but yet effective procedure to extract and quantitate important plant hormones and other signaling compounds.
It is easy to perform and most of the tools you've seen can be easily made from standard laboratory material. So that's it. Thanks for watching and good luck with your experiments.
