Metabolomic Analysis of Barley by Gas Chromatography/Mass Spectrometry

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

1. Preparation of standards (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.

Discussion

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.......

Materials

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