Consecutive digestion of archaeological skeletal proteomes with multiple proteases

Ancient skeletal proteomes are increasingly utilised for phylogenetic and evolutionary analysis. These proteomes are, however, often small and with low sequence coverage. We expand on previous observations which have shown that parallel digestion of Pleistocene skeletal proteomes increases proteome size and protein sequence coverage. Furthermore, we demonstrate that the consecutive digestion of a skeletal proteome using two proteases, particularly Glu-C or chymotrypsin followed by trypsin digestion, enables the recovery of alternative proteome components not reachable through trypsin digestion alone. The sequential utilisation of several proteases is a promising avenue for the study of highly degraded ancient proteomes for phylogenetic purposes.