Successive Protein Extraction Using Hydroxylamine to Increase the Depth of Proteome Coverage in Fresh, Forensic, and Archaeological Bones

Proteomics is continually being applied to a wider range of applications, now including the analysis of archaeological samples and anatomical specimens, particularly collagen-containing tissues such as bones and teeth. Here, we present the application of a chemical digestion-based proteomics sample preparation protocol to the analysis of fresh, anatomical, and archaeological samples. We describe and discuss two protocols: one that uses hydroxylamine as an additional step of the proteomic workflow, applied to the insoluble fraction, and another that applies hydroxylamine directly on demineralized bones and teeth. We demonstrate the additional information that can be extracted using both protocols, including an increase in the sequence coverage and number of peptides detected in modern and archaeological samples and an increase in the number of proteins identified in archaeological samples. By targeting research related to collagens or extracellular matrix proteins, the use of this protocol will open new insights, considering both fresh and ancient mineralized samples.