Dec 02, 2024
Neutralisation acid etching of enamel for proteomic amelogenin sex estimation
- 1University of York
Protocol Citation: Charllotte Blacka 2024. Neutralisation acid etching of enamel for proteomic amelogenin sex estimation. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn6qr6l5d/v1
Manuscript citation:
Blacka, C, Dowle, A, Lisowski, M, Alexander, M, Hendy, J, Penkman, K and Mosely, J. 2024. “Rapid Amelogenin Sex Estimation of Human and Cattle Remains Using Untargeted Evosep-timsTOF Mass Spectrometry.” Authorea Inc. https://doi.org/10.22541/au.172599306.61984507/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 use this protocol and it's working
Created: September 20, 2024
Last Modified: December 02, 2024
Protocol Integer ID: 108103
Keywords: Amelogenin, Palaeoproteomics, Proteomics, Acid Etching, Enamel, Sex Estimation, Sex Determination
Funders Acknowledgements:
Natural Environment Research Council
Grant ID: NE/S00713X/1
Abstract
Minimally invasive and rapid sampling of enamel proteins for sex estimation via mass spectrometric analysis of sexually dimorphic amelogenin proteins is becoming increasingly important in bioarchaeological studies. Here, an acid etching method followed by neutralisation by base addition to the acid etch, and subsequent Evotip clean-up, is outlined.
Materials
(Optional) 3% H2O2
5% HCl
5% NH4OH
0.2 mL PCR tube
LoBind tubes
Evotips
Acid Etch Preparation
Acid Etch Preparation
If tooth is visibly dirty, it can be rinsed briefly with 3% H2O2 (v/v) in water.
If there is no exposed enamel to sample, the tooth can be drilled, sanded or etched with 5% HCl for a few minutes to expose enamel for acid etch sampling.
Pipette 60 μL of 5% HCl into the cap of a 0.2 mL PCR tube, forming a convex meniscus.
Place the exposed enamel of the tooth in contact with the 5% HCl (v/v) in water and maintain contact for 2 minutes.
2m
Close the cap of the 0.2 mL PCR tube to transfer the 5% HCl (v/v) in water into the tube, and pipette 45 μL of 5% NH4OH (v/v) in water into the PCR tube. A white precipitate should form upon the addition of the NH4OH.
Vortex/mix the sample for ~5 seconds.
5s
Centrifuge the sample at 13,250 rcf for 3 minutes.
3m
Transfer the supernatant to a 0.5 mL LoBind tube and store in the freezer until Evotip clean-up.
Evotip Clean-Up
Evotip Clean-Up
6m 30s
6m 30s
Wash dry Evotips with 20 μL of solvent B (acetonitrile containing 0.1% (v/v) formic acid) and centrifuge for 60 seconds at 800 rcf.
1m
Soak the Evotips in 100 μL of 1-propanol until the tips are pale white (approximately 20 seconds).
20s
Equilibrate the Evotips with 20 μL of solvent A (aqueous 0.1% (v/v) formic acid) and centrifuge for 60 seconds at 800 rcf.
1m
Transfer the full acid etch supernatant to the Evotip and centrifuge for 60 seconds at 800 rcf.
1m
Wash the tips with 50 μL of solvent A and centrifuge for 60 seconds at 800 rcf.
1m
Repeat step 13 twice more.
2m
Transfer 100 μL of solvent A to the tips and centrifuge for 10 seconds at 800 rcf, ensuring the tips stay wet.
10s
Load the Evotips onto the Evosep LC.
Protocol references
Blacka, C, Dowle, A, Lisowski, M, Alexander, M, Hendy, J, Penkman, K and Mosely, J. 2024. “Rapid Amelogenin Sex Estimation of Human and Cattle Remains Using Untargeted Evosep-timsTOF Mass Spectrometry.” Authorea Inc. https://doi.org/10.22541/au.172599306.61984507/v1.
Stewart NA, Molina GF, Issa JPM, et al. The identification of peptides by nanoLC-MS/MS from human surface tooth enamel following a simple acid etch extraction. RSC Adv. 2016;6(66):61673-61679. doi:10.1039/C6RA05120K
Stewart NA, Gerlach RF, Gowland RL, Gron KJ, Montgomery J. Sex determination of human remains from peptides in tooth enamel. Proc Natl Acad Sci U S A. 2017;114(52):13649-13654. doi:10.1073/pnas.1714926115