Palaeoproteomics protocol - arid environment samples
Louise Le Meillour
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.
Steps
MilliQ Water 1mL
Acetic Acid 2.8µL
Solutions to prepare before starting
EDTA 500mL 0.5Molarity (M) 7.4
NH4HCO3 200mL 50millimolar (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.
Iodoacetamide solution 1mL 1Molarity (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.
Dithiothreitol (DTT) 1mL 1Mass Percent
Trypsin
According to manufacturer
MilliQ water + Acetic acid 100µL
Trypsin 100µg
Divide into pellets 10µL
Store -20°C
Chemical preparation of samples
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 1mL
Store 4°C
Put on a mixing carousel to allow contact with every “particle” of bone/tooth
Change the solution once. Centrifuge 13400x g
Collect the supernatants in a tube labelled with the sample code.
Homogenise the mixture (Vortex 1min)
Store 4°C
Solubilisation
Add ammonium bicarbonate to each sample 500-300µL 50Molarity (M)
Thermomixer 350rpm
Reduction - Alkylation
For every 100µL of sample, add DTT 1µL 1Molarity (M) (Final concentration ~10mM)
Thermomixer 450rpm
Allow the samples to come to room temperature (on the bench, approx. 30 min)
Add Iodoacetamide solution 1.6µL 1Molarity (M) (final concentration approx. 15mM)
Incubate for 30 min in the dark (protected by foil)
Enzymatic digestion
Add 1µL of trypsin prepared at 1µg/µL to 300 μL of solubilisation solution
ThermoMixer 350rpm
Prepare the formic acid which will be used to stop the digestion:
- Pure formic acid
10µL - MilliQ water
90µL - Adjust the volume to be prepared according to the number of samples
Stop the digestion by adding 1µL of prepared 10% formic acid.
centrifuge ``
Place between 40 and 100 μL of each sample in an insert dedicated to the LC-MS/MS analyses with an electrospray source.
