SPIN enables high throughput species identification of archaeological bone by proteomics.

Species determination based on genetic evidence is an indispensable tool in archaeology, forensics, ecology, and food authentication. Most available analytical approaches involve compromises with regard to the number of detectable species, high cost due to low throughput, or a labor-intensive manual process. Here, we introduce "Species by Proteome INvestigation" (SPIN), a shotgun proteomics workflow for analyzing archaeological bone capable of querying over 150 mammalian species by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Rapid peptide chromatography and data-independent acquisition (DIA) with throughput of 200 samples per day reduce expensive MS time, whereas streamlined sample preparation and automated data interpretation save labor costs. We confirm the successful classification of known reference bones, including domestic species and great apes, beyond the taxonomic resolution of the conventional peptide mass fingerprinting (PMF)-based Zooarchaeology by Mass Spectrometry (ZooMS) method. In a blinded study of degraded Iron-Age material from Scandinavia, SPIN produces reproducible results between replicates, which are consistent with morphological analysis. Finally, we demonstrate the high throughput capabilities of the method in a high-degradation context by analyzing more than two hundred Middle and Upper Palaeolithic bones from Southern European sites with late Neanderthal occupation. While this initial study is focused on modern and archaeological mammalian bone, SPIN will be open and expandable to other biological tissues and taxa.

Palaeoproteomic identification of breast milk protein residues from the archaeological skeletal remains of a neonatal dog.

Accurate postmortem estimation of breastfeeding status for archaeological or forensic neonatal remains is difficult. Confident identification of milk-specific proteins associated with these remains would provide direct evidence of breast milk consumption. We used liquid chromatography coupled to tandem mass spectrometry (MS) to confidently identify beta-lactoglobulin-1 (LGB1) and whey acidic protein (WAP), major whey proteins associated with a neonatal dog (Canis lupus familiaris) skeleton (430-960 cal AD), from an archaeological site in Hokkaido, Japan. The age at death of the individual was estimated to be approximately two weeks after birth. Protein residues extracted from rib and vertebra fragments were analyzed and identified by matching tandem MS spectra against the dog reference proteome. A total of 200 dog protein groups were detected and at least one peptide from canine LGB1 and two peptides from canine WAP were confidently identified. These milk proteins most probably originated from the mother's breast milk, ingested by the neonate just before it died. We suggest the milk diffused outside the digestive apparatus during decomposition, and, by being absorbed into the bones, it partially preserved. The result of this study suggests that proteomic analysis can be used for postmortem reconstruction of the breastfeeding status at the time of death of neonatal mammalian, by analyzing their skeletal archaeological remains. This method is also applicable to forensic and wildlife studies.

Species identification of archaeological skin objects from Danish bogs: comparison between mass spectrometry-based peptide sequencing and microscopy-based methods.

Denmark has an extraordinarily large and well-preserved collection of archaeological skin garments found in peat bogs, dated to approximately 920 BC - AD 775. These objects provide not only the possibility to study prehistoric skin costume and technologies, but also to investigate the animal species used for the production of skin garments. Until recently, species identification of archaeological skin was primarily performed by light and scanning electron microscopy or the analysis of ancient DNA. However, the efficacy of these methods can be limited due to the harsh, mostly acidic environment of peat bogs leading to morphological and molecular degradation within the samples. We compared species assignment results of twelve archaeological skin samples from Danish bogs using Mass Spectrometry (MS)-based peptide sequencing, against results obtained using light and scanning electron microscopy. While it was difficult to obtain reliable results using microscopy, MS enabled the identification of several species-diagnostic peptides, mostly from collagen and keratins, allowing confident species discrimination even among taxonomically close organisms, such as sheep and goat. Unlike previous MS-based methods, mostly relying on peptide fingerprinting, the shotgun sequencing approach we describe aims to identify the complete extracted ancient proteome, without preselected specific targets. As an example, we report the identification, in one of the samples, of two peptides uniquely assigned to bovine foetal haemoglobin, indicating the production of skin from a calf slaughtered within the first months of its life. We conclude that MS-based peptide sequencing is a reliable method for species identification of samples from bogs. The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium with the dataset identifier PXD001029.