Mar 25, 2025
Version 2
Untargeted Metabolomics: Extraction of Plasma Metabolites V.2
- 1CEA
- MetaboHub-IDF
External link: https://joliot.cea.fr/drf/joliot/Pages/Entites_de_recherche/medicaments_technologies_sante/SPI/lims.aspx
Protocol Citation: florence castelli, emeline.chu-van CHU-VAN 2025. Untargeted Metabolomics: Extraction of Plasma Metabolites . protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4qo18vo5/v2Version created by emeline.chu-van CHU-VAN
Manuscript citation:
N/A
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: February 19, 2024
Last Modified: March 25, 2025
Protocol Integer ID: 124951
Keywords: METABOHUB, large cohorts, Human health, Biomarker discovery
Funders Acknowledgements:
METABOHUB
Grant ID: 11-INBS-0010
MetEx+
Grant ID: 21-ESRE-0035
Disclaimer
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Abstract
The extraction of plasma metabolites for untargeted metabolomics involves methanolic protein precipitation from a fixed volume of fasting plasma, aiming to maximize coverage of the plasma 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.
Image Attribution
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Guidelines
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Protocol materials
ColchicineMerck MilliporeSigma (Sigma-Aldrich)Catalog #C9754
In 2 steps
ampicillinMerck MilliporeSigma (Sigma-Aldrich)Catalog #59349
In 2 steps
Formic acid 99.0+% qualite LC/MS optimaFisher ScientificCatalog #10780320
In 2 steps
D-Glucose-1-*13*CMerck MilliporeSigma (Sigma-Aldrich)Catalog #297046
In 2 steps
Ammonia solution 25% for HPLMerck MilliporeSigma (Sigma-Aldrich)Catalog #5438300100
In 2 steps
Ethylmalonic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #102687
In 2 steps
Metformine (1,1-Dimethylbiguanide hydrochloride)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D150959
In 2 steps
AtropineMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0132
In 2 steps
RoxithromycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #R4393
In 2 steps
2-methyl-4-chlorophenoxyacetic acid (MCPA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #45555
Step 10
Alanine-C13Merck MilliporeSigma (Sigma-Aldrich)Catalog #489867
In 2 steps
Acetonitrile ULC-MSBiosolveCatalog #0001204101BS
In 2 steps
Imipramine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I7379
In 2 steps
2-aminoanthraceneMerck MilliporeSigma (Sigma-Aldrich)Catalog #A38800
In 2 steps
prednisoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6254
In 2 steps
Methanol LC-MS CHROMASOLVFisher ScientificCatalog #15614740
Step 10
water LC-MS ChromasolvFisher ScientificCatalog #15665350
In 2 steps
Amiloride hydrochloride hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7410
In 2 steps
DihydrostreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #D7253
In 2 steps
L-Aspartic acid-*15*NMerck MilliporeSigma (Sigma-Aldrich)Catalog #332135
In 2 steps
DimetridazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #31707
Step 10
2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A0326
Step 10
AMMONIUM CARBONATE, 99.999%Merck MilliporeSigma (Sigma-Aldrich)Catalog #379999
In 2 steps
DinosebMerck MilliporeSigma (Sigma-Aldrich)Catalog #45453
Step 10
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
Randomisation
Reorganize plasma samples in random order on dry ice
Store at -80 °C until aliquoting
Sample aliquoting and quality control sample constitution
Sample aliquoting and quality control sample constitution
Thaw plasma samples (keep them fresh on ice)
Aliquoting : 50µL (or 25µL) depending on available volume
Create a quality control (QC) : pool of a same volume (~20µL) of all samples
Aliquoting QC : 50µL (or more) depending on available volume
Store at -80 °C until sample extraction
Sample extraction
Sample extraction
Thaw plasma samples (keep them fresh on ice)
Performe metabolite extraction twice (HILIC and C18) from 25µL or 50µL of plasma
Add 4 volumes (100µL or 200 µL) of cold MeOH containing internal standards (IS) at 37.5 ng/mL
extraction solvent :
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 00:00:05
5s
Incubate on ice 01:30:00 until complete protein precipitation
1h 30m
20000 x g, 4°C, 00:15:00
15m
Collect supernatants
Dry supernatants under a nitrogen stream using a
Equipment
TurboVap LV
NAME
Evaporator
TYPE
BIOTAGE
BRAND
TurboVap® LV
SKU
30 °C
Store at -80 °C until analysis
sample reconstitution
sample reconstitution
15m 5s
15m 5s
Resuspend dried extracts in 3 volumes of Ammonium carbonate 10mM pH10.5 (with Ammonia solution 25%)+ ES* / Acetonitrile (40:60, v/v) for ZIC-pHILIC analysis
or ultrapure water / acetonitrile (95:5, v/v), containing 0.1% formic acid and ES* for C18 analysis.
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
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
Vortex 00:00:05
5s
20000 x g, 4°C, 00:15:00
15m
Transfer supernatant into 0.2 mL vials
QC recontitution
QC recontitution
15m 5s
15m 5s
Resuspend dried extracts in 3 volumes of Ammonium carbonate 10mM pH10.5 (with Ammonia solution 25%)+ ES* / Acetonitrile (40:60, v/v) for ZIC-pHILIC analysis
or ultrapure water / acetonitrile (95:5, v/v), containing 0.1% formic acid and ES* for C18 analysis.
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
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
Vortex 00:00:05
5s
20000 x g, 4°C, 00:15:00
15m
Transfer supernatant into 0.2 mL vials
Prepare 2-fold, 4-fold and 8-fold diluted QC samples
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