Jun 10, 2025

Public workspaceExtraction Protocol for untargeted LC-MS/MS - Microbial Cultures

DOI
https://dx.doi.org/10.17504/protocols.io.ewov19417lr2/v1
  • Mauricio Caraballo1
  • 1University of California San Diego
Mauricio Caraballo
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DOI: https://dx.doi.org/10.17504/protocols.io.ewov19417lr2/v1
Protocol Citation: Mauricio Caraballo 2025. Extraction Protocol for untargeted LC-MS/MS - Microbial Cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov19417lr2/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
This is a working protocol and can be adapted to your purposes.
Created: April 24, 2024
Last Modified: June 10, 2025
Protocol Integer ID: 98893
Keywords: Microbial culture, Metabolomics, LC-MS/MS, extraction of bacterial small molecule, microbial cultures this protocol, extraction protocol for untargeted lc, microbial culture, bacterial small molecule, extraction protocol, extraction, m, untargeted lc
Funders Acknowledgements:
Gordon and Betty Moore Foundation
Grant ID: GBMF12120
Abstract
This protocol details the extraction of bacterial small molecules for untargeted LC-MS/MS.
Guidelines
Definitions (optional)
Metadata:
Sample information.

Untargeted LC-MS/MS:
Liquid chromatography tandem mass spectrometry, not targeting specific molecules.
Materials
ReagentWater Optima™ LC/MS Grade Fisher Chemical™Fisher ScientificCatalog #W6-4 ReagentMethanol Optima™ LC/MS Grade Fisher ChemicalFisher ScientificCatalog #A456-4 Extraction solvent: 50% methanol → 50:50 v/v of LC grade methanol/ LC grade water

Troubleshooting
Metadata - sample information
Prepare metadata table according to ReDU2-MS2 Sample Information Template [for more information about the ReDu, see the Preprint from our lab: Repository-scale Co- and Re-analysis of Tandem Mass Spectrometry Data as well as the ReDu platform].
Fill the metadata table with all the applicable information. Information from LC-MS/MS can be included later on after data has been acquired.
Specifically, from each sample we will need at least the NCBITaxonomy (e.g. 1313|Streptococcus pneumoniae).
Additional columns can be created to include information related to the specificity of the experiments (e.g. microbial interactions: co-cultures vs monocultures).
Sample preparation and microbial extraction
1h 45m

Note
Exometabolome: extraction procedure performed on supernatant from microbial cultures [See Pinu & Villas-Boas, 2017].

Briefly, for liquid cultures:
Separate microbial culture from cells either by centrifugation or filtration.
Add internal standard to monitor extraction procedure (although it can be optional, the addition of a known compound will be useful to guarantee the efficiency of the extraction procedure).
Quenching (deactivate enzymatic activity) by freeze-drying or any other drying process (such as vacuum-drying): at this step, samples can be shipped to us as they are dry;
If not quenching but direct extraction is performed, it can be done via liquid-liquid extraction or SPE (solid-phase extraction) [the extracted metabolites will depend on the sorbent material used in SPE].
For semi-solid cultures (eg. containing agar), the following protocol can be used:

Note
If the culture is performed using 96-deep well plates, then the extraction can be performed using a 96-well SPE plate.

After the incubation period freeze/thaw the plate (10-15mins, 3 times) to lyse the cells.

  • After the incubation period, freeze/thaw the plate (Duration00:10:00 -Duration00:15:00 ) to lyse the cells (1/3).
  • After the incubation period, freeze/thaw the plate (Duration00:10:00 -Duration00:15:00 ) to lyse the cells (2/3).
  • After the incubation period, freeze/thaw the plate (Duration00:10:00 -Duration00:15:00 ) to lyse the cells (3/3).
1h 15m
Add Amount1 mL methanol and sonicate (Duration00:15:00 ).

15m
Centrifuge (Centrifigation865 x g, 00:15:00 ) to separate cell pellets from supernatant
15m
Centrifigation
Transfer the supernatant to a new 96-well plate and store at Temperature-80 °C if the extraction will not be performed immediately. Also, if the cell pellets will be extracted separately, they can be stored at Temperature-80 °C until extraction.
Temperature
Prepare the 96-well SPE plate before performing the extraction. See the SPE steps described below.
The following are the steps for SPE:
Note
Specific protocols can vary from providers, so follow recommendations from the providers of the purchased product: Phenomenex, Sigma-Aldrich or Waters.

Briefly, the steps are:
Condition the cartridge with methanol (Amount2 mL ).
Equilibrate with a similar solvent as the sample. In this case, water (Amount2 mL ) as the supernatant is aqueous.
Load the sample (Amount1 mL of supernatant is fine for a 100mg SPE cartridge).
Wash the sample (Amount1 mL of water).
Elute (Amount1 mL of methanol).
Optional: recondition cartridges for another SPE.
Now the extracts (Amount1 mL of methanol containing the extracted metabolites) are in a 96-deep well plate.
You can dry them under vacuum. Dried samples will be suitable for shipping to the Dorrestein Lab.

Note
Endometabolome: extraction procedure performed on filtered microbial cells (pellets) [For optional protocols, such as for B. subtilis, see Meyer et al., 2013. This review Pinu et al., 2017 might be also helpful];

Include, together with the samples, blanks of the culture media used in each experiment (extracted under exact conditions as the exometabolome).
At least biological replicates (3-6) from each sample. We run technical replicates for each sample. Ship dry extracts if possible.
If shipping standards together with the microbial extracts is possible, please include them as well as their information in the metadata table.
If possible, use Deepwell Plates, 96-well, Polypropylene, 2000µL [e.g. E951033600]. Include the map of the samples’ name/label.
Protocol references
Acknowledgements
This work was supported by the Gordon and Betty Moore Foundation, GBMF12120 and https://doi.org/10.37807/GBMF12120.