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.

Insights into the Differential Preservation of Bone Proteomes in Inhumed and Entombed Cadavers from Italian Forensic Caseworks

“…The presence of a single collagen-binding protein in this group, namely, procollagen C-endopeptidase enhancer 1, suggests that NCPs with a high affinity for the mineral matrix may be more resistant to hydrolysis than collagens and collagen-binding proteins. This result further confirms previous findings on the successful recovery of these NCPs in archeological contexts, even when only a minimal amount of intact collagen is available …”

“…This result further confirms previous findings on the successful recovery of these NCPs in archeological contexts, even when only a minimal amount of intact collagen is available. 57 …”

“…This result further confirms previous findings on the successful recovery of these NCPs in archeological contexts, even when only a minimal amount of intact collagen is available. 57 Considering the proteins correlated uniquely with AAD (Supplementary Figure S2), the best positive correlation was shown by the collagen alpha-1(XII) chain. The surprising behavior of COCA1 could be linked to the protective role of the zinc lining: In fact, the older individuals present in this study were placed in zinc-lined coffins.…”

Paleoproteomics

“…When choosing an extraction method, sample type, size, and preservation should be considered, as well as the complexity of the proteome, the protein(s) of interest, and the amount of protein needed for analysis. The postdepositional history of the sample should also be taken into account, as well as potential chemical modifications introduced during the chosen extraction protocol. − In addition, because proteomic analyses often require less sample material than other methods, such as ancient DNA analysis, stable isotope analysis, and radiocarbon dating, protein extractions can often be performed on the leftover material or byproducts of these protocols. ,− Combining such protocols is desirable, as it reduces sampling demands on irreplaceable material.…”

A primer for ZooMS applications in archaeology

“…Even less material is required for ZooMS, which has been shown to reliably yield identifiable collagen spectra from bones with as little as 0.26% N ( 60 ). As such, ZooMS can generally be applied to a wider range of archaeological remains, including from challenging environments and deep time that would otherwise be ill suited for other methods ( 61 ).…”