Jun 26, 2024

Public workspaceMultiplatform Plant Metabolomics Analysis Protocol for Arabidopsis thaliana

DOI
dx.doi.org/10.17504/protocols.io.n92ld8pn9v5b/v1
  • 1School of Biological Sciences, The University of Queensland, St Lucia, QLD 4072, Australia;
  • 2Q-MAP, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD 4072, Australia
Akila Wijerathna Yapa
Open access
DOI: dx.doi.org/10.17504/protocols.io.n92ld8pn9v5b/v1
Protocol Citation: Akila Wijerathna Yapa, Gabriele Netzel, Venea Dara Daygon, Terra Stark 2024. Multiplatform Plant Metabolomics Analysis Protocol for Arabidopsis thaliana. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8pn9v5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 17, 2024
Last Modified: June 26, 2024
Protocol Integer ID: 100011
Keywords: Metabolomics, HPLC, LCMS, GCMS, Arabidopsis thaliana
Abstract
In the pursuit of comprehensive metabolomic profiling, a multiplatform analysis protocol has been developed for Arabidopsis thaliana, utilizing an array of analytical techniques to quantify a diverse set of intracellular metabolites. This protocol encompasses methods for the extraction and purification of metabolites, followed by their analysis using high-performance liquid chromatography (HPLC) for amino acids, liquid chromatography-tandem mass spectrometry (LC-MS/MS) for central carbon metabolism, and gas chromatography-tandem mass spectrometry (GC-MS/MS) for open profiling of amino acids, organic acids, sugars, and fatty acids. Each method is meticulously designed to ensure precise quantification and identification of metabolites critical for understanding the metabolic networks in Arabidopsis thaliana. This protocol not only outlines the steps for metabolite extraction and analysis but also details the preparation of reagents and solutions, instrumental settings, and the analytical conditions required for optimal detection and quantification. The integration of these platforms provides a robust framework for metabolic studies, contributing significantly to the field of plant metabolomics by enabling detailed metabolic profiling with high accuracy and reproducibility.
Image Attribution
Image made using Biorender (Agreement number: PO26Z2LCDS)
Materials
1. Extraction and Purification of Intracellular Metabolites

1. 1 Materials and Supplies
  • 0.5 mm glass beads
  • Dry ice
  • 2 ml centrifuge tubes
  • 1 ml clear glass vials
  • 300 μl glass inserts
  • 2 ml reinforced tubes with screw caps (Scientific Specialties Inc. Cat No. 2320-00 and 2001-00) for bead beating

1.2 Equipment
  • Vortex mixer
  • Analytical balance
  • Pipettes (1 ml, 100 μL)
  • Centrifuge (with cooling capacity to 4°C)
  • Mini bead beater (Omni BR24 bead beater)
  • Vacuum concentrator
  • Freeze drier

1.3 Chemicals and Reagents
  • Methanol: Solvent for extraction and purification of metabolites.
  • Ethanol: Used for prechilling equipment.
  • Millipore filtered distilled water (MQ water): Provides a pure water source for extraction.
  • 100% Acetonitrile (LC grade): Used for reconstitution of samples before analysis.
  • Chloroform: Solvent for biphasic separation to remove lipids and proteins.
  • Azidothymidine (AZT): Internal standard for quantification.



2. HPLC — Amino Acid Analysis
2.1 Materials and supplies

Consumables
  • 1.5 mL centrifuge tubes
  • 1.8 mL HPLC glass vials
  • HPLC glass insert
  • Agilent Zorbax Extend C-18 column (3.5 um, 4.6 x 150 mm, Agilent PN: 763953-902)
  • Guard column (SecurityGuard Gemini C18, Phenomenex PN: AJO-7597)
For sample digestion:
  • Digestion vials
  • 0.2 uM syringe filters
  • Syringe with Luer lock system

Equipment

  • HPLC System with FLD
  • Vortex mixer
  • Centrifuge (with cooling capacity to 4ºC)
  • pH meter
  • Filtering and degassing apparatus with 0.45 um nylon and PTFE membrane filters
  • Magnetic stirrer
  • Oven, incubator or heat block (with heating capacity of 110ºC)
  • Fume cupboard


2.2 Chemicals and Reagents


Sample digestion:
  • High purity water (18.2 MΩ.cm at 25 ºC)
  • 6M hydrochloric acid
  • Phenol
  • 6M Sodium hydroxide
  • Nitrogen gas
  • Lithium hydroxide monohydrate (Sigma 62528-50G
Derivatisation and HPLC analysis:
  • Internal standard - α-aminobutyric acid, norvaline and sarcosine 500 µM each
  • External standard - amino acid standards 500 µM each and containing 250 µM of internal standards.
  • Reducing agent - 0.5% (v/v) 3-mercaptopropionic acid (Sigma M5801) in borate buffer (0.4 N, pH 10.2, Agilent PN: 5061-3339)
  • Alkylating agent - 120 mM iodoacetic acid (Sigma, I4386) in 140 mM NaOH
  • OPA reagent - 10 mg o-pthalaldehyde/mL in 3-mercaptopropionic acid, (Agilent PN: 5061-3335)
  • FMOC reagent - 2.5 mg 9-fluorenylmethyl chloroformate/mL in acetonitrile (Agilent PN:5061-3337)
  • Sodium phosphate dibasic (Na2HPO4)
  • Sodium azide (NaN3)
  • Hydrochloric acid
  • Acetonitrile (HPLC grade)
  • Methanol (HPLC grade)

2.3 Preparation of solutions and reagents

External standards (calibration points)
Stock solution of external standards is prepared with 500 µM concentration (L1) of each amino acid analysed, containing 250 µM internal standard (ABU, NVal, Sar). The solution is aliquoted at 50 µL in PCR tubes and stored at -80ºC freezer until use. The following concentrations are usually analysed and used as calibration points.
standard Concentration (µM)
L1 500
L2 250
L3 125
L7  7.81
L8  3.91
L9  1.95
Mobile phases
Mobile phase A (10x solution): 400 mM Na2HPO4, 0.2%NaN3. Adjust to pH 7.8 using HCl. Vacuum filter. Store in 4°C and dilute to 1x with MQ water before use. Note: precipitates may form during storage. Mix well or apply heat to resuspend in solution.
Mobile phase B: 45% Acetonitrile, 45% Methanol, 10% water

Derivatisation reagents
Borate Solution: Prepare Borate solution by mixing 995 µL Borate buffer and 5 µL 3-mercaptopropionic acid.
FMOC solution: by mixing 30 µL of 10x stock FMOC reagent (made in—house) and 270 µl acetonitrile.



3. LC-MS/MS — Central Carbon Metabolism (CCM)

3.1 Materials
  • 2 mL HPLC glass screw cap vials with inserts
  • Pipettes (5 mL, 1 mL, 200 µL)

3.2 Chemicals and Reagents

  • Azidothymidine (AZT) as internal standard
  • Glacial acetic acid
  • Tritbutylamine (TBA)
  • Millipore filtered distilled water (MQ water)
  • External standards (calibration points): Individual chemicals are prepared in 200 µM concentration with water and stored at -80ºC freezer. Stock solutions are serially diluted from 200 µM to 1.5nM and added with 5µM AZT.
  • Mobile Phases A: 7.5mM Tritbutylamine, acetic acid to pH 4.95.
  • Mobile Phase B: 100% Acetonitrile
  • Rinse solution – 10% isopropanol



4. GC-MS/MS — Open profiling—amino acids, organic acids, sugars, fatty acids

4.1 Materials
  • 1.5 mL centrifuge tubes
  • 1.8 mL HPLC glass vials
  • HPLC glass insert
  • Agilent DB-5 column (30m, 0.25mm x 1um, Agilent PN: 122-5033)

4.2 Chemicals and Reagents
  • Methoxyamine HCl (Merck #89803)
  • Pyridine (Merck # 360570)
  • BSTFA +1%TMCS (Macharey Nagel)
  • 13C- D-sorbitol (Merck #605514)

4.3 Equipment
  • Vortex mixer
  • Analytical balance
  • Pipettes (1 ml, 100 μL)
  • Centrifuge (with cooling capacity to 4°C)
  • Vacuum concentrator


1. Extraction and Purification of Intracellular Metabolites
1. Extraction and Purification of Intracellular Metabolites
Weigh approximately 25 mg of freeze-dried Arabidopsis thaliana tissues into bead beating tubes. Record the exact weights. Keep samples frozen. Temperature0 °C

Add approximately 200 μl of 0.5 mm silica glass beads to each sample. Prechill the bead beater (OMNI Bead Ruptor Elite NE486LUA) to Temperature-1 °C with ethanol and dry ice.
Extract tissue samples in the bead beater using the following settings:
  • Speed: 7.00 m/s
  • Pulses: 3 x 45 sec
  • Interval: 30 sec between pulses
Add 1 ml methanol to the ground samples. Vortex mix.
Add 0.4 ml chloroform to the samples. Vortex mix. Sonicate in an ice bath for 10 min. ALiquot 500 μl of samples for GC-MS open profiling (Step 23).
Add 500 μl MQ water with 500 nM AZT. Vortex mix.
Centrifuge at 16,000 RCF for 5 minutes at Temperature4 °C . Transfer approximately 1 ml of the aqueous layer to new 2 ml Eppendorf tubes, avoiding the interface layer containing proteins.

Evaporate methanol in the samples using a vacuum concentrator until about 400 μl of liquid (water fraction) remains. This step ensures low levels of methanol in preparation for freeze drying.
Freeze dry the samples overnight.
Reconstitute in 200 μl of 2% acetonitrile solution (5x concentrated).
Let stand for 10 minutes on ice. Centrifuge at 16,000 RCF at Temperature4 °C for 5 minutes, then transfer 200 μl into HPLC glass vial insert. Samples can be used directly for LCMS and HPLC analysis, or stored at Temperature-80 °C freezer.


Untitled.png
Schematic diagram of Multiplatform Plant Metabolomics Analysis Protocol for Arabidopsis thaliana
1. Extraction and Purification of Intracellular Metabolites
2. HPLC — Amino Acid Analysis
3. LC-MS/MS — Central Carbon Metabolism (CCM)
4. GC-MS/MS — Open profiling—amino acids, organic acids, sugars, fatty acids


2. HPLC — Amino Acid Analysis
2. HPLC — Amino Acid Analysis
25 µL of crude solution of samples are added to 25 µL of 1 mM internal standards.
Instrumentation
Instrument: Thermo Scientific Vanquish Core UHPLC with FLD detector
Column: Agilent Zorbax Extend C-18 column 3.5 um, 4.6 x 150 mm (PN: 763953-902)
Guard Column: Phenomenex SecurityGuard Gemini C18 (PN: AJO-7597)
Detector FLD 1: OPA-derivatised amino acids detected at 340ex and 450em nm
Detector FLD 2: FMOC-derivatised amino acids detected at 260ex and 325em nm


20240605_114019.jpg
Thermo Scientific Vanquish Core UHPLC with FLD detector

Derivatisation
Aliquot FMOC reagent, OPA reagent, Borate buffer, Iodoacetic acid and Mobile phase A into HPLC vials. Take note of the position in the HPLC autosampler. Derivatisation is performed as previously published [Valgepea et al 2017]. The following steps are programmed in a high-performance autosampler.
1 µL of sample (which has been diluted 1:1 with internal standard), is added into 3 µL of 0.5% (v/v) 3-mercaptopropionic acid (Sigma M5801) in borate buffer (0.4 N, pH 10.2, Agilent PN: 5061-3339), mixed and incubated for 20 s at Temperature4 °C , to reduce free cystines.

To alkylate reduced cysteines, 1 µL of 120 mM iodoacetic acid (Sigma, I4386) in 140 mM NaOH is added, mixed and incubated for 20 s at Temperature4 °C .

1.5 µL of OPA reagent is then added to derivatise primary amino acids. The reaction was mixed and incubated for 20s at Temperature4 °C

FMOC reagent (1 µL) is added, mixed and incubated for 20 s at Temperature4 °C to derivatise the secondary amino acids.

The pH is then lowered by adding 50 µL of Mobile phase A (pH 7). The whole 57.5 µL are then injected to the UHPLC.
UHPLC parameters
The UHPLC gradient:
AB
Mobile phase B concentration (%) Time (min)
2 – 30% 0 – 14
30 – 25% 14.1 – 15
40 – 45% 15.1 – 18
50 – 60% 18.1 – 20
100% 20.1 – 25
2% 25.1 – 27
Column Temperature: 37 ºC
Flow rate: 1.8 mL/min
Derivatised amino acids are monitored using a fluorescence detector. OPA-derivatised amino acids were detected at 340ex and 450em nm from 1-18 min, and FMOC-derivatised amino acids at 260ex and 325emnm from 18-21 min.
Quantifications are based on standard curves derived from serial dilutions of a mixed amino acid standard. The upper and lower limits of quantification are 1000 and 1.95 µM, respectively. Chromatograms are integrated using Chromeleon software.



thumbnail_image001.png
Chromatogram


3. LC-MS/MS — Central Carbon Metabolism (CCM)
3. LC-MS/MS — Central Carbon Metabolism (CCM)
Data acquisition was performed using negative ionization mode, and all analyses were executed on a Shimadzu 8060 LC-MS/MS system.

Instrument: Shimadzu 8060 LC MS-MS
Column: Phenomenex Gemini NX-C18 3µm x 150mm x 2mm (Part No. 00F-4453-B0) fitted with
Phenomenex SecurityGuard column Gemini-NX C18 4 x 2.0mm ID (AJ0-8367).


20240605_110549.jpg
Shimadzu 8060 LC MS-MS System

CCM method compound list

image.png
The Central Carbohydrate Metabolic Network.
Metabolites targeted in LC-MS/MS in this protocol are underlined.


Multiple reaction monitoring (MRM) transitions (Q1/Q3)
List for identification and quantification of CCMs.

ABCDEF
Q1 Quantifier ionQualifier ionPrecursor CE
Compound Namem/z(1)m/z(2)Ref.(1) m/z(2)Target Collision EnergyRT (min)
2-Keto-3-Deoxy-6-Phosphogluconate257.297.0578.816.925.797
3- Phospho-D-Glycerate18596.9578.9515.624.751
3-Dehydroquinic Acid189.05171.2109.0513.26.801
3-Dehydroshikimic Acid171.05109.05127.119.78.627
3-Hydroxybutyrate103.3594113.39.295
3-Hydroxybutyrate Coenzyme A852.15408.05158.8540.236.5
6-Phosphogluconic Acid275.196.978.951724.39
α-Ketoglutarate145.210157.111.923.349
Acetoacetyl Coenzyme A850.15766.1158.8528.937
Acetolactate131.2587.1542.059.716.646
Acetyl Coenzyme A807.9408.1461.137.238.372
Acetylphosphate139.278.86320.820.589
Aconitic Acid173.185.05129.1513.225.982
Adenosine Diphosphate426134.1159.123.425.84
Adenosine Diphosphate741.95620408.0517.624.711
Adenosine Monophosphate346.057996.9524.717.662
Adenosine Triphosphate505.9158.95408.0530.635.585
Adipic Acid145.25101.0583.151521.075
Anthranilic Acid136.2592.16516.622.283
Azidothymidine (Internal standard)266.1223.254211.315.887
Citrate19187.05111.0518.225.437
Coenzyme A766408.05419.136.237.604
Creatine Phosphate210.05799721.721.911
Cytidine Monophosphate322.1596.957921.611.485
Cytidine Triphosphate482159.1384.130.134.235
D-Erythrose 4-Phosphate199.196.957910.312.129
Dihydroxyacetone Phosphate169.2977910.712.734
D-Ribose 5-Phosphate229.1977913.88.969
D-Ribulose 5-Phosphate229.1977911.611.163
D-Xylulose 5-Phosphate229.1977911.610.806
Flavin Adenine Dinucleotide784.15437.1346.128.430.44
Flavin Adenine Mononucleotide454.99778.8526.424.281
Fructose 1,6-Bisphosphate339.196.95792225.161
Fructose 6-Phosphate25996.9579.0516.89.345
Fumarate115.271.052711.424.3
Glucose 1-Phosphate25979.05241.1523.19.93
Glucose 6-Phosphate259.196.978.918.18.195
Glyceraldehyde 3-Phosphate169.196.978.9109.075
Glycerol 3-Phosphate171.179.0596.8523.39.695
Glycolic Acid75.347.0544.9514.26.514
Guanosine Diphosphate442344.1159.0520.524.96
Guanosine Monophosphate362.0579.05211.1524.515.234
Guanosine Triphosphate522159.142433.435.019
Isocitrate191.173173.221.925.633
Lactate89.34345.0514.48.684
Malate133.2115.1711622.114
Malonyl Coenzyme A852808.140826.120.1
Nicotinamide Adenine Dinucleotide (Oxidised)662.1540.15273.0515.914.168
Nicotinamide Adenine Dinucleotide (Reduced)664408.1397.132.826.152
Nicotinamide Adenine Dinucleotide Phosphate (Reduced)744159622.15335.308
Para-Amino Benzoic Acid136.0592NA15.513.471
Phenylpyruvate163.359110111.739.974
Phosphoenolpyruvate167.3578.962.921.825.169
Propionyl Coenzyme A821.95408158.934.437
Pyruvate87.24331.6512.513.161
Ribulose 1,5-Bisphosphate308.99778.821.625.017
Sedoheptulose 7-Phosphate28996.9578.9NA9.269
Shikimate-3-Phosphate25396.9578.9517.123.763
Shikimic Acid173.0593.05111.0521.65.864
Succinate117.257398.9514.319.499
Succinate D6 (Internal standard)12176.9102.11419.438
Succinyl-Coenzyme A866426.1339.237.238.5
Uridine Diphosphate403159.1111.0526.225.04
Uridine Diphosphate Glucuronic Acid578.95403323.123.634.754
Uridine Diphosphate N-Acetylglucosamine606.05385.05159.12824.437
Uridine Monophosphate323.0596.9579.052314.326
Uridine Triphosphate482.95159385.0527.435.445

MS/MS Settings
  • Acquisition settings: negative ionisation mode
  • Nebulizing Gas flow: 3 L/min
  • Heating gas flow: 10 L/min
  • Drying gas flow: 10 L/min
  • Interface temperature: Temperature300 °C
  • Desolvation line temperature: Temperature250 °C

LC gradient protocol
AB
Time Mobile phase B (%)
8 2
22 20
32 27
47 50
48 98
54 98
60 2
image.png
LC Gradient

An injection volume of 5 µL was utilized for each sample, with a flow rate maintained at 0.3 mL/min throughout the procedure. The mobile phases consisted of Mobile Phase A: 7.5 mM Tritbutylamine adjusted with acetic acid to a pH of 4.95, and Mobile Phase B: 100% acetonitrile. The chromatographic gradient was established as follows: from 0 to 8 minutes, 2% B; 8 to 22 minutes, a gradient increase from 2% to 20% B; 22 to 32 minutes, further increased to 27% B; 32 to 47 minutes, elevated to 50% B; a sharp rise to 98% B at 47 to 48 minutes, held constant at 98% B until 54 minutes, and then reduced back to 2% B from 54 to 60 minutes.

  • Total analysis time: 60 min
  • Flow: 0.3 mL /min
  • Injection volume: 5ul
  • Oven temperature: Temperature45 °C

4. GC-MS/MS — Open profiling—amino acids, organic acids, sugars, fatty acids
4. GC-MS/MS — Open profiling—amino acids, organic acids, sugars, fatty acids
To the 500uL samples prepared by DD and AP, add 5uL of 500uM 13C sorbitol. Vortex
Dry aliquots of this extract, by adding 10µl aliquots into a glass insert and drying in a speed vacuum.
Before analysis, perform a final quick dry down by adding 25µl methanol as samples must be completely dry before derivatising with methoxyamine and BSTFA + 1%TMCS.
Derivatise with methoxyamine (meox) (30mg/ml in pyridine) and BSTFA + 1%TMCS.
Add 25ul meox solution and incubate at Temperature37 °C for 2 hours. Centrifuge for 10 seconds.

Add 25uL BSTFA+ 1%TMCS reagent and incubate at Temperature37 °C for 30 minutes. Leave at room temperature for 2 hours before injecting in GCMS system.

GC-MS analysis was performed on a Shimadzu GC/MS-TQ8050 NX system.  1 µL of derivatised sample was injected into the GC inlet set at Temperature280 °C in split mode of 1:10. Chromatographic separation was achieved using an Agilent DB-5 ms capillary column (30 m × 0.25 mm × 1 µm). Oven conditions were set at Temperature100 °C starting temperature, held for 4 min, then ramped at 10 °C/min to Temperature320 °C and held for 11 min. Helium was used as the carrier gas at a flow rate of 1 mL/min.

20240605_110602.jpg
Shimadzu GC/MS-TQ8050 NX system

Compounds were fragmented by electron impact (EI) ionization and analysed in full scan and MRM mode using the Shimadzu Smart Metabolites Database.  (https://www.shimadzu.com/an/gcms/metabolites/index.html) 
A high-quality matrix was manually curated using the Shimadzu LabSolutions Insight GCMS program (v.3.7 SP3, Shimadzu Corporation), where metabolite targets were removed from the dataset if they were not present in all samples.
Protocol references
Valgepea, K., et al. Arginine deiminase pathway provides ATP and boosts growth of the gas-fermenting acetogen Clostridium autoethanogenum. Metab Eng, (2017). 41: p. 202-211. https://doi.org/10.1016/j.ymben.2017.04.007

Wood, J.C., et al. Characterisation of acetogen formatotrophic potential using Eubacterium limosum. Appl Microbiol Biotechnol 107, 4507–4518 (2023). https://doi.org/10.1007/s00253-023-12600-6

Heffernan, J., et al. Adaptive laboratory evolution of Clostridium autoethanogenum to metabolize CO2 and H2 enhances growth rates in chemostat and unravels proteome and metabolome alterations. Microbial Biotechnology, (2024) 17, e14452. https://doi.org/10.1111/1751-7915.14452

Forwood, D.L., Innes, D.J., Parra, M.C. et al. Feeding an unsalable carrot total-mixed ration altered bacterial amino acid degradation in the rumen of lambs. Sci Rep 13, 6942 (2023). https://doi.org/10.1038/s41598-023-34181-0