Multiplatform Plant Metabolomics Analysis Protocol for Arabidopsis thaliana

Weigh approximately 25 mg of freeze-dried Arabidopsis thaliana tissues into bead beating tubes. Record the exact weights. Keep samples frozen. °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 -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 4°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 4°Cfor 5 minutes, then transfer 200 μl into HPLC glass vial insert. Samples can be used directly for LCMS and HPLC analysis, or stored at -80°Cfreezer.

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 340_ex and 450_emnm

Detector FLD 2: FMOC-derivatised amino acids detected at 260_ex and 325_emnm

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 4°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 4°C.

1.5 µL of OPA reagent is then added to derivatise primary amino acids. The reaction was mixed and incubated for 20s at 4°C

FMOC reagent (1 µL) is added, mixed and incubated for 20 s at 4°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 :

A	B
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 340_ex and 450_em nm from 1-18 min, and FMOC-derivatised amino acids at 260_ex and 325_emnm 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.

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).

CCM method compound list

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

A	B	C	D	E	F
	Q1	Quantifier ion	Qualifier ion	Precursor CE
Compound Name	m/z(1)	m/z(2)	Ref.(1) m/z(2)	Target Collision Energy	RT (min)
2-Keto-3-Deoxy-6-Phosphogluconate	257.2	97.05	78.8	16.9	25.797
3- Phospho-D-Glycerate	185	96.95	78.95	15.6	24.751
3-Dehydroquinic Acid	189.05	171.2	109.05	13.2	6.801
3-Dehydroshikimic Acid	171.05	109.05	127.1	19.7	8.627
3-Hydroxybutyrate	103.3	59	41	13.3	9.295
3-Hydroxybutyrate Coenzyme A	852.15	408.05	158.85	40.2	36.5
6-Phosphogluconic Acid	275.1	96.9	78.95	17	24.39
α-Ketoglutarate	145.2	101	57.1	11.9	23.349
Acetoacetyl Coenzyme A	850.15	766.1	158.85	28.9	37
Acetolactate	131.25	87.15	42.05	9.7	16.646
Acetyl Coenzyme A	807.9	408.1	461.1	37.2	38.372
Acetylphosphate	139.2	78.8	63	20.8	20.589
Aconitic Acid	173.1	85.05	129.15	13.2	25.982
Adenosine Diphosphate	426	134.1	159.1	23.4	25.84
Adenosine Diphosphate	741.95	620	408.05	17.6	24.711
Adenosine Monophosphate	346.05	79	96.95	24.7	17.662
Adenosine Triphosphate	505.9	158.95	408.05	30.6	35.585
Adipic Acid	145.25	101.05	83.15	15	21.075
Anthranilic Acid	136.25	92.1	65	16.6	22.283
Azidothymidine (Internal standard)	266.1	223.25	42	11.3	15.887
Citrate	191	87.05	111.05	18.2	25.437
Coenzyme A	766	408.05	419.1	36.2	37.604
Creatine Phosphate	210.05	79	97	21.7	21.911
Cytidine Monophosphate	322.15	96.95	79	21.6	11.485
Cytidine Triphosphate	482	159.1	384.1	30.1	34.235
D-Erythrose 4-Phosphate	199.1	96.95	79	10.3	12.129
Dihydroxyacetone Phosphate	169.2	97	79	10.7	12.734
D-Ribose 5-Phosphate	229.1	97	79	13.8	8.969
D-Ribulose 5-Phosphate	229.1	97	79	11.6	11.163
D-Xylulose 5-Phosphate	229.1	97	79	11.6	10.806
Flavin Adenine Dinucleotide	784.15	437.1	346.1	28.4	30.44
Flavin Adenine Mononucleotide	454.9	97	78.85	26.4	24.281
Fructose 1,6-Bisphosphate	339.1	96.95	79	22	25.161
Fructose 6-Phosphate	259	96.95	79.05	16.8	9.345
Fumarate	115.2	71.05	27	11.4	24.3
Glucose 1-Phosphate	259	79.05	241.15	23.1	9.93
Glucose 6-Phosphate	259.1	96.9	78.9	18.1	8.195
Glyceraldehyde 3-Phosphate	169.1	96.9	78.9	10	9.075
Glycerol 3-Phosphate	171.1	79.05	96.85	23.3	9.695
Glycolic Acid	75.3	47.05	44.95	14.2	6.514
Guanosine Diphosphate	442	344.1	159.05	20.5	24.96
Guanosine Monophosphate	362.05	79.05	211.15	24.5	15.234
Guanosine Triphosphate	522	159.1	424	33.4	35.019
Isocitrate	191.1	73	173.2	21.9	25.633
Lactate	89.3	43	45.05	14.4	8.684
Malate	133.2	115.1	71	16	22.114
Malonyl Coenzyme A	852	808.1	408	26.1	20.1
Nicotinamide Adenine Dinucleotide (Oxidised)	662.1	540.15	273.05	15.9	14.168
Nicotinamide Adenine Dinucleotide (Reduced)	664	408.1	397.1	32.8	26.152
Nicotinamide Adenine Dinucleotide Phosphate (Reduced)	744	159	622.1	53	35.308
Para-Amino Benzoic Acid	136.05	92	NA	15.5	13.471
Phenylpyruvate	163.35	91	101	11.7	39.974
Phosphoenolpyruvate	167.35	78.9	62.9	21.8	25.169
Propionyl Coenzyme A	821.95	408	158.9	34.4	37
Pyruvate	87.2	43	31.65	12.5	13.161
Ribulose 1,5-Bisphosphate	308.9	97	78.8	21.6	25.017
Sedoheptulose 7-Phosphate	289	96.95	78.9	NA	9.269
Shikimate-3-Phosphate	253	96.95	78.95	17.1	23.763
Shikimic Acid	173.05	93.05	111.05	21.6	5.864
Succinate	117.25	73	98.95	14.3	19.499
Succinate D6 (Internal standard)	121	76.9	102.1	14	19.438
Succinyl-Coenzyme A	866	426.1	339.2	37.2	38.5
Uridine Diphosphate	403	159.1	111.05	26.2	25.04
Uridine Diphosphate Glucuronic Acid	578.95	403	323.1	23.6	34.754
Uridine Diphosphate N-Acetylglucosamine	606.05	385.05	159.1	28	24.437
Uridine Monophosphate	323.05	96.95	79.05	23	14.326
Uridine Triphosphate	482.95	159	385.05	27.4	35.445

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: 300°C
• Desolvation line temperature: 250°C

LC gradient protocol

A	B
Time	Mobile phase B (%)
8	2
22	20
32	27
47	50
48	98
54	98
60	2

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: 45°C

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 37°C for 2 hours. Centrifuge for 10 seconds.

Add 25uL BSTFA+ 1%TMCS reagent and incubate at 37°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 280°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 100°C starting temperature, held for 4 min, then ramped at 10 °C/min to 320°C and held for 11 min. Helium was used as the carrier gas at a flow rate of 1 mL/min.

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) 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.