The effect of eraser sampling for proteomic analysis on Palaeolithic bone surface microtopography

Abstract

Bone surface modifications are crucial for understanding human subsistence and dietary behaviour, and can inform about the techniques employed in the production and use of bone tools. Permission to destructively sample such unique artefacts is not always granted. The recent development of non-destructive proteomic extraction techniques has provided some alternatives for the analysis of rare and culturally significant artefacts, including bone tools and personal ornaments. The Eraser Extraction Method (EEM), first developed for ZooMS analysis of parchment, has recently been applied to bone and ivory specimens. To test the potential impact of the EEM on ancient bone surfaces, we analyse six anthropogenically modified Palaeolithic bone specimens from Bacho Kiro Cave (Bulgaria) through a controlled sampling experiment using qualitative and 3D quantitative microscopy. Although the overall bone topography is generally preserved, our findings demonstrate a slight flattening of the microtopography alongside the formation of micro-striations associated with the use of the eraser for all bone specimens. Such modifications are similar to ancient use-wear traces. We therefore consider the EEM a destructive sampling approach for Palaeolithic bone surfaces. Together with low ZooMS success rates in some of the reported studies, the EEM might not be a suitable approach to taxonomically identify Pleistocene bone specimens.

Figure 1

Description of the experimental workflow. The location of ROIs (cut and control) was defined on each bone specimen included in the study. The macro- and microscopic surface topography of the bone surface of each area was visually inspected using photos by digital microscopy (ZEISS, Smartzoom 5) and measurements by confocal disc-scanning microscopy (μsurf mobile, Nanofocus AG) before and after EEM. Cut and control areas were sampled using EEM while the downward force applied during sampling was measured via an instrumented stage. Each sample collected was analysed through peptide mass fingerprinting (n = 12). Animal silhouette is not to scale and derives from http://phylopic.org.

Figure 2

Distribution of the peak forces applied during the EEM of each sampling cut area (in yellow) and control area (in red) for each bone specimen. The reference peak force measurements (eraser on paper) is shown in grey. The insert in the top right is an example of how peak forces were acquired from force data. The mean peak force (red line) for each surface area consists of the maximum force values (red markers) for each eraser and bone contact during the 2 min of the EEM event. Dashed lines equal to + 1 and − 1 SD.

Figure 3

Micrographs of the control area of CC7-379 using automated digital microscopy (ZEISS, Smartzoom 5), (a) before the use of EEM, (b) after the use of EEM. The white arrow highlights the orientation of the micro-striations which follow the orientation of the erasing movement. In addition, we note the removal of surface residues, visible in particular in the top-left corner as the removal of dark-stained regions (white dashed line).

Figure 4

Matched before and after EEM pairwise scatterplot for each ISO 25178 surface texture parameter measured in this study (Sa, Spc, Sha and Smrk1). Lines represent equivalent parameter values after and before EEM. Each specimen is represented by different symbols, cut areas are in yellow and control areas are in red. 2D depictions of low and high values for each surface texture parameter are indicated on each plot.

Figure 5

Bone surface microtopography of the specimen CC7-379, before (left) and after (right) EEM, using confocal disc-scanning microscopy. 3D surface models (a,b,e,f) and 2D intensity micrographs (c,d,g,h) of control (a–d) and cut areas (e–h). Orientation of eraser movements are indicated by the black arrows. Depth of the bone microtopography is color-coded with blue indicating the lowest valleys and white the highest peaks. We note the generation of microstriations after the use of EEM with some examples indicated by white arrows. We note the presence of a residue in the middle of the bone surface (a) which has been removed with the use of EEM (b) and is potentially related to the formation of the deeper traces located near its initial position.