Nov 08, 2024
Extraction Protocol for untargeted LC-MS/MS - Animal tissues
- Mauricio Caraballo1,
- Marwa Tawfik1,
- Janet Tseng1,
- Monica Wu1
- 1University of California San Diego
- Mauricio Caraballo: MSCollaboratory Director
- Marwa Tawfik: MSCollaboratory visitor
- Janet Tseng: undergraduate student
- Monica Wu: Undergraduate student
Protocol Citation: Mauricio Caraballo, Marwa Tawfik, Janet Tseng, Monica Wu 2024. Extraction Protocol for untargeted LC-MS/MS - Animal tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8x8blmk/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: In development
We are still developing and optimizing this protocol
Created: March 19, 2024
Last Modified: November 08, 2024
Protocol Integer ID: 96958
Keywords: Metabolomics, Untargeted LC-MS/MS
Funders Acknowledgement:
Gordon and Betty Moore Foundation
Grant ID: GBMF12120
Abstract
This protocol details the preparation of animal tissue samples for untargeted metabolomics analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). It covers sample collection, homogenization, metabolite extraction, and preparation for LC-MS/MS analysis.
Protocol materials
Water, Optima™ LC/MS Grade, Fisher Chemical™Fisher ScientificCatalog #W64
Step 2
Methanol, Optima™ LC/MS Grade, Fisher ChemicalFisher ScientificCatalog #A456-4
Step 2
Description
Description
This protocol details the preparation of animal tissue samples for untargeted metabolomics analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). It covers sample collection, homogenization, metabolite extraction, and preparation for LC-MS/MS analysis. This protocol can be adapted to other sample types. This is not a final document, not peer-reviewed and it can be updated.
Solvents
Solvents
Water, Optima™ LC/MS Grade, Fisher Chemical™Fisher ScientificCatalog #W64
Methanol, Optima™ LC/MS Grade, Fisher ChemicalFisher ScientificCatalog #A456-4
Note
Additionally, prepare the following solutions:
- Extraction solvent: 50% methanol:water → 50:50 v/v of LC grade methanol/ LC grade water.
- Resuspension solvent (50:50 v/v methanol:water) containing 1uM sulfadimethoxine (CAS 122-11-2) as an internal standard.
Equipment
Equipment
Equipment
TissueLyser II
NAME
Bead Mill
TYPE
QIAGEN
BRAND
85300
SKU
LINK
Equipment
Legend RT
NAME
Refrigerated Benchtop Centrifuge
TYPE
Sorvall
BRAND
sorvall legend rt
SKU
LINK
Equipment
Centrifuge 5418
NAME
Centrifuge
TYPE
Eppendorf US
BRAND
Catalog No. 5401000137
SKU
LINK
Equipment
Sonicator
NAME
Branson
BRAND
B2510
SKU
Consumables
Consumables
2 mL Qiagen sample tubes (catalog no. 990381)
Stainless Steel Beads (Qiagen, Inc., 5 mm diameter, Cat. No. NC9257481)
Shallow polypropylene 96-well plate (Cat. No. 951040048)
Deep polypropylene 96-well plate (Cat. No. 951033405)
Seal plates (Cat. No. ZAFPE50) Note: for LC-MS/MS acquisition
AlumaSeal CS (Sealing Foil, Excel Scientific) (Cat. No. FC100) Note: for long time storage
Metal tweezers
Bead dispenser
Note
Wash the stainless steel beads with LC-MS grade water followed by LC-MS methanol in a glass Petri dish then allow to dry overnight.
Sample description
Sample description
Liver, stomach and intestine preserved in Ethanol 95% from fish (threespine stickleback, Gasterosteus aculeatus).
Note
Worms (fish parasite) can be extracted following this protocol
Note
In case samples were put in tubes without ethanol: Shake (vortex can be used) the tube without touching/manipulation of the samples and transfer to a new empty tube.
Note
Transferring beads from the Petri dish or big beaker to the beads dispenser using aluminum foil to cover either of them and to create a small mouth to either the big beaker or the Petri dish to avoid falling of beads.
Sample preparation
Sample preparation
Samples are received in plastic centrifugation tubes containing 1mL of 95% Ethanol.
Note
If ink labels are inside the tube, carefully remove them using tweezers disinfected with methanol every time. Make sure the tube is labeled from the outside via sticker or marker.
Dry samples using Centrivap.
Label empty sample tube.
Add 1 bead with the beads dispenser into each empty sample tube.
Add 1 mL of extraction solvent (50% methanol:water) per tube.
Homogenize at 30 Hz for 8 min in a Qiagen TissueLyzer II.
Allow to extract at 4 °C for 1 h.
Note
This step helps with protein precipitation
Centrifuge at 14000 rpm, 4°C, 00:15:00
15m
Transfer at total of 800 uL of supernatant split in 2 labeled deep polypropylene 96-well plates (400 uL for each plate).
Note
- Label plates before transferring any sample.
- One of the plates will be used as a backup plate (storage under -80 °C).
Dry plates using Centrivap.
Seal plates and store under -80 °C until LC–MS/MS analysis.
Prior to LC-MS/MS analysis, reconstitute one of the plates with 200uL of resuspension solvent (50% methanol/water solution containing 1uM sulfadimethoxine as an internal standard).
Seal plate and sonicate 00:15:00
15m
Hand-shake or vortex
Note
This guarantees complete solubilization of the sample
Transfer to a shallow polypropylene 96-well plate
Note
If particles or turbid solution are observed, centrifuge plate 2000 rpm, 4°C, 00:15:00 and transfer to a new shallow polypropylene 96-well plate.
This shallow polypropylene 96-well plate will be used for LC-MS/MS acquisition.
References
References
Acknowledgements
This work was supported by the Gordon and Betty Moore Foundation, GBMF12120
and https://doi.org/10.37807/GBMF12120.