State of the art and challenges

Paleoproteomics is currently the only approach providing access to genetic data from time periods too chronologically remote, or geographic areas too warm, to allow ancient DNA preservation. Ancient protein sequence variation has proven to be large enough to allow confident phylogenetic placement of extinct species otherwise recalcitrant to ancient DNA analysis. We have recently demonstrated that an almost complete set of ancient proteins, i.e. a palaeoproteome, can be consistently retrieved from dental enamel specimens ~2 millions of years old, from temperate and tropical areas. Furthermore, recent methodological and computational advances in palaeoproteomics have now allowed the used of ancient proteins for the phylogenetic placement of long-extinct species.

We believe that the systematic application of palaeoproteomics to palaeontological, palaeoanthropological and archaeological studies in the ~12million-year-old to 40-thousand-year-old time-range will enable confident molecular-based phylogenetic placement of multiple enigmatic hominoid species and better reconstruction of the practical knowledge behind the first evidence of modern human abstract thinking.

Research lines and themes

The PUSHH research activity will be divided in two Research Lines: “APPLICATIONS” and “TECHNOLOGIES”.

The APPLICATIONS research line includes four themes and covers PhD projects 1-9.

The TECHNOLOGIES research line only includes the "Methods" theme and covers PhD projects 10-14.

Through the PUSHH ETN research programme, we aim to create a multidisciplinary training environment that brings together palaeontologists, palaeoanthropologists, archaeologists, as well as biologists, engineers, analytical chemists, around a common hub developing MS-based ancient protein sequencing and applying it to ancient biological materials. We will develop a strategic training platform to equip the next generation of palaeoanthropologists, palaeontologists and archaeologists with the skills to exploit the latest biomolecular technologies.

Only a close interaction and collaboration among museum curators, restorers and analytical scientists can lead to the development of innovative procedures for sample collection and preparation best fulfilling both analytical and conservation requirements.

Details about Each Theme


The ‘Hominoid’ theme encompasses research into the evolution of apes, which were much more diverse and widely distributed during the Miocene than nowadays. As a result of the decimated current diversity of the group, their fragmentary fossil record, and the high levels of homoplasy documented by previous studies, many disagreements persist regarding the phylogeny of fossil apes. By providing deep-time molecular data for Miocene apes, palaeoproteomics offers the prospect to provide anchoring points for key extinct taxa, ultimately resulting in a much better resolved phylogeny of the group, with implications for the adequate reconstruction of ancestral morphotypes, including the chimp-human last common ancestor from which hominins evolved. Furthermore, palaeoproteomics would also allow for testing specific hypothesis of relationship among species, as well as potential interactions among branch length and life traits, such as BMI.


The ‘Hominin’ theme focuses on our evolution since the split from chimpanzees. There are many novel traits that appear across the last five or more million years, but on the other hand there is continuity in many aspects. The fossil record indicates that human evolution is not a gradual anagenetic process with one species evolving from another, but involves abundant branching (speciation) and extinction, with multiple lineages coexisting at the same time. It has been suggested that there are some phases, in the Pliocene, and the Plio-Pleistocene, when the rate of phenotypic evolution in hominins may have been particularly high. Palaeoproteomics has the potential to provide fundamental contributions to answer questions of primary relevance, such as: “Was Homo erectus the direct ancestor of Homo sapiens?”, or “Are there actually several species of early human represented by what we are now calling Homo erectus?”.


The ‘Neanderthals and modern humans’ theme will involve using an MS-based ancient protein analysis relying on peptide mass fingerprinting (PMF, also conventionally referred to as “ZooMS” in archaeozoology). We will use ZooMS to determine the biological species of origin of fragments too small, or too altered, to be assigned to a species by visual inspection. In particular, these fragments have important implication for recent human evolution and prehistory, and have been retrieved from caves known to be occupied by anatomically modern humans, Neanderthals and Denisovans. We will also use ZooMS to study organic compounds from stone tools and pigments used by Neanderthals and modern humans.


The ‘Commercial assay’ theme, although thematically distant from the other applications, will use the same state-of-the-art technological solutions developed to address the analytical challenges associated with ancient protein analysis to deliver an accurate, convenient and effective assay of protein damage characterisation in industrial production. It includes a single research project, which will involve working with DEVRO - a world-leader in artificial sausage casing production. Artificial sausage casing is made of collagen usually derived from cattle and pig skin. During the sausage casing production process, collagen is subject to a series of harsh acidic and alkaline steps inducing permanent and irreversible biochemical damage in the collagen. DEVRO is interested in accurately characterising the amount and the type of damage in the collagen it processes, because this affects the final mechanical properties of its end product. Currently, DEVRO measures its collagen damage using a rudimentary ammonia assay, quantifying the amount of ammonia released as the product of deamidation of asparagine and glutamine in controlled acidic conditions. This assay has no resolution concerning the extent of each deamidation site and it is completely blind to other types of biochemical damage the production treatments can induce in collagen.


The projects in the second innovation area Technologies will serve to create and use new methods to advance investigation of ancient protein residues implementing solutions not attempted before. These results and insights will be thoroughly contextualised into existing traditions of palaeoanthropological, palaeontological and archaeological material investigation and safeguard. PUSHH will offer training in proteomics, mass spectrometry, bioinformatics and conservation of organic materials in biological and cultural heritage. It will also provide ESRs with the skills required to contextualise these results within existing research frameworks. Finally, the methodological development PUSHH pursues will also be crucial to minimise the amount of material needed to proceed to palaeoproteomic analysis. This will make the application of palaeoproteomic analysis more acceptable and sustainable in a larger number of palaeontological and archaeological contexts.