Name: Metabolomic Analysis of Barley by Gas Chromatography/Mass Spectrometry (Video) | JoVE Journal
Uploaded: 2024-11-08T13:50:00.000+00:00
Duration: 8 min 15 s
Description: Learn metabolomic analysis of barley using GC/MS to study drought stress effects on grain. This improved method enhances compound identification for agricultural research and food security applications.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

Funding was provided by the USDA ARS Pulse Crop Health Initiative and the Idaho Barley Commission.

1. Preparation of standards (Figure 2)
<img alt="chemical structure diagram of heptacosane, 5α-cholestan-3β-ol, phenyl-β-D-glucopyranoside, 4-chloro-L-phenylalanine" class="xfigimg" src="/files/ftp_upload/67175/67175fig02.jpg" data-altupdatedat="1788840098000" data-alt="Figure 2"> 
Figure 2: Chemical structures for the four internal standards used in this method. These four standards are used for the semiquantitation of metabolites in the different barley fractions. <a href="https://www.jove.com/files/ftp_upload/67175/67175fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Prepare Standard 1 by dissolving n-heptacosane (50 mg, ≥97% purity) in n-hexane (100 mL, ≥95% purity).</li>
	<li>Prepare Standard 2 by dissolving 5α-cholestan-3β-ol (60 mg, ≥95% purity) in dichloromethane (CH2Cl2, 100 mL, ≥99% purity).</li>
	<li>Prepare Standard 3 by dissolving phenyl-β-D-glucopyranoside (80 mg, ≥95% purity) in deionized water (DI H2O, 100 mL).</li>
	<li>Prepare Standard 4 by dissolving 4-chloro-L-phenylalanine (40 mg, ≥97% purity) in DI H2O (100 mL).</li>
</ol>
2. Initial fractionation
<ol>
	<li>Load barley flour (200 mg) into a Solid Phase Extraction (SPE) solvent reservoir. Soak the flour with methanol (200 µL, ACS grade or higher purity) for 20 min to macerate. Remove the methanol through evaporation under vacuum for 30 min (Figure 3).</li>
	<li>Once dried, attach the column to a vacuum manifold. Extract non-polar components (Fraction A) with CH2Cl2 (4 mL) into a 10 mL vial and run the vacuum manifold until the flour is dry.</li>
	<li>Extract the polar components (Fraction B) by adding a mixture of methanol and DI H2O (10 mL, 8:2 v/v). Collect into a 10 mL vial and run the vacuum manifold until the flour is dry.</li>
</ol>
<img alt="Vacuum manifold system setup for SPE, sample purification, chromatographic analysis in laboratory." class="xfigimg" src="/files/ftp_upload/67175/67175fig03.jpg" data-altupdatedat="1788840098000" data-alt="Figure 3"> 
Figure 3: The vacuum manifold setup. This setup is used to dry the methanol-soaked barley flour. <a href="https://www.jove.com/files/ftp_upload/67175/67175fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Processing of Fraction A to produce Fractions 1 and 2
<ol>
	<li>Add standard 1 and standard 2 (100 µL each) to Fraction A and mix by repeated pipetting. Fully evaporate the solvent under reduced pressure using a rotatory evaporator.</li>
	<li>Redissolve the sample in methyl tert-butyl ether (MTBE, 500 µL, ACS grade or higher purity). Add methanol (300 µL) and sodium methoxide (14.6 mg, 97%). Allow transesterification to proceed for 90 min at room temperature under magnetic stirring.</li>
	<li>Add aqueous hydrochloric acid (2 mL, 0.35 M, ACS grade or higher purity). Mix the acidified solution with CH2Cl2 (1 mL). Collect the contents of the vial with a long Pasteur pipette and allow the phases to separate in the pipette.</li>
	<li>Transfer the lower (organic) phase to a new vial. Wash the organic phase by adding aqueous hydrochloric acid (2 mL, 0.35 M). Collect the contents of the vial with a long Pasteur pipette and allow the phases to separate in the pipette.</li>
	<li>Transfer the lower (organic) phase to a new vial. Evaporate the lower (organic) phase to dryness on a rotary evaporator.</li>
	<li>Add anhydrous sodium sulfate (200 mg, ACS grade or higher purity) to an SPE column. Dissolve the sample from step 3.5 in CH2Cl2 (250 µL) and load onto the SPE column.</li>
	<li>Elute Fraction 1 from the SPE column with three portions of n-hexane and MTBE (2.04 mL, 50:1 v/v) into a 10 mL vial. Evaporate the solution containing Fraction 1 (FAMEs and hydrocarbons) to dryness on a rotary evaporator. Redissolve the sample in n-hexane (300 µL), and transfer to an autosampler vial for use on the GC/MS.</li>
	<li>Elute pre-fraction 2 from the SPE column (after elution of Fraction 1 in step 3.7 has been performed) with three subsequent portions of n-hexane and MTBE (2 mL, 7:3 v/v) into a 10 mL vial. Evaporate pre-fraction 2 to dryness on a rotary evaporator.</li>
	<li>Using the same vial, redissolve pre-Fraction 2 in anhydrous pyridine (250 µL, 99.8%) (transfer pyridine using the argon balloon method-see below) and add N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA, 50 µL, Reagent grade). 
	NOTE: All anhydrous solvents are transferred using the argon balloon method.
	<ol>
		<li>Using zip ties, secure a nitrile 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.</li>
		<li>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 (Figure 4).</li>
	</ol>
	</li>
	<li>Flush the sample vial with a gentle stream of anhydrous argon, cap, seal with parafilm, and place in a 70 °C oil bath under magnetic stirring for 15 min.</li>
	<li>Following the silylation, cool Fraction 2 (TMS-derivatized: fatty acids, fatty alcohols, hydroxy FAMEs, hydroxy fatty acids, phenols, and sterols) at room temperature for 5 min. Load Fraction 2 into an autosampler vial.</li>
</ol>
<img alt="Laboratory gas cylinder setup with gloves for safety, showcasing gas flow experiment or apparatus." class="xfigimg" src="/files/ftp_upload/67175/67175fig04.jpg" data-altupdatedat="1788840098000" data-alt="Figure 4"> 
Figure 4: The argon balloon setup. <a href="https://www.jove.com/files/ftp_upload/67175/67175fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Processing of Fraction B to produce Fractions 3 and 4
<ol>
	<li>Add Standards 3 and 4 (300 µL each) to Fraction B and mix by repeated pipetting. Transfer 2 mL of this solution to a new vial to be processed into Fraction 3 and then transfer 4 mL of this solution to a separate vial to be processed into Fraction 4, starting from the evaporation of pre-fraction 4 in step 4.4. Evaporate pre-fraction 3 to dryness on a rotary evaporator, followed by coevaporation with anhydrous pyridine (2 x 1.5 mL). 
	NOTE: Coevaporation is used to remove trace amounts of water as anhydrous pyridine forms an azeotrope.</li>
	<li>Redissolve pre-fraction 3 in anhydrous pyridine (200 µL) and add N-trimethylsilyimidazole (TMSIM, 100 µL, 97%). Flush the sample vial with a gentle stream of anhydrous argon, cap, seal with parafilm, and heat for 20 min in a 70 °C oil bath under magnetic stirring. 
	NOTE: TMSIM is a mild silylating agent and will preferentially silylate -OH groups over amine groups8.</li>
	<li>Following the silylation, allow the solution to cool at room temperature for 5 min, add n-hexane (200 µL) and DI H2O (400 µL), and stir. Transfer the upper, organic phase, i.e., Fraction 3 (TMS-derivatized sugars and polyols), to an autosampler vial using a long Pasteur pipette.</li>
	<li>Evaporate pre-fraction 4, from step 4.1, to dryness on a rotary evaporator, followed by coevaporation with anhydrous pyridine (2 x 1.5 mL). Redissolve the sample in anhydrous pyridine (250 µL) and add methoxyamine hydrochloride (5 mg, 98%). Flush the sample vial with argon, cap, seal with parafilm, and oximate for 30 min in a 70 °C oil bath under magnetic stirring.</li>
	<li>Allow the solution to cool at room temperature for 5 min, add MSTFA (100 µL), gently flush the sample vial with anhydrous argon, cap, seal with parafilm, and silylate for 20 min in a 70 °C oil bath under magnetic stirring.</li>
	<li>Allow the solution to cool at room temperature for 5 min, then add n-hexane and DI H2O (800 µL, 5:3 v/v). Collect the lower (aqueous) phase using a long Pasteur pipette and wash with 4 x 500 µL of n-hexane, collecting the lower phase and discarding the upper phase every time. Evaporate the aqueous phase to dryness on a rotary evaporator, and coevaporate the remaining material with anhydrous acetonitrile (2 x 1.5 mL, ACS grade or higher purity).</li>
	<li>Redissolve the sample in anhydrous acetonitrile (250 µL) and add MSTFA (50 µL). Gently flush the sample vial with anhydrous argon, cap, seal with parafilm, and heat for 60 min in a 70 °C oil bath under magnetic stirring.</li>
	<li>Allow the solution to cool at room temperature for 5 min, then transfer Fraction 4 (TMS-derivatized carboxylic acids, amino acids, amines) to an autosampler vial.</li>
</ol>
5. GC/MS methods
<ol>
	<li>Load samples onto a GC/MS with the following column characteristics: 15 m x 0.25 mm internal diameter, coated with a 0.25 µm nonpolar film.</li>
	<li>Use the following injection volumes for Fractions 1-4, respectively: 1.0, 1.0, 0.5, and 0.5 µL. Run injected samples with a split flow (1:10 split ratio) of 14 mL/min helium at an injection temperature of 250 °C.</li>
	<li>For Fractions 1 and 2, ramp the column temperature from 100 °C to 300 °C at 4 °C/min with a 10 min hold. 
	NOTE: The total run time is 65 min.</li>
	<li>For Fraction 3, heat and hold the column at 300 °C for the entirety of the run. 
	NOTE: The total run time is 70 min.</li>
	<li>For Fraction 4, heat the column from 70 °C to 142 °C at 2.5 °C/min with a 10 min hold, followed by heating from 142 °C to 235 °C at 4 °C/min with a 10 min hold. Finally, bake the column at 320 °C for 8 min. 
	NOTE: The total run time is 102 min.</li>
</ol>
6. Metabolite identification
<ol>
	<li>Perform metabolite identification by comparing each compound's mass spectrum to the NIST mass spectral libraries using chromatography software with search index matching. Select a candidate peak using the computer cursor; then, view the spectra. Right-click on the spectra to initiate the library search. 
	NOTE: This step can be automated depending on the instrument software.</li>
	<li>Semiquantitate each sample by normalizing the peak areas to the standard by dividing the peak area of the sample peak by the peak area of the standard for each fraction. 
	NOTE: This allows for comparison of metabolite levels across fractions and samples even when sample processing varies.</li>
</ol>

Derivatization and Separation of Barley Flour Metabolites into Four Fractions

<ol><li>Verstegen, H., Köneke, O., Korzun, V., von Broock, R. The world importance of barley and challenges to further improvements. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aVerstegen%2C+H+%22Biotechnological+approaches+to+barley+improvement.+Biotechnology+in+Agriculture+and+Forestry%22">Biotechnological approaches to barley improvement. Biotechnology in Agriculture and Forestry</a>. Kumlehn, J., Stein, N. 69, Springer. Berlin, Heidelberg. 3-19 (2014).</li>
<li>Agribusiness handbook: Barley malt beer. , Agriculture Organization of the United Nations. Rome. 
 <a target="_blank" href="https://www.fao.org/4/i1003e/i1003e00.htm">https://www.fao.org/4/i1003e/i1003e00.htm</a> (2009).</li>
<li>Jena, J., et al. Role of legumes in cropping system for soil ecosystem improvement. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aJena%2C+J+%22Ecosystem+services%3A+types%2C+management+and+benefits%22">Ecosystem services: types, management and benefits</a>. , Nova Science Publishers, Inc. New York. 1-21 (2022).</li>
<li>Ahmed, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+changes+in+grain+ultrastructure%2C+amylase%2C+protein+and+amino+acid+profiles+between+Tibetan+wild+and+cultivated+barleys+under+drought+and+salinity+alone+and+combined+stress%5BTitle%5D)+AND+%22Food+Chem%22%5BJournal%5D)">Differential changes in grain ultrastructure, amylase, protein and amino acid profiles between Tibetan wild and cultivated barleys under drought and salinity alone and combined stress.</a> Food Chem. 141 (3), 2743-2750 (2013).</li>
<li>Marchev, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolomics+and+health%3A+from+nutritional+crops+and+plant-based+pharmaceuticals+to+profiling+of+human+biofluids%5BTitle%5D)+AND+%22Cell+Mol+Life+Sci%22%5BJournal%5D)">Metabolomics and health: from nutritional crops and plant-based pharmaceuticals to profiling of human biofluids.</a> Cell Mol Life Sci. 78 (19-20), 6487-6503 (2021).</li>
<li>Kusch, P. Gas chromatography: Derivatization, sample preparation, application. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKusch%2C+P+%22IntechOpen%22">IntechOpen</a>. , (2019).</li>
<li>Haas, M., Lamour, S., Trapp, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+an+advanced+derivatization+protocol+for+the+unambiguous+identification+of+monosaccharides+in+complex+mixtures+by+gas+and+liquid+chromatography%5BTitle%5D)+AND+%22J+Chromatogr+A%22%5BJournal%5D)">Development of an advanced derivatization protocol for the unambiguous identification of monosaccharides in complex mixtures by gas and liquid chromatography.</a> J Chromatogr A. 1568, 160-167 (2018).</li>
<li>Moldoveanu, S. C., David, V. Derivatization methods in GC and GC/MS. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMoldoveanu%2C+SC+%22Gas+chromatography-derivatization%2C+sample+preparation%2C+application%22">Gas chromatography-derivatization, sample preparation, application</a>. , IntechOpen. (2018).</li>
<li>Kim, B. R., Sung, G. H., Kim, J. -J., Yoon, Y. -J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+development+of+rapid%2C+practical+and+selective+process+for+preparation+of+z-oximes%5BTitle%5D)+AND+%22Journal+of+the+Korean+Chemical+Society%22%5BJournal%5D)">A development of rapid, practical and selective process for preparation of z-oximes.</a> Journal of the Korean Chemical Society. 57 (2), 295-299 (2013).</li>
<li>Miyagawa, H., Bamba, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+sequential+derivatization+with+concurrent+methods+for+GC%2FMS-based+metabolomics%5BTitle%5D)+AND+%22J+Biosci+Bioeng%22%5BJournal%5D)">Comparison of sequential derivatization with concurrent methods for GC/MS-based metabolomics.</a> J Biosci Bioeng. 127 (2), 160-168 (2019).</li>
<li>Yi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MeOx-TMS+derivatization+for+GC-MS+metabolic+profiling+of+urine+and+application+in+the+discrimination+between+normal+C57BL%2F6J+and+type+2+diabetic+KK-Ay+mice%5BTitle%5D)+AND+%22Analytical+Methods%22%5BJournal%5D)">MeOx-TMS derivatization for GC-MS metabolic profiling of urine and application in the discrimination between normal C57BL/6J and type 2 diabetic KK-Ay mice.</a> Analytical Methods. 6 (12), 4380-4387 (2014).</li>
<li>Röhlig, R. M., Eder, J., Engel, K. -H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolite+profiling+of+maize+grain%3A+differentiation+due+to+genetics+and+environment%5BTitle%5D)+AND+%22Metabolomics%22%5BJournal%5D)">Metabolite profiling of maize grain: differentiation due to genetics and environment.</a> Metabolomics. 5, 459-477 (2009).</li>
<li>Cook, B. I., Mankin, J. S., Anchukaitis, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Climate+change+and+drought%3A+From+past+to+future%5BTitle%5D)+AND+%22Current+Climate+Change+Reports%22%5BJournal%5D)">Climate change and drought: From past to future.</a> Current Climate Change Reports. 4, 164-179 (2018).</li>
<li>Samarah, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+drought+stress+on+growth+and+yield+of+barley%5BTitle%5D)+AND+%22Agronomy+for+Sustainable+Development%22%5BJournal%5D)">Effects of drought stress on growth and yield of barley.</a> Agronomy for Sustainable Development. 25 (1), 145-149 (2005).</li>
</ol>

The authors have no conflicts of interest to declare.

We disclose a successful method for the extraction of metabolites from barley grain for metabolic analysis. Several steps in this extraction protocol require particular attention to achieve satisfactory results. In addition, care also needs to be taken during the analysis stage, especially given the number and different properties of the derivatized products.
In order for the metabolite derivatizations to be effective, it is necessary to exclude moisture from solvents and reaction vessels. For example, if a residue continues to form an oily film following a rotary evaporation step, an additional coevaporation step, using a suitable anhydrous solvent, should be added. Likewise, it is important to run silylation reactions under argon to exclude atmospheric moisture, which would otherwise sequester the minute amounts of the silylating reagent. If moisture continues to be a problem, one can use oven-dried reaction vessels to minimize moisture levels. 
Additionally, we have found it to be important to keep the time between extractions, derivatizations, and GC/MS injections to a minimum, to reduce the exposure of reactive metabolite derivatives to moisture. Furthermore, we have modified the previously reported protocols that served as starting points for the method presented herein to increase efficiency. The present method uses methoxyamine hydrochloride in anhydrous pyridine rather than hydroxylammonium hydrochloride in methanol as used in other studies9. Hydroxylammonium hydrochloride in methanol may sequester the silylating reagent used in the subsequent step, which can reduce the efficiency of that step. The methoxyamine hydrochloride, however, lacks reactive hydroxyl groups and is not expected to interfere with and sequester silylating reagents used in the following steps. We use this reagent mixture for the oximation of metabolites as has been done in other studies10,11.
Sample carryover and contamination are common issues that can be resolved through the following instrument maintenance protocol. Siloxane contaminants were observed periodically. We found that by replacing worn septa on the needle wash vials, the siloxane peaks disappeared. Carryover from previous runs is also a concern as evidenced by the occasional presence of intense sucrose derivative peaks in blank runs. We found that silylated and oximated derivates leave behind residues in the GC/MS, which can be difficult to remove. Because these contaminating derivatized peaks are occurring at the same retention time within a particular method, the contamination is likely located at the inlet, needle, or liner. A solution to this problem is to run a more aggressive needle wash regimen, including both protic and aprotic solvents. This procedure covers a broader spectrum of solubility and helps to remove additional contaminants.
The protocol above is adapted from R&#246;hlig's protocol, which explored the metabolites of corn12. We expect the presented protocol for fractionation, derivatization, and identification of barley metabolites to have broad applications for researchers working in this field. We intend to use this method to learn about how barley metabolites are influenced by drought and intercropping. In addition, the barley protocol presented here can be adapted to other grains. As the climate warms, more places are experiencing droughts, especially the western U.S13. Grains, such as barley, are a major source of food in the U.S, and droughts can change how these grains grow14. Being able to learn how important food sources are influenced by drought can help preserve these food sources.

Barley is the fourth most produced grain worldwide1. With climate change increasing the frequency and intensity of droughts, the productivity of this crop is expected to drop significantly as severe drought stress can decrease the grain yield by nearly an order of magnitude2. Agronomic practices that diversify the landscape are hypothesized to increase the resilience -- the tendency to maintain yields and soil properties of cultivated land. Intercropping, the cultivation of two crops simultaneously in the same field, is one such practice, especially when nitrogen-fixing pulse crops, like lentils or peas, are included3.
Plant metabolites are small molecules found in plant tissues and cells, such as sugars, amino acids, and lipids, that can be used to understand plant responses to physical and environmental stress. Previous studies have shown certain metabolites in barley, like proline and certain sugars, are present at a higher level in drought conditions, but a full metabolomic study has yet to be done for both drought stress and intercropped conditions4. What remains unclear is how the metabolome of the grain changes due to the biotic stress of intercropping and environmental stress arising from drought. The metabolome -- the total metabolic profile of the grain -- is important for both the end applications of the barley and for identifying the biochemical responses that allow plants to adapt to changes in their environment5.
This study describes the method development and preliminary results of metabolomic analysis of barley grown under drought-stressed and intercropped conditions using gas chromatography/mass spectrometry (GC/MS). For analysis, the barley metabolites were fractionated and derivatized according to polarity into four fractions (Figure 1). The main constituents of the fractions are as follows: Fraction 1: fatty acid methyl esters (FAMEs) and hydrocarbons; Fraction 2: silylated free fatty acids, fatty alcohols, hydroxy FAMEs, phenols, and sterols; Fraction 3: silylated sugars and polyols; and Fraction 4: silylated hydrophilic acids, amino acids, and amines. Thus, Fractions 1 and 2 predominantly contain non-polar metabolites, while Fractions 3 and 4 predominantly contain polar metabolites.
First, metabolites are extracted from the ground barley grain into polar and non-polar solvents. Further fractionation is performed to enable better separation of metabolites in the mass spectra. Additionally, the boiling points of many of the metabolites are too high to allow for volatilization by GC, requiring further derivatization. Thus, metabolites containing hydroxyl groups were silylated into the corresponding trimethylsilyl (TMS) ethers6, while fatty acid esters were converted into fatty acid methyl esters. Additionally, sugars were oximated into their straight-chain form, reducing the number of possible isomers present for each sugar7. Figure 1 shows a flow chart for the fractionation and derivatization of the metabolites in barley flour. The protocol below provides more detail on the specific steps shown in this figure.
<img alt="Barley flour extraction diagram, detailing lipid and polar extraction processes and compounds." class="xfigimg" src="/files/ftp_upload/67175/67175fig01v2.jpg" data-altupdatedat="1788840098000" data-alt="Figure 1"> 
Figure 1: A flow diagram for the fractionation and derivatization of barley metabolites. Abbreviations: MeOH = methanol; NaOMe = sodium methoxide; TMSIM = N-trimethylsilyimidazole; MeONH2 = methoxyamine hydrochloride; MSTFA = N-methyl-N-(trimethylsilyl)trifluoroacetamide; DI H2O = deionized water; MTBE = methyl tert-butyl ether; SPE = solid phase extraction. <a href="https://www.jove.com/files/ftp_upload/67175/67175fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>4-chloro-L-phenylalanine</td><td>Alfa Aesar</td><td>15470329</td><td>97%</td></tr><tr><td>5&amp;alpha;-cholestan-3&amp;beta;-ol</td><td>Sigma</td><td>D6128-10G</td><td>95%</td></tr><tr><td>Anhydrous acetonitrile</td><td>Sigma</td><td>360457</td><td>ACS (Dried over 3&amp;Aring; molecular sieves)</td></tr><tr><td>Anhydrous Pyridine</td><td>Sigma</td><td>PX2012</td><td>99.8%</td></tr><tr><td>Anhydrous Sodium Sulfate</td><td>EMD</td><td>SX0760-1</td><td>ACS</td></tr><tr><td>Argon</td><td></td><td></td><td></td></tr><tr><td>Barley flour&amp;nbsp;</td><td></td><td></td><td>Grown at the Aberdeen Research and Extension Center, University of Idaho</td></tr><tr><td>Deionized Water</td><td></td><td></td><td></td></tr><tr><td>Dichloromethane</td><td>TCI</td><td>D3478</td><td>99%</td></tr><tr><td>Hydrochloric Acid</td><td>EMD</td><td>HX0603-3</td><td>ACS</td></tr><tr><td>ISQ 7000 mass spectrometer</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>Long Pastuer pipet</td><td></td><td></td><td></td></tr><tr><td>Methanol</td><td>Macron</td><td>3016-16</td><td>ACS</td></tr><tr><td>Methoxylamine hydrochloride</td><td>Thermo</td><td>A19188.06</td><td>98%</td></tr><tr><td>Methyl tert-butyl ether</td><td>VWR</td><td>BDH1139-4LG</td><td>ACS</td></tr><tr><td>n-Heptacosane</td><td>Thermo</td><td>L07796.MD</td><td>97%</td></tr><tr><td>n-Hexane</td><td>Sigma</td><td>HX0295-6</td><td>95%</td></tr><tr><td>N-methyl-N-trimethylsilyl-trifluoroacetamide</td><td>Restek</td><td>35600</td><td>Reagent</td></tr><tr><td>N-trimethylsilyimidazole</td><td>Alfa Aesar</td><td>A12512</td><td>97%</td></tr><tr><td>phenyl-&amp;beta;-D-glucopyranoside</td><td>Sigma</td><td>292710-5G</td><td>95%</td></tr><tr><td>Rotary Evaporator</td><td></td><td></td><td></td></tr><tr><td>Sodium Methoxide</td><td>Merck</td><td>8.06538.0005</td><td>97%</td></tr><tr><td>SPE Column</td><td>Agilent</td><td>12113010</td><td></td></tr><tr><td>SPE Solvent Resevoir Cartridge</td><td>Agilent</td><td>12131014</td><td></td></tr><tr><td>TG-SQC column</td><td></td><td></td><td></td></tr><tr><td>Trace 1300 gas chromatograph</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>Vacuum Manifold</td><td></td><td></td><td></td></tr></tbody></table>

metabolomic analysis of barley by gas chromatography/mass spectrometry

Metabolomic Analysis of Barley by Gas Chromatography/Mass Spectrometry

Climate change increases drought risk to agriculture and impacts both food nutrient content and overall food security. Metabolomics is one way to observe and quantify the impacts of drought on grain and other agricultural products. The identified metabolites may allow for the identification of the biochemical response that allows the plant to tolerate stressful environments. The methodology presented herein allowed for the total metabolomic analysis of barley flour using gas chromatography/mass spectrometry (GC/MS). 
Barley flour metabolite extracts were fractionated into four fractions based on polarity. To allow for analysis by GC/MS, metabolites were derivatized to increase volatility and metabolite separation: fatty acids esters were derivatized into fatty acid methyl esters; sugars were oximated into their straight chain form; and metabolites with hydroxyl groups were converted to their corresponding silyl ethers. The derivatized samples were injected into the GC/MS and the generated mass spectra were used for metabolite identification by comparing the generated spectra to the National Institute of Standards and Technology (NIST) Tandem Mass Spectra library. The method described here can also be used to examine the total metabolome for other plants, furthering our understanding of the biochemical responses of stressed plants.

The developed protocol successfully derivatizes and resolves the many metabolites present in the barley flour. Each peak on the chromatogram is associated with a compound identified from its mass spectra. The three most prominent peaks in Fraction 1 of the sample were octadecadienoic acid methyl ester, hexadecanoic acid methyl ester, and octadecenoic acid. The three most prominent peaks in Fraction 2 of the sample were TMS-derivatized palmitic acid, octadecanoic acid, and n-hexadecanoic acid. The three most prominent peaks in Fraction 3 of the sample were TMS-derivatized sucrose, glycerol, and β-D-glucopyranose. The three most prominent peaks in Fraction 4 of the sample were TMS-derivatized sucrose, malic acid, and glycerol. A complete list of metabolites identified by the chemical library search is provided in Supplemental Table S1. From the mass spectra, one observes that the fatty acid esters have been converted to the corresponding methyl esters and TMS-derivatized sugars and acids are ubiquitous. Likewise, (derivatized) metabolites are well-sorted by polarity between the four fractions. The four chromatograms are shown in Figure 5, Figure 6, Figure 7, and Figure 8. 
<img alt="Chromatography result, mass spectrometry, intensity vs. time graph, protein separation analysis." class="xfigimg" src="/files/ftp_upload/67175/67175fig05.jpg" data-altupdatedat="1788840098000" data-alt="Figure 5"> 
Figure 5: Fraction 1 chromatogram. <a href="https://www.jove.com/files/ftp_upload/67175/67175fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Chromatography TIC graph; intensity vs. time; protein analysis; spectrometry result; data peaks." class="xfigimg" src="/files/ftp_upload/67175/67175fig06.jpg" data-altupdatedat="1788840098000" data-alt="Figure 6"> 
Figure 6: Fraction 2 chromatogram. <a href="https://www.jove.com/files/ftp_upload/67175/67175fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Gas chromatography graph, showing compound peaks, used for sample analysis and separation process." class="xfigimg" src="/files/ftp_upload/67175/67175fig07.jpg" data-altupdatedat="1788840098000" data-alt="Figure 7"> 
Figure 7: Fraction 3 chromatogram. <a href="https://www.jove.com/files/ftp_upload/67175/67175fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Chromatography result graph; intensity vs. time; peak detection; analytical chemistry data analysis." class="xfigimg" src="/files/ftp_upload/67175/67175fig08.jpg" data-altupdatedat="1788840098000" data-alt="Figure 8"> 
Figure 8: Fraction 4 chromatogram. <a href="https://www.jove.com/files/ftp_upload/67175/67175fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Table S1: A complete list of metabolites identified by the chemical library search. <a href="https://www.jove.com/files/ftp_upload/67175/Supplementary%20Fraction%20compound%20list.zip" target="_blank">Please click here to download this File.</a>

A method for metabolomic analysis of barley is presented. The method entails fractionation and derivatization of metabolites and analysis thereof by gas chromatography/mass spectrometry (GC/MS). Metabolomic analysis can be used to determine the effect of intercropping and drought stress on the grain.

Climate change increases drought risk to agriculture and impacts both food nutrient content and overall food security. Metabolomics is one way to observe ...

metabolomic-analysis-of-barley-by-gas-chromatographymass-spectrometry

Research

JoVE Journal

Environment

The urease method of sample preparation for GC/MS analysis of intermediary metabolites is presented by its inventor.  The method allows  one-step  follow-up of newborn screening for inborn errors by tandem mass spectrometry by quantifying carbohydrates, organic and amino acids all in a single process.

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism.  This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

This article describes the application of untargeted metabolomics, transcriptomics and multivariate statistical analysis to grape berry transcripts and metabolites in order to gain insight into the terroir concept, i.e., the impact of the environment on berry quality traits.

The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

This protocol describes how to prepare Drosophila larvae for GC-MS-based metabolomic analysis.

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

System-wide analysis of multiple biomolecules is crucial to gain functional and mechanistic insights into biological processes. Hereby, an extensive protocol is described for high throughput extraction of lipids, metabolites, proteins and starch from a single sample harvested from synchronized Chlamydomonas culture.

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Learn metabolomic analysis of barley using GC/MS to study drought stress effects on grain. This improved method enhances compound identification for agricultural research and food security applications.

Metabolomic Analysis of Barley by Gas Chromatography/Mass Spectrometry

Explore More Videos