Increasing sustainability in palaeoproteomics by optimizing digestion times for large-scale archaeological bone analyses

Palaeoproteomic analysis of skeletal proteomes is used to provide taxonomic identifications for an increasing number of archaeological specimens. The success rate depends on a range of taphonomic factors and differences in the extraction protocols employed. By analyzing 12 archaeological bone specimens from two archaeological sites, we demonstrate that reducing digestion duration from 18 to 3 hours has no measurable impact on the obtained taxonomic identifications. Peptide marker recovery, COL1 sequence coverage, or proteome complexity are also not significantly impacted. Although we observe minor differences in sequence coverage and glutamine deamidation, these are not consistent across our dataset. A 6-fold reduction in digestion time reduces electricity consumption, and therefore CO2 emission intensities. We furthermore demonstrate that working in 96-well plates further reduces electricity consumption by 60%, in comparison to individual microtubes. Reducing digestion time therefore h..., Six bones from La Draga (Spain, Holocene, samples LD_01 to LD_06) and Bayisha Karst Cave (China, Pleistocene, samples BKC_07 to BKC_12) were sampled for this study. Initial sampling was divided into three sub-samples for the three digestion durations tested here (site code_sample number_3h, site code_sample number_6h, and site code_sample number_18h). Samples were then processed according to the ZooMS protocol: they were demineralised in 0.6 M hydrochloric acid (HCl) for 24 hours. The HCl supernatant was then removed and samples were rinsed thrice in 100 µL ammonium bicarbonate (50 mM, NH4HCO3, hereafter AmBic) for subsequent gelatinisation in a final volume of 100 µL AmBic for one hour at 65°C. Following gelatinisation, the 100 µL of the AmBic solution was transferred to a new microtube, to which 0.8 µg trypsin (Promega) was added for incubation at 37°C, with mild agitation at 300 rpm (VWR, Thermal Shake lite). Digestion occurred for either 3, 6, or 18 hours. To stop trypsin digestion,..., , # Increasing sustainability in palaeoproteomics by optimizing digestion times for large-scale archaeological bone analyses

[https://doi.org/10.5061/dryad.cz8w9gj8j](https://doi.org/10.5061/dryad.cz8w9gj8j)

## Description of the data and file structure

Data deposited on Dryad are structured as follows:

1. Digestion_time_Datasheet.csv containing all information concerning sample names, experimental information (sampling amount), and the palaeoproteomics methods data tested in this study (ZooMS and SPIN).
2. Electicitymeasurement.csv concerning all data gathered during the measurement of electricity consumption of the three digestion times tested in the paper.
3. Three folders: Full proteome MQ (txt files generated after the MaxQuant search against Bos taurus full proteome); msd_files_3replicates (.msd files of all LC-MS/MS raw data) and a SPIN MQ (txt files generated after the MaxQuant search against the SPIN database).
4. Four R code markdowns with statistical analyses of the pap...