Dec 10, 2025
Untargeted Metabolomics : Extraction of Tissue Metabolites
- 1CEA
- MetaboHub-IDF
- MetaboHUB
External link: https://joliot.cea.fr/drf/joliot/Pages/Entites_de_recherche/medicaments_technologies_sante/SPI/lims.aspx
Protocol Citation: Emeline CHU-VAN, florence castelli 2025. Untargeted Metabolomics : Extraction of Tissue Metabolites. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvowex9l4o/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: June 12, 2025
Last Modified: December 10, 2025
Protocol Integer ID: 220040
Keywords: METABOHUB, large cohorts, human health, biomarker discovery, untargeted metabolomic, extraction of tissue metabolite, internal metabolite standards in the extraction process, tissue metabolome, internal metabolite standard, coverage of tissue metabolome, tissue metabolite, accurate comparison of relative metabolite abundance, relative metabolite abundance, methanolic protein precipitation from tissue, controlled extraction process, extraction process, extraction, methanolic protein precipitation, samples within the large cohort
Funders Acknowledgements:
MetEx+
Grant ID: 21-ESRE-0035
METABOHUB
Grant ID: 11-INBS-0010
Abstract
The extraction of tissue metabolites for untargeted metabolomics involves methanolic protein precipitation from tissue samples, aiming to maximize coverage of tissue metabolome. The proposed protocol includes both external and internal metabolite standards in the extraction process to answert to our quality control (QC) procedures. The expected outcome is a normalized and controlled extraction process, allowing for the accurate comparison of relative metabolite abundances across all samples within the large cohorts.
Materials
Freezer -80°C :
Vortex shaker :
Centrifuge :
Ultrasonic processor :
plate reader (562 nm)
Evaporator :
Sharp scissors :
Lobind microtubes :
Micropipettes :
Protocol materials
Water LC-MS ChromasolvFisher ScientificCatalog #15665350
Methanol LC-MS ChromasolvFisher ScientificCatalog #15614740
DimetridazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #31707
2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A0326
2-methyl-4-chlorophenoxyacetic acid (MCPA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #45555
DinosebMerck MilliporeSigma (Sigma-Aldrich)Catalog #45453
Pierce BCA protein assayThermo ScientificCatalog #23227
Alanine-C13Merck MilliporeSigma (Sigma-Aldrich)Catalog #489867
ampicillinMerck MilliporeSigma (Sigma-Aldrich)Catalog #59349
RoxithromycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #R4393
ColchicineMerck MilliporeSigma (Sigma-Aldrich)Catalog #C9754
2-aminoanthraceneMerck MilliporeSigma (Sigma-Aldrich)Catalog #A38800
Formic acid 99.0+% qualite LC/MS optimaFisher ScientificCatalog #10780320
D-Glucose-1-*13*CMerck MilliporeSigma (Sigma-Aldrich)Catalog #297046
prednisoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6254
DihydrostreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #D7253
Metformine (1,1-Dimethylbiguanide hydrochloride)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D150959
Acetonitrile ULC-MSBiosolveCatalog #0001204101BS
Imipramine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I7379
L-Aspartic acid-*15*NMerck MilliporeSigma (Sigma-Aldrich)Catalog #332135
Ethylmalonic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #102687
Amiloride hydrochloride hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7410
AtropineMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0132
AMMONIUM CARBONATE, 99.999%Merck MilliporeSigma (Sigma-Aldrich)Catalog #379999
Ammonia solution 25% for HPLMerck MilliporeSigma (Sigma-Aldrich)Catalog #5438300100
Safety warnings
This protocol involves the use of volatile solvents that may pose health risks. Therefore, it is essential to handle these solvents within a designated chemical fume hood and to wear appropriate personal protective equipment (gloves, goggles, lab coat, etc.).
Wear appropriate clothing, closed shoes, lab coat, glasses, gloves, extractor hood
Use of CMR solvents, use of nitrogen
Wear your Personal Protective Equipment (PPE) at least a lab coat, protective eyewear, gloves, long pants and closed toe shoes
Ethics statement
For experiments involving humans, the CPP (commission de protection des personnes physique) agreement.
Before start
To analyze plasma samples, it is essential to collect all samples using the same anticoagulant (preferably EDTA for LC-HRMS analyses)
For human samples analyses in France, a CODECOH ("Déclaration ou autorisation des activités de conservation et de préparation à des fins scientifiques d'éléments issus du corps humain") must be submitted at least 2-3 months in advance to ensure approval before sample reception.
Randomisation
Reorganize plasma samples in random order on dry ice (the same randomization is applied for the entire process (sample prep - LC-MS analysis - data treatment)
Store at -80 °C until extraction
Sample extraction
Thaw samples
Transfer samples in eppendorf tubes (weight < 100 mg, if not cut samples)
Add 170 µL of water
Water LC-MS ChromasolvFisher ScientificCatalog #15665350
Cut into small pieces with small scissors
Sonicate samples with :
Equipment
Vibracell VCX 130
NAME
Ultrasonic processor
TYPE
Sonics
BRAND
432-0135
SKU
with probe 2 mm during 10 s five times at 40% ampl
Transfer 20 µL of homogenate in Lobind tube for future protein assay (BCA)
Add 350 µL of MeOH with internal standards to 150 µL remaining volume
extraction solvent :
MeOH containing Internal standards at 37.5 ng/mL
Methanol LC-MS ChromasolvFisher ScientificCatalog #15614740
Internal standards :
DimetridazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #31707
2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A0326
2-methyl-4-chlorophenoxyacetic acid (MCPA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #45555
DinosebMerck MilliporeSigma (Sigma-Aldrich)Catalog #45453
Vortex 5 s
Incubate on ice 90 min until complete protein precipitation
20000 x g, 5°C 15 min
Transfer supernatants :
150 µL for Hilicneg analysis
150 µL for C18pos analysis
Remaining volume for QCpool (about 150 µL )
Aliquote QCpool (150 µL )
Dry supernatants under a nitrogen stream at 30 °C using a
Equipment
TurboVap LV
NAME
Evaporator
TYPE
BIOTAGE
BRAND
TurboVap® LV
SKU
Store at -20 °C until analysis.
Protein assay (BCA kit)
Pierce BCA protein assayThermo ScientificCatalog #23227
Standards (BSA) preparation (in lobind tubes if samples are in lobind tubes)
Diluent = water
Well plate map (duplicates for all) :
| 1 | 2 | 3 | 4 | 5 | |
| A | Blank | std A | std A | sample 1 | sample 1 |
| B | Blank | std B | std B | sample 2 | sample 2 |
| C | Blank | std C | std C | sample 3 | sample 3 |
| D | Blank | std D | std D | sample 4 | sample 4 |
| E | Blank | std E | std E | sample 5 | sample 5 |
| F | Blank | std F | std F | sample 6 | sample 6 |
| G | Blank | std G | std G | sample 7 | sample 7 |
| H | Blank | std H | std H | sample 8 | sample 8 |
| 6 | 7 | 8 | 9 | 10 | |
| A | sample 9 | sample 9 | sample 17 | sample 17 | sample 25 |
| B | sample 10 | sample 10 | sample 18 | sample 18 | sample 26 |
| C | sample 11 | sample 11 | sample 19 | sample 19 | sample 27 |
| D | sample 12 | sample 12 | sample 20 | sample 20 | sample 28 |
| E | sample 13 | sample 13 | sample 21 | sample 21 | sample 29 |
| F | sample 14 | sample 14 | sample 22 | sample 22 | sample 30 |
| G | sample 15 | sample 15 | sample 23 | sample 23 | sample 31 |
| H | sample 16 | sample 16 | sample 24 | sample 24 | sample 32 |
| 11 | 12 | |
| A | sample 25 | sample 33 |
| B | sample 26 | sample 33 |
| C | sample 27 | sample 34 |
| D | sample 28 | sample 34 |
| E | sample 29 | sample 35 |
| F | sample 30 | sample 35 |
| G | sample 31 | H2O |
| H | sample 32 | H2O |
Colored reagent preparation :
Reagent B diluted 50-fold in reagent A
total amount = (standards + samples) x replicates x (volume of colored reagent per sample)
Blank = colored reagent only
Typical dilution of sample according to matrix :
- cell pellets = 5-fold
- stools = 50-fold
- organs = 5-fold or more if samples weigh around 50 - 100 mg
Pipette 20 µL of each blank / standard / sample in well plate
Add 200 µL of colored reagent
Incubate the plate 100 rpm, 40°C 20 min
Read the plate at 562 nm (reading between 540 and 590 nm can be used).
Expected results
Substract the average absorbance measurement of the blank replicates from the measurements of all other replicates (standards / samples).
curve fitting = quadratic
Expected result
Results are validated if :
- CV < 15% for standards and samples
- exactitude < 15% for standards
- Calculated concentrations for samples are std A > conc > std G
Samples reconstitution for Hilic analysis in 2 steps
Resuspend dried extracts :
According to protein assay, samples are reconstituted with a ratio 1 mg proteins / 50 µL.
if the final volumes are lower than 75 µL or heigher than 300 µL, the ratio can be adapted.
reagents for mobile phases :
Water LC-MS ChromasolvFisher ScientificCatalog #15665350
AMMONIUM CARBONATE, 99.999%Merck MilliporeSigma (Sigma-Aldrich)Catalog #379999
Ammonia solution 25% for HPLMerck MilliporeSigma (Sigma-Aldrich)Catalog #5438300100
Acetonitrile ULC-MSBiosolveCatalog #0001204101BS
*ES (external standards : mixture of 14 authentic chemical standards covering the mass range of interest):
D-Glucose-1-*13*CMerck MilliporeSigma (Sigma-Aldrich)Catalog #297046
L-Aspartic acid-*15*NMerck MilliporeSigma (Sigma-Aldrich)Catalog #332135
Ethylmalonic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #102687
Amiloride hydrochloride hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7410
prednisoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6254
Metformine (1,1-Dimethylbiguanide hydrochloride)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D150959
AtropineMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0132
ColchicineMerck MilliporeSigma (Sigma-Aldrich)Catalog #C9754
Imipramine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I7379
Alanine-C13Merck MilliporeSigma (Sigma-Aldrich)Catalog #489867
2-aminoanthraceneMerck MilliporeSigma (Sigma-Aldrich)Catalog #A38800
ampicillinMerck MilliporeSigma (Sigma-Aldrich)Catalog #59349
DihydrostreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #D7253
RoxithromycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #R4393
mobile phases preparation for Hilic analysis :
mobile phase A = Ammonium carbonate 10 mM - pH 10.5
in 1L of water add 0.9606 mg ammonium carbonate
adjust pH at 10.5 with ammonia solution
mobile phase B = 100% acétonitrile
first step = 40% (of final volume) of mobile phase A containing ES* 2.5x
Vortex 5 s
second step = 60% (of final volume) of mobile phase B
Vortex 5 s
20000 x g, 4°C 15 min
Transfer supernatant into 0.2 mL vials
Samples reconstitution for C18 analysis in 1 step
Resuspend dried extracts :
According to protein assay, samples are reconstituted with a ratio 1 mg proteins / 50 µL.
if the final volumes are lower than 75 µL or heigher than 300 µL, the ratio can be adapted.
reagents for mobile phases :
Water LC-MS ChromasolvFisher ScientificCatalog #15665350
Acetonitrile ULC-MSBiosolveCatalog #0001204101BS
Formic acid 99.0+% qualite LC/MS optimaFisher ScientificCatalog #10780320
*ES (external standards : mixture of 14 authentic chemical standards covering the mass range of interest):
D-Glucose-1-*13*CMerck MilliporeSigma (Sigma-Aldrich)Catalog #297046
L-Aspartic acid-*15*NMerck MilliporeSigma (Sigma-Aldrich)Catalog #332135
Ethylmalonic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #102687
Amiloride hydrochloride hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7410
prednisoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6254
Metformine (1,1-Dimethylbiguanide hydrochloride)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D150959
AtropineMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0132
ColchicineMerck MilliporeSigma (Sigma-Aldrich)Catalog #C9754
Imipramine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I7379
Alanine-C13Merck MilliporeSigma (Sigma-Aldrich)Catalog #489867
2-aminoanthraceneMerck MilliporeSigma (Sigma-Aldrich)Catalog #A38800
ampicillinMerck MilliporeSigma (Sigma-Aldrich)Catalog #59349
DihydrostreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #D7253
RoxithromycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #R4393
Mobile phases preparation for C18 analysis :
mobile phase A = Water 0.1% HCOOH
add 1 mL formic acid in 1L water
mobile phase B = Acétonitrile 0.1% HCOOH
add 1 mL formic acid in 1L acetonitrile
add a mix mobile phase A / mobile phase B / ES* (90/5/5)
Vortex 5 s
20000 x g, 5°C 15 min
Transfer supernatant into 0.2 mL vials
QC reconstition
According to protein assay, samples are reconstituted with a ratio 1 mg proteins / 50 µL.
For QC reconstitution, final volume = average of all sample volumes
Hilic analysis - reconstitution in 2 steps
C18 analysis - reconstitution in 1 step
Prepare 2-fold, 4-fold and 8-fold diluted QC samples (in mobile phases)
Protocol references
Boudah et al. Annotation of the human serum metabolome by coupling three liquid chromatography methods to high-resolution mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Sep 1:966:34-47. DOI: 10.1016/j.jchromb.2014.04.025