Summary of Study ST002012

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001276. The data can be accessed directly via it's Project DOI: 10.21228/M8S70F This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST002012
Study Title	Untargeted primary metabolite profiling in Arabidopsis thaliana
Study Summary	The goal of this project is to compare the primary metabolite profile in different tissue types of the model plant Arabidopsis thaliana. Specifically, plants were grown hydroponically under the long-day (16hr light/day) condition at 21C. Tissue samples, including leaves, inflorescences, and roots were harvest 4 1/2 weeks post sowing. Untargeted primary metabolites profiling was carried out using GCTOF.
Institute	Salk Institute for Biological Studies
Department	PBIO
Laboratory	Joanne Chory
Last Name	Wu
First Name	Xuelin
Address	Salk Institute for Biological Studies
Email	xwu@salk.edu
Phone	858-453-4100, x1128
Submit Date	2021-12-09
Raw Data Available	Yes
Raw Data File Type(s)	cdf
Analysis Type Detail	GC-MS
Release Date	2022-01-02
Release Version	1

Select appropriate tab below to view additional metadata details:

Sample Preparation:

Sampleprep ID:	SP002104
Sampleprep Summary:	Extraction of Plant Samples: Leaves 1. References: Fiehn O (2006) Metabolite Profiling in Arabidopsis. In: Arabidopsis Protocols 2nd edition. Salinas J, Sanchez-Serrano JJ (eds.), Methods in Molecular Biology ser., Humana Press, Totowa NJ, 439-447 Weckwerth W, Wenzel K, Fiehn O (2004) Process for the integrated extraction, identification and quantification of metabolites, proteins and RNA to reveal their co-regulation in biochemical networks. Proteomics 4, 78-83 2. Starting material: Arabidopsis rosette leaves: 20±5 mg fresh weight sample OR 2 mg dry weight sample Rice leaves: 10±3 mg fresh weight sample OR 1 mg dry weight sample Other plant organs not validated but advised to use this SOP in a similar manner 3. Equipment: Eppendorf pipettes: 1-200µL and 100-1000µL Eppendorf tubes 2.0mL, uncoloured (Cat. No. 022363204 ) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Precision balance with accuracy ± 0.1 mg ThermoElectron Neslab RTE 740 cooling bath at –20°C MiniVortexer, VWR. Speed vacuum concentration system (Labconco Centrivap cold trap) Ball mill MM301 (Retsch corp.) Metal balls (Retsch 3 mm I.D. Cat. No 22.455.0002 or 5 mm I.D. Cat. No 22.455.0003) Large tweezers Dewar cold gloves 2 liquid nitrogen dewars pH paper 5-10 (EMD Chem. Inc.) Crushed ice Nitrogen line with pipette tip Cork borer (Ø diameter of cylinder related to the amount to be used. 4 mm I.D. for Arabidopsis leaves.) 4. Chemicals Methanol, LCMS grade Chloroform, HPLC grade Liquid nitrogen Water, Millipore (pure) 5. Harvest Procedure NOTE: No weighing of plant material needed unless the experimental design suggests marked changes for the ratio of leaf disk diameter to fresh weight, e.g. in drought stress experiments. In such cases, leaves need to be lyophilized and weighed prior to extraction Label Eppendorf tubes according to the sample name defined in SetupX and put a grinding ball in each. Using the cork borer take one or two disks per leaf and immediately transfer to an Eppendorf tube. Close the tube and immediately place into the dewar of liquid nitrogen. 6. Preparation of extraction mix and material Check the pH of methanol (pH7) Switch on the ThermoElectron Neslab RTE 740 cooling bath at to pre-cool at –18°C to -22°C Make the extraction solution by mixing methanol, chloroform, and water in proportions of 5 : 2 : 2 Rinse the extraction solution for 5 min with nitrogen, making sure that the nitrogen line was flushed out of air before using it for degassing the extraction solvent solution. 7. Homogenisation and extraction Take the Eppendorf tubes from the liquid nitrogen and place into the tube-holder of the grinder being careful to compensate for weight, maintaining equilibrium. Shake for 30s at a frequency of 25 s-1 and check that the leaves have been ground into a fine powder. Repeat if necessary, submerging in liquid nitrogen first. After grinding add 1mL of pre-chilled extraction solution to each tube one by one to prevent even partial thawing of the sample. Store all samples on ice while finishing this step. Vortex the sample for 10s and shake on the Orbital Mixing Chilling/Heating Plate for 6 minutes at 4°C Centrifuge for 2min at 14000 rcf using the centrifuge Eppendorf 5415 D. Remove the whole supernatant in two 500uL portions, saving one as a backup. Dry one portion in the Labconco Centrivap cold trap concentrator to complete dryness and submit for derivitization. 8. Problems To prevent contamination disposable material is used. Control pH from extraction mix. 8. Quality assurance For each sequence perform one blank negative control by applying the total procedure (i.e. all materials and plastic ware) without biological sample. Also perform all tasks once samples/standards have come to room temperature. Finally, include a pure MSTFA vial for liner conditioning steps. 9. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules.

Sampleprep ID:

SP002104

Sampleprep Summary:

Extraction of Plant Samples: Leaves 1. References: Fiehn O (2006) Metabolite Profiling in Arabidopsis. In: Arabidopsis Protocols 2nd edition. Salinas J, Sanchez-Serrano JJ (eds.), Methods in Molecular Biology ser., Humana Press, Totowa NJ, 439-447 Weckwerth W, Wenzel K, Fiehn O (2004) Process for the integrated extraction, identification and quantification of metabolites, proteins and RNA to reveal their co-regulation in biochemical networks. Proteomics 4, 78-83 2. Starting material: Arabidopsis rosette leaves: 20±5 mg fresh weight sample OR 2 mg dry weight sample Rice leaves: 10±3 mg fresh weight sample OR 1 mg dry weight sample Other plant organs not validated but advised to use this SOP in a similar manner 3. Equipment: Eppendorf pipettes: 1-200µL and 100-1000µL Eppendorf tubes 2.0mL, uncoloured (Cat. No. 022363204 ) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Precision balance with accuracy ± 0.1 mg ThermoElectron Neslab RTE 740 cooling bath at –20°C MiniVortexer, VWR. Speed vacuum concentration system (Labconco Centrivap cold trap) Ball mill MM301 (Retsch corp.) Metal balls (Retsch 3 mm I.D. Cat. No 22.455.0002 or 5 mm I.D. Cat. No 22.455.0003) Large tweezers Dewar cold gloves 2 liquid nitrogen dewars pH paper 5-10 (EMD Chem. Inc.) Crushed ice Nitrogen line with pipette tip Cork borer (Ø diameter of cylinder related to the amount to be used. 4 mm I.D. for Arabidopsis leaves.) 4. Chemicals Methanol, LCMS grade Chloroform, HPLC grade Liquid nitrogen Water, Millipore (pure) 5. Harvest Procedure NOTE: No weighing of plant material needed unless the experimental design suggests marked changes for the ratio of leaf disk diameter to fresh weight, e.g. in drought stress experiments. In such cases, leaves need to be lyophilized and weighed prior to extraction Label Eppendorf tubes according to the sample name defined in SetupX and put a grinding ball in each. Using the cork borer take one or two disks per leaf and immediately transfer to an Eppendorf tube. Close the tube and immediately place into the dewar of liquid nitrogen. 6. Preparation of extraction mix and material Check the pH of methanol (pH7) Switch on the ThermoElectron Neslab RTE 740 cooling bath at to pre-cool at –18°C to -22°C Make the extraction solution by mixing methanol, chloroform, and water in proportions of 5 : 2 : 2 Rinse the extraction solution for 5 min with nitrogen, making sure that the nitrogen line was flushed out of air before using it for degassing the extraction solvent solution. 7. Homogenisation and extraction Take the Eppendorf tubes from the liquid nitrogen and place into the tube-holder of the grinder being careful to compensate for weight, maintaining equilibrium. Shake for 30s at a frequency of 25 s-1 and check that the leaves have been ground into a fine powder. Repeat if necessary, submerging in liquid nitrogen first. After grinding add 1mL of pre-chilled extraction solution to each tube one by one to prevent even partial thawing of the sample. Store all samples on ice while finishing this step. Vortex the sample for 10s and shake on the Orbital Mixing Chilling/Heating Plate for 6 minutes at 4°C Centrifuge for 2min at 14000 rcf using the centrifuge Eppendorf 5415 D. Remove the whole supernatant in two 500uL portions, saving one as a backup. Dry one portion in the Labconco Centrivap cold trap concentrator to complete dryness and submit for derivitization. 8. Problems To prevent contamination disposable material is used. Control pH from extraction mix. 8. Quality assurance For each sequence perform one blank negative control by applying the total procedure (i.e. all materials and plastic ware) without biological sample. Also perform all tasks once samples/standards have come to room temperature. Finally, include a pure MSTFA vial for liner conditioning steps. 9. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules.