SPIN enables high throughput species identification of archaeological bone by proteomics

Abstract

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.

Neanderthal subsistence, taphonomy and chronology at Salzgitter‐Lebenstedt (Germany): a multifaceted analysis of morphologically unidentifiable bone

“…In the future, additional proteomic sequencing methods [e.g. SPIN (Species by Proteome INvestigation); Rüther et al ., 2022] will also become available to provide more fine‐grained taxonomic identifications (e.g. Bos or Bison ) to allow further environmental and behavioural insights.…”

Extinct species identification from late middle Pleistocene and earlier Upper Pleistocene bone fragments and tools not recognizable from their osteomorphological study by an enhanced proteomics protocol

“…Remarkably, on 5 mg of bone powder only, we were able to identifying proteins, genus, and PTMs from Pleistocene bones. Very recently Rüther and colleagues (Rüther et al, 2022) have developed a new method "Species by Proteome INvestigation" (SPIN), which allows high-throughput bone proteome analysis. In this paper the SPIN method is compared to different preparation methods including the FASP method but not the eFASP method, which is better than FASP.…”

Paleoproteomics

“…DIA offers the potential to extend the dynamic range of MS/MS by generating data from more peptides, and especially lower abundance peptides, while also improving reproducibility and quantification. A DIA-based approach has recently been integrated into a new paleoproteomics workflow known as species by proteome investigation (SPIN), which enables rapid mammalian species assignment using LC–MS/MS . Although currently limited in scope due to its computational complexity, further improvements in DIA development, improved databases, and the application of machine learning may soon allow DIA to become more mainstream in ancient protein studies.…”

“…Because many of the NCPs and some of the collagens have higher mutation rates than COL1, they are better targets for phylogenetic reconstructions, especially between closely related species. , Analyses of bone and dentine proteomes have successfully aided in resolving the phylogenetic relationships of extinct megafauna, ,,, including archaic hominins. , In addition, bone proteome-scale analyses using new high-throughput LC–MS/MS workflows, such as SPIN, show great promise for enabling genus and species-level taxonomic assignments of nondiagnostic bone …”