Feb 14, 2025
DES foodcrust protein extraction protocol
- Joannes Dekker1,2,3,
- Max Ramsøe1
- 1UCPH;
- 2University of York;
- 3University of Turin
Protocol Citation: Joannes Dekker, Max Ramsøe 2025. DES foodcrust protein extraction protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk9r6wv5r/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
We have successfully used this protocol for the extraction of proteins from both experimental and archaeological foodcrusts. However, we acknowledge there may be room for optimisation.
Created: February 04, 2025
Last Modified: February 14, 2025
Protocol Integer ID: 119549
Keywords: Palaeoproteomics, DES, foodcrust, organic residue analysis
Funders Acknowledgements:
EU Horizon 2020 programme
Grant ID: 956351
Abstract
This protocol is an adaptation of the DES protocol developed by Manasji Chowdhury for the extraction of proteins from ceramic. This version has been designed for the extraction of proteins from foodcrust.
The protocol consists of protein extraction from foodcrusts by incubating in a GuHCl and urea DES mixture, followed by reduction and alkylation, C18 StageTip desalting, sequential enzymatic digestion using trypsin and chymotrypsin. Lastly, the samples are cleaned up with Evotips in order to prepare them for loading on a Evosep LC system coupled to a mass spectrometer.
Materials
Equipment:
- Heating block
- Vortex
- Centrifuge
- Speedvac
Reagents:
- Urea
- Guanidine hydrochloride (GuHCl)
- Purified water
- TCEP (Tris-(2-carboxyethyl)-phosphine), concentration: 100 mM
- CAA (Chloroacetamide), concentration: 100 mM
- MeOH (methanol), 100%
- ACN (Acetonitrile)
- TFA (Trifluoroacetic acid), concentration: 0.1% v/v
- ABC (Ammonium bicarbonate), concentration: 50 mM.
- FA (Formic acid), concentration 0.1% v/v
- Trypsin Gold-Mass Spec Grade, 100ugPromegaCatalog #V5280
- Chymotrypsin, Sequencing Grade, 25ugPromegaCatalog #V1061
Consumables:
- C18 StageTips
- Evotips
- Eppendorf protein LoBind tubes, 1.5 mL
DES extraction
DES extraction
5h 35m
5h 35m
To make the DES solution mix urea and GuHCl in a 2:1 molar ratio.
Heat to 75-80°C until a clear liquid is obtained. This can take around an hour. Wait for additional 20 minutes until after it is molten to give it time to mix.
Note: the DES solution will crash out once it cools down. Once cooled, it can be reheated, but making the DES solution fresh is recommended.
1h 20m
Add 300 µL of the DES solution to 10-20 mg foodcrust.
Vortex to mix the DES and the foodcrust, then incubate in a heating block at 65°C for 4 hours.
Note: as the DES solution will rapidly cool down and crash out, it is recommended to preheat the pipette tips and work rapidly when pipetting.
4h
Take the samples off the heating block, add 500 µL of purified water to each sample and vortex to thoroughly homogenize the samples.
Centrifuge for a minimum of 15 minutes at 15000 rcf.
15m
Reduction and alkylation
Reduction and alkylation
10m
10m
Add 15 µL of 100 mM TCEP and 100 mM CAA solution to each sample, then vortex to mix, centrifuge at 15000 rcf for 1 minute and incubate in a heating block at 99°C for 10 min.
10m
C18 StageTip cleaning
C18 StageTip cleaning
7m 30s
7m 30s
Desalting of the samples using C18 StageTips.
Conditions the StageTips by loading each tip with 150 µL 100% MeOH, then centrifuge until all methanol has flowed through. This can usually be achieved by centrifuging at 1000 rcf for 1 minute.
1m
Load 150 µL 80% ACN + 0.1% TFA on the tips and again centrifuge until complete flowthrough is achieved. Typically by centrifuging at 1000 rcf for 2 minutes.
2m
Load 150 µL 0.1% TFA. Centrifuge until completion, typically at 1000 rcf for 2 minutes.
2m
Load 150 µL of one sample, centrifuge at 1000 rcf until completion, then load another 150 µL of the sample and repeat until all the sample has been loaded on the StageTip. This typically requires 6 loadings.
Note: depending on the sample, it may take considerably longer for the sample to be pushed through the StageTip. A single loading can take up to 20 minutes, although a centrifugation time 5-10 frequently sufficed. Ensuring no leftover foodcrust pellet is transferred to the stagetip is imperative to ensure the tips do not block up and the time needed to spin in limited.
Wash the StageTips by loading 150 µL 0.1% TFA andd centrifuging until completion, usually 2:30 minute at 1000 rcf.
Repeat this for a total of 2 washes.
2m 30s
Elute the proteins by loading 50 µL of 50% ACN + 0.1% TFA and centrifuging at 700 rcf for 3 minutes. Ensure to capture the flowthrough as that fraction will contain the extracted proteins.
Digestion
Digestion
1d 12h
1d 12h
Before digestion, dry down the samples to near dry, but still wet, by incubating the samples in a speedvac for 45 min at 45°C. Then, resolubilise the samples in 100 µL 50 mM ABC.
Note: a P1000 pipette tip placed with tip side facing up slots perfectly into a 1.5 mL protein loBind tube allowing for evaporation through the filter while limiting potential contamination during this step
If you wish to use a protein assay, such as a Bradford or BCA assay to quantify the amount of protein in your sample, take a 10 µL aliquot at this step.
For a sequential digestion, first add 1 µL if a 0.4 µg/µL chymotrypsin solution and incubate at 25°C for 18 hours.
18h
Following chymotryptic digestion, add 1 µL of a 0.4 µg/µL trypsin solution and incubate at 37°C for 18 hours.
18h
After finishing the tryptic digestion, centrifuge the samples for 1 minute at 13000 rcf.
Then add 10 µL of 5% v/v TFA to acidify the samples.
Evotip loading
Evotip loading
After digestion we loaded our samples on Evotips in accordance with the Evosep Evotip loading protocol to facilitate loading on the Evosep One LC system. However, this section can easily be replaced by other forms of C18 desalting, such as the earlier described StageTip cleaning, or ZipTips.
Load 20 µL of Solvent B consisting of 0.1% FA in ACN on the Evotips. Then centrifuge for 60 seconds at 700 rcf.
Place the filtered tips of the Evotips in isopropanol until the filters change from ivory white to a pale white, usually for a minimum of 10s.
Note: An easy way to do this is by filling the bottom of an empty pipette tip box with isopropanol and then transferring the insert with the tips.
Load 20 µL of Solvent A consisting of 0.1% FA in water on top of each Evotip, then centrifuge at 700 rcf for 60 seconds.
Load 20 µL of sample onto each Evotip and centrifuge again at 700 rcf for 60 seconds.
Wash the Evotips by loading 20 µL Solvent A consisting of 0.1% FA and centrifuging at 700 rcf for 60 seconds.
Repeat for a total of 3 washes.
Note: A larger volume of wash buffer can be used if the samples are very dirty, just ensure that no flowthrough touch the tips in the bottom of the box
Load 100-150 µL 0.1% FA onto the samples, then centrifuge at 700 rcf for 10 seconds. The solution should now sit directly on top of the filter. The samples are now ready for short term storage.
Note: For longer storage it is important that the filters of the Evotips remain wet. This can be achieved by filling the bottom of the tip box with 0.1% FA so that it touches the filters.
Protocol references
Our DES protocol for protein extraction from foodcrusts is based on the following DES protocol designed for the extraction of proteins from ceramic:
CITATION
For the use of StageTips in protein extraction:
CITATION
LINK
https://doi.org/The loading protocol for Evotips as described by their manufacturer can be found here: https://www.evosep.com/wp-content/uploads/2020/03/Sample-loading-protocol.pdf
Citations
Pal Chowdhury M, Makarewicz C, Piezonka H, Buckley M. Novel Deep Eutectic Solvent-Based Protein Extraction Method for Pottery Residues and Archeological Implications.
https://doi.org/10.1021/acs.jproteome.2c00340Rappsilber J, Mann M, Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.
https://doi.org/Acknowledgements
We would like to thank Dr. Manasji Chowdhury for his helpful feedback in applying DES.