Digging deeper into ancient skeletal proteomes through consecutive digestion with multiple proteases

An increasing number of studies utilise the recovery of ancient skeletal proteomes for phylogenetic and evolutionary analysis. Although these studies manage to extract and analyse ancient peptides, the recovered proteomes are generally small in size and with low protein sequence coverage. We expand on previous observations which have shown that the parallel digestion and analysis of Pleistocene skeletal proteomes increases overall proteome size and protein sequence coverage. Furthermore, we demonstrate that the consecutive digestion of a skeletal proteome using two proteases, particularly the combination of Glu-C or chymotrypsin followed by trypsin digestion, enables the recovery of alternative proteome components not reachable through trypsin digestion alone. The proteomes preserved in Pleistocene skeletal specimens are larger than previously anticipated, but unlocking this protein sequence information requires adaptation of extraction and protein digestion protocols. The sequential utilisation of several proteases is, in this regard, a promising avenue for the study of highly degraded but unique hominin proteomes for phylogenetic purposes. Palaeoproteomic analysis of archaeological materials, such as hominin skeletal elements, show great promise in studying past organisms and evolutionary relationships. However, as most proteomic methods are inherently destructive, it is essential to aim to recover as much information as possible from every sample. Currently, digestion with trypsin is the standard approach in most palaeoproteomic studies. We find that parallel or consecutive digestion with multiple proteases can improve proteome size and coverage for both Holocene and Pleistocene bone specimens. This allows for recovery of more proteomic data from a sample and maximises the chance of recovering phylogenetically relevant information. [Display omitted] •Proteins in archaeological skeletal material provide an opportunity to study the past beyond the preservation of ancient DNA.•Choice of protease during protein extraction can significantly affect the reconstructed proteome.•Using alternative proteases parallel to trypsin can increase the number of extracted proteins and peptides.•Consecutive digestion with two proteases can improve proteome size and coverage.•Digestion with multiple proteases therefore increases the amount of information that is gained from archaeological specimens.