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, in particular, collagen-containing tissues such as bones and teeth. Here we present the application of a chemi-cal digestion-based proteomics sample preparation protocol to the analysis of fresh, anatomical and archaeological samples. We are describing and discussing two protocols, one using hydroxylamine as an additional step of the proteomic workflow, applied to the insoluble fraction, and another focusing directly on demineralized bones and teeth. We demonstrate the additional information that can be extracted using both protocols, including increasing the sequence coverage and number of peptides detected in modern and archaeological samples, and increasing the number of proteins identified in archaeological samples. Targeting research related to collagens or extracellular matrix proteins, the use of this protocol will open new insights considering both fresh and ancient mineral-ized samples.