Research into the palaeobiology of extinct taxa through ancient DNA and proteomics has been mostly limited to Plio-Pleistocene fossils1–9, due to molecular breakdown over time, which is exacerbated in tropical settings1–3. Here we sample small proteomes from the interior enamel of fossils at palaeontological sites from the Pleistocene to the Oligocene in the Turkana Basin, Kenya, which has produced a rich record of Cenozoic mammalian evolution10. Through a mass-spectrometry-based proteomic workflow, and using criteria to locate diagenetiforms derived from enamel, we recover fragments of enamelin, ameloblastin, matrix metalloprotease-20 and dentin matrix acidic phosphoprotein 1 from an Early Miocene rhinocerotid and several proboscideans collected from the sites of Buluk (16 million years ago; Ma) and Loperot (18 Ma). Diagenetiform counts decline in progressively older fossils, and we observe variability in Early Miocene preservation across sites. Phylogenetic analyses reveal the contribution of these sequences to the systematic placement of extinct taxa, although we caution that this approach must account for sparse fragments, uncertainty in fragment identification and possible sequence diagenesis. We identify likely modifications that support the ancient age of these proteins, and some of the oldest examples of advanced glycation end-products yet known. The discovery of protein sequences within dense enamel tissues in one of the persistently warmest regions on Earth promises the discovery of much older proteomes that will aid in the study of the palaeobiology and evolutionary relationships of extinct taxa.
