Dec 10, 2025
Untargeted Metabolomics : Extraction of Cell Metabolites
This protocol is a draft, published without a DOI.
- Emeline CHU-VAN1,
- florence castelli1
- 1CEA
- MetaboHub-IDF
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 Cell Metabolites. protocols.io https://protocols.io/view/untargeted-metabolomics-extraction-of-cell-metabol-f7rubrm6x
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: April 29, 2025
Last Modified: December 10, 2025
Protocol Integer ID: 192020
Keywords: METABOHUB , large cohorts, human health, biomarker discovery, untargeted metabolomic, extraction of cell metabolite, internal metabolite standards in the extraction process, cell metabolome, internal metabolite standard, accurate comparison of relative metabolite abundance, coverage of cell metabolome, cell metabolite, relative metabolite abundance, methanolic protein precipitation from cell pellet, controlled extraction process, extraction process, extraction, methanolic protein precipitation, cell pellet, samples within the large cohort
Funders Acknowledgements:
MetEx+
Grant ID: 21-ESRE-0035
METABOHUB
Grant ID: 11-INBS-0010
Abstract
The extraction of cell metabolites for untargeted metabolomics involves methanolic protein precipitation from cell pellets, aiming to maximize coverage of cell 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
DinosebMerck MilliporeSigma (Sigma-Aldrich)Catalog #45453
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
Pierce BCA protein assayThermo ScientificCatalog #23227
ampicillinMerck MilliporeSigma (Sigma-Aldrich)Catalog #59349
DihydrostreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #D7253
D-Glucose-1-*13*CMerck MilliporeSigma (Sigma-Aldrich)Catalog #297046
Amiloride hydrochloride hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7410
ColchicineMerck MilliporeSigma (Sigma-Aldrich)Catalog #C9754
Imipramine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I7379
Alanine-C13Merck MilliporeSigma (Sigma-Aldrich)Catalog #489867
Ammonia solution 25% for HPLMerck MilliporeSigma (Sigma-Aldrich)Catalog #5438300100
L-Aspartic acid-*15*NMerck MilliporeSigma (Sigma-Aldrich)Catalog #332135
Metformine (1,1-Dimethylbiguanide hydrochloride)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D150959
AMMONIUM CARBONATE, 99.999%Merck MilliporeSigma (Sigma-Aldrich)Catalog #379999
2-aminoanthraceneMerck MilliporeSigma (Sigma-Aldrich)Catalog #A38800
Acetonitrile ULC-MSBiosolveCatalog #0001204101BS
prednisoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6254
AtropineMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0132
RoxithromycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #R4393
Ethylmalonic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #102687
Formic acid 99.0+% qualite LC/MS optimaFisher ScientificCatalog #10780320
Troubleshooting
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
Store at -80 °C until extraction
Sample extraction
Extraction for "cellules adhérentes"
Note
Samples were first extracted directly into the flasks, then scraped and transferred to tubes.
The volume of extraction solvent is 3 mL per sample.
Store at -80 °C until using
extraction solvent :
30% H2O
70% MeOH containing Internal standards at 37.5 ng/mL
Water LC-MS ChromasolvFisher ScientificCatalog #15665350
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
Thaw samples
Vortex 5 s
2900 x g, 4°C 10 min
Transfer supernant for Hilicneg - C18pos analysis and QCpool
1 mL
/!\ Keep pellets for future protein assay (BCA)
Aliquoting QCpool : 1 mL (or more) depending on available volume
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
Extraction for "cellules non adhérentes"
Thaw samples (pellets)
Add 170 µL of Water
Water LC-MS ChromasolvFisher ScientificCatalog #15665350
Scrap samples to put them into suspension
Sonicate samples with :
Equipment
Vibracell VCX 130
NAME
Ultrasonic processor
TYPE
Sonics
BRAND
432-0135
SKU
With probe 2 mm during 10 s at 40% ampl
Transfer 20 µL of homogenate in Lobind tube for future protein assay (BCA)
Add 350 µL of MeOH with internal standards (cf step3) to 150 µL remaining volume
Vortex 5s
Incubate on ice 90 min until complete protein precipitation
20000 x g, 4°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)
- In case of "cellules adhérentes" samples : reconstitute pellets in 150 µL of water (sonication step if needed) and process protein assay.
- In case of "cellules non adhérentes" samples : process to protein assay directly.
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 = add 40% (of final volume) of mobile phase A containing ES* 2.5x
Vortex 5 s
second step = add 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 for LC-MS analysis.
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 5s
20000 x g, 4°C 15 min
Transfer supernatant into 0.2 mL vials for LC-MS analysis.
QC reconstitution
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