Nov 09, 2023
Palaeoproteomics protocol - arid environment samples
- Louise Le Meillour1
- 1Muséum national d'Histoire naturelle
- Metabolomics Protocols & WorkflowsTech. support email: [email protected]
Protocol Citation: Louise Le Meillour 2023. Palaeoproteomics protocol - arid environment samples. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmn1kog3p/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. It has been developed as part of a palaeoproteomics study (Le Meillour et al. 2018) of African zooarchaeological remains.
Created: June 23, 2022
Last Modified: November 09, 2023
Protocol Integer ID: 65160
Keywords: protocols for protein extraction, skeletal remain, proteomic, protein extraction, vertebrates in archaeological context, extracted protein, proteomic approach, integrity of bone protein, bone protein, preserved bone, palaeoproteomics protocol, protein, taxonomic identification, palaeoproteomic, mass spectrometry, chronological repartitions of mammal, collagen content, successful detection of peptide, preservation of the organic phase, trypsin digestion, contrasted collagen content, vertebrate, archaeological context, arid environment, unidentified splinter, involving trypsin digestion, old site of toteng
Funders Acknowledgements:
Muséum national d'Histoire naturelle
Grant ID: ATM ProtéArch
Agence Nationale de la Recherche
Grant ID: ANR-10-LABX-0003-BCDiv
Abstract
The analysis of the skeletal remains of vertebrates in archaeological contexts provides information about human-animal relationship and their environment. Their taxonomic identification based on macroscopic observation is not always possible due to fragmentation and poor preservation. In recent years, proteomics has emerged as an alternative but there is clearly a lack of data in arid environment where diagenesis rapidly affects the integrity of bone proteins. Here, we report the efficiency of three protocols for protein extraction. The protocols used harsh (1 M HCl and 0.6 M HCl) and soft (Tris-EDTA) decalcification agents and were tested on unidentified splinters from the 2000 years-old site of Toteng, Botswana. The preservation of the organic phase was first estimated using attenuated total reflectance Fourier transform infrared spectroscopy and a set of samples with contrasted collagen contents were selected for palaeoproteomics. The extracted proteins were submitted to a bottom-up proteomic approach involving trypsin digestion followed by ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS). Our results identify Tris-EDTA buffer as the most suitable decalcification protocol for poorly preserved bones and propose a collagen content threshold of ~3% weight content for successful detection of peptides. This approach, combined with biogeographical and chronological repartitions of mammals in Africa allows refining taxonomic attributions for four out of nine splinters, leading to species identification. Data are available via ProteomeXchange with identifier PXD010725.
Materials
Solutions: (steps to make them in the protocol)
- EDTA Buffer 0.5M
- NH4HCO350mM
- Iodoacetamide 1M (/!\ protect from light)
- DTT 1M (/!\ highly toxic)
- Trypsin 1µg/µL
- 10% Formic acid
Troubleshooting
Safety warnings
DTT Highly toxic, need to be under fume hood to handle it
Iodoacetamide needs to be protected from light
Solutions to prepare before starting
2h
EDTA 500 mL 0.5 Molarity (M) 7.4
Safety information
Under a fume hood
Note
pH meter in the solution and bar magnet
1h
NH4HCO3 200 mL 50 millimolar (mM)
Weigh 0.7906 g of ammonium bicarbonate (Sigma Aldrich). Add the weighed powder to 100 mL of milliQ water. Shake manually until the crystals are dissolved. Make up to 200 mL. Store at room temperature.
20m
Iodoacetamide solution 1 mL 1 Molarity (M)
Weigh 184.9 mg of iodoacetamide and add to 1 mL of milliQ water in a tube covered with foil or brown microtube. Shake manually until the crystals are dissolved. Can be stored at -20°C.
10m
Dithiothreitol (DTT) 1 mL 1 Mass Percent
10m
Trypsin
According to manufacturer
10m
MilliQ water + Acetic acid 100 µL
Trypsin 100 µg
Note
Mix in the tube by ups and downs
Divide into pellets 10 µL
Store -20 °C
Safety information
Do not thaw the trypsin more than 3 times
Chemical preparation of samples
1d
Sampling
Weigh empty tubes
Prepare paper and EtOH for cleaning between samples
Use ultrasounds to clean diamond head of dremel in the end
Decalcification
Add EDTA to each sample 1 mL
Store 4 °C
Put on a mixing carousel to allow contact with every “particle” of bone/tooth
Change the solution once. Centrifuge 13400 x g, 00:01:00
Collect the supernatants in a tube labelled with the sample code.
Homogenise the mixture (Vortex 1min)
Store 4 °C
Note
Decalcification is completed when only a bone phantom remains (should resemble wet cotton candy)
1m
Solubilisation
Add ammonium bicarbonate to each sample 500-300 µL 50 Molarity (M)
Thermomixer 350 rpm, 67°C, 03:00:00
Reduction - Alkylation
For every 100µL of sample, add DTT 1 µL 1 Molarity (M) (Final concentration ~10mM)
Thermomixer 450 rpm, 50°C, 01:00:00
Allow the samples to come to room temperature (on the bench, approx. 30 min)
Add Iodoacetamide solution 1.6 µL 1 Molarity (M) (final concentration approx. 15mM)
Incubate for 30 min in the dark (protected by foil)
Enzymatic digestion
Add 1 µL of trypsin prepared at 1 µg/µL to 300 μL of solubilisation solution
ThermoMixer 350 rpm, 37°C, 18:00:00 Trypsin can act in only 3 to 5h
Prepare the formic acid which will be used to stop the digestion:
- Pure formic acid 10 µL
- MilliQ water 90 µL
- Adjust the volume to be prepared according to the number of samples
Stop the digestion by adding 1 µL of prepared 10% formic acid.
centrifuge , 00:10:00
10m
Place between 40 and 100 μL of each sample in an insert dedicated to the LC-MS/MS analyses with an electrospray source.
Protocol references
Le Meillour, L., Zazzo, A., Lesur, J., Cersoy, S., Marie, A., Lebon, M., ... & Zirah, S. (2018). Identification of degraded bone and tooth splinters from arid environments using palaeoproteomics. Palaeogeography, Palaeoclimatology, Palaeoecology, 511, 472-482.