Sep 19, 2024

Public workspacePlasma/serum sample preparation for untargeted MS-based metabolomic

This protocol is a draft, published without a DOI.
  • 1Université Clermont Auvergne, INRAE, UNH, Plateforme d’Exploration du Métabolisme, MetaboHUB Clermont, Clermont-Ferrand, France;
  • 2INRAE
  • MetaboHUB-Clermont PFEM
Estelle Pujos-Guillot
Icon indicating open access to content
QR code linking to this content
External link: https://eng-pfem.isc.inrae.fr/
Protocol Citation: Carole MIGNÉ, Charlotte JOLY, Stéphanie DURAND, Estelle Pujos-Guillot 2024. Plasma/serum sample preparation for untargeted MS-based metabolomic. Protocol exchange https://protocols.io/view/plasma-serum-sample-preparation-for-untargeted-ms-c263yhgn
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: In development
We are still developing and optimizing this protocol
Created: October 09, 2023
Last Modified: September 19, 2024
Protocol Integer ID: 89019
Keywords: serum, plasma, preparation, untargeted metabolomic analysis, mass spectrometry
Funders Acknowledgement:
Agence Nationale de la Recherche
Grant ID: ANR-11-INBS-0010 MetaboHUB
Agence Nationale de la Recherche au titre de France 2030
Grant ID: ANR- 21-ESRE-0035
Abstract
The following protocol describes the preparation of serum and plasma samples for non-targeted analysis by mass spectrometry coupled to liquid or gas chromatography, as proposed by the PFEM (MetaboHUB-Clermont).
Protocol materials
ReagentULC/MS grade methanolBiosolveCatalog #0013684101BS
Step 5
ReagentMilliQ water
In 2 steps
Sample preparation
Sample preparation
40m
40m
Thaw the samples according to these conditions depending on the analysis to be carried out :

LCMSGCMS
Room temperature4°C overnight
Samples thawing conditions


Add Amount100 µL of serum or plasmaSamplesample in Eppendorf tubes.

Prepare blank sample for GCMS analysis : add Amount100 µL of ReagentMilliQ waterContributed by users in Eppendorf tube

Prepare Amount2 mL of an internal standard solution [13C1]-L-valine (Concentration0.2 mg/mL ) in ReagentMilliQ waterContributed by users , store at Temperature-20 °C for GCMS analysis

Extract protein by adding ice-cold methanol to each sample under the following conditions :ReagentULC/MS grade methanolBiosolveCatalog #0013684101BS


MatrixVol MeOH LCMSVol MeOH GCMS
blankNA200 µL (plasma) 400 µL (serum)
plasma200 µL200 µL
serum200 µL400 µL
Methanol volume to be added to extract proteins

Mix and placed the samples at Temperature-20 °C for Duration00:30:00 .

30m
Centrifuge samples Centrifigation15493 x g, 4°C, 00:10:00 (Sigma 3-16PK, Fischer Bioblock Scientific).

The supernatant is divided into three aliquots. A intermediate pooled QC sample is prepared by mixing Amount10 µL from each extracted samples (per day or per batch).

Sample Type Vol supernatant LCMS (µL) Vol supernatant Plasma GCMSVol supernatant Serum GCMS (µL)Vol internal standard GCMS (µL)
Pool QC45200NANA
Safety 4520030010
Sample4520030010
BlankNA200 (milliQ water)300 (milliQ water)10
Distribution of extracted samples


10m
Dry samples completely using a EZ2.3 genevac system (Biopharma Technologies France).

Equipment
new equipment
NAME
Genevac EZ-2
BRAND
EZ-2
SKU
https://www.spscientific.com/Products/Centrifugal_Evaporators___Sample_Concentrators/Genevac/EZ-2_Series/EZ-2_Series/
SPECIFICATIONS
The following program is used:
ALCMSGCMS
ProgramHPLCHPLC
Final Stage 10 min50 min
Reduce Odour OffOff
Lamp OffOn (under 30°C)
Drying program used

Store samples for LCMS analysis at Temperature-80 °C until further analysis

Derivatisation for GCMS analysis :
Prepare a fresh methoxylamine solution (Concentration15 mg/mL ) in pyridine. The volume of this solution depends on the number of samples to be analysed (Amount80 µL for each samples)

Dissolve the dry samples in adding Amount80 µL of methoxylamine solution to each vial.

Vortex vigorously for 1 min and incube at Temperature37 °C for Duration24:00:00 (in order to inhibit the cyclization of reducing sugars and the decarboxylation of α-keto acids).

1d
Add Amount80 µL of BSTFA (1%TMCS) in the mixture and derivatize at Temperature70 °C for Duration01:00:00

1h
Waiting a few minutes and transfer Amount50 µL of derivatized mixture in a glass vial containing Amount100 µL of heptane prior to injection.
Store blank and biological samples at Temperature4 °C until further analysis
As well as a sample pool is formed from Amount10 µL of each extracted and derivatized sample to monitor the drift of the spectrometer during GCMS analysis.
Transfer Amount50 µL of sample pool in a glass vial containing Amount100 µL of heptane prior to injection, repeat this step 8 times (preprocessing analysis)
Store pool samples at Temperature4 °C until further analysis
Protocol references

CITATION
Pereira, H., Martin, JF., Joly, C. et al. (2009). Development and validation of a UPLC/MS method for a nutritional metabolomic study of human plasma. Metabolomics .
LINK
https://doi.org/10.1007/s11306-009-0188-9

Citations
Pereira, H., Martin, JF., Joly, C. et al. . Development and validation of a UPLC/MS method for a nutritional metabolomic study of human plasma
https://doi.org/10.1007/s11306-009-0188-9