Proteome Variation with Collagen Yield in Ancient Bone

Abstract

Isotope analyses are some of the most common analytical methods applied to ancient bone, aiding the interpretation of past diets and chronology. For this, the evaluation of "collagen yield" (as defined in radiocarbon dating and stable isotope research) is a routine step that allows for the selection of specimens that are deemed adequate for subsequent analyses, with samples containing less than ∼1% "collagen yield" normally being used for isotopic analysis but discounted for radiocarbon dating. The aims of this study were to use proteomic methods of MALDI-TOF (matrix assisted laser desorption ionization time-of-fligh mass spectrometry) and LC-ESI-MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry) to investigate the endogeneity of the dominant proteinaceous biomolecules within samples that are typically considered to contain poorly preserved protein. Taking 29 archaeological samples, we evaluated the proteome variability between different acid-soluble fractions removed prior to protein gelatinization and considered waste as part of the radiocarbon dating process. We then correlated these proteomes against the commonly used "collagen yield" proxy for preservation. We found that these waste fractions contained a significant amount of both collagenous and noncollagenous proteins (NCPs) but that the abundance of these was not correlated with the acquired "collagen yield". Rather than a depleted protein load as would be expected from a low "collagen yield", the variety of the extracted NCPs was comparable with that commonly obtained from ancient samples and included informative proteins useful for species identification, phylogenetic studies, and potentially even for isotopic analyses, given further method developments. Additionally, we did not observe any correlation between "collagen yield" and peptide mass fingerprint success or between the different fractions taken from the same sample but at different radiocarbon pretreatment stages. Overall, these findings highlight the value in retaining and analyzing sample fractions that are otherwise discarded as waste during the radiocarbon dating process but more importantly, that low "collagen yield" specimens that are often misinterpreted by archaeologists as being devoid of protein can still yield useful molecular sequence-based information.

Keywords: NCPs; ancient bone; collagen; proteomics; radiocarbon dating; stable isotopes.

Figure 1

Schematic representation of the treatments and analyses to which samples have been subjected. “R/T” indicates room temperature, “O/N” indicates overnight.

Figure 2

Scatterplot for the percentage of “AG Collagen yield” (X axis) and for the number of NCPs (Y axis) identified in the dataset. Different colors and symbols (legend) represent different species. Species identifications achieved using both ZooMS and LC–ESI–MS/MS proteomics data are indicated with full shapes, whereas species identifications achieved uniquely with LC–ESI–MS/MS proteomics (and where ZooMS analyses failed) are indicated with empty shapes.

Figure 3

Scatterplot for the percentage of “AG collagen yield” (X axis) and for the total spectrum counts for (A) COL1A1 and B) COL1A2 (Y axis) identified in the dataset. Different colors and symbols (legend) represent different species. Species identifications achieved using both ZooMS and LC–ESI–MS/MS proteomics data are indicated with full shapes, whereas species identifications achieved uniquely with LC–ESI–MS/MS proteomics (and where ZooMS analyses failed) are indicated with empty shapes.

Figure 4

Scatterplot for the percentage of “AG Collagen Yield” (X axis) and for the total spectrum counts for biglycan (Y axis) identified in the dataset. Different colors and symbols (legend) represent different species. Species identifications achieved using both ZooMS and LC–ESI–MS/MS proteomic data are indicated with full shapes, whereas species identifications achieved uniquely with LC–ESI–MS/MS proteomics (and where ZooMS analyses failed) are indicated with empty shapes.

Figure 5

Bar plot representing (A) number of NCPs and (B) COL1A1 total spectrum count obtained from fraction A (red) and fraction B (blue) of samples subjected to a prewashing step with HCl. Sample names are indicated to the side of the two bar plots.

Figure 6

STRING association network of the NCPs extracted from TD1.A and TD1.B. The line thickness indicates the strength of data support (edge confidence). Proteins marked with the star symbol were identified in both sample fractions A and B (second prewash and final overnight HCl incubation).