Beyond DNA

Beyond DNA: How proteins let us get up
close and personal to our ancient relatives
Palaeoproteomics, a new technology that studies the proteins of ancient

remains, is shaking up history. Not only can we now peer further back in
time, but the technique is also letting us see our past in a new way.
Ancient DNA teased out from the fossilised bones and teeth of human species has
transformed what we know about our ancestors. Over the past two decades,
analysis of genetic material has not only revealed new human species, but it’s also
allowed archaeologists to recreate what our ancestors looked like, thousands of
years after they went extinct.
But it can’t give us the whole story because DNA is fragile – it breaks down over
time to the point where its code becomes unintelligible. This has meant that many
ancient bones can’t be analysed genetically, therefore much of the human family
tree has remained hidden from view.
But over the past few years, new insights into our ancient ancestors have come
from the proteins locked inside fossilised remains. Proteins can survive much
longer than DNA, and advances in lab techniques, such as mass spectrometry, have
increased researchers’ ability to detect and characterise tiny amounts of them.
Most promising of all is ‘shotgun proteomics’, a technique that creates a profile of
all the proteins inside a fossilised bone or tooth. These ‘protein fingerprints’ have
already proven their ability to identify which species of ancient human fossilised
bones belonged to, even when DNA evidence has been lost.
It means we’re on the cusp of a ‘palaeoproteomics’ revolution that promises to

provide an unprecedented view of who our ancient relatives were and how they
lived.
Paleoproteomics, the study of ancient proteins, is a rapidly growing field

at the intersection of molecular biology, paleontology, archaeology,
paleoecology, and history. Paleoproteomics research leverages the
longevity and diversity of proteins to explore fundamental questions
about the past. While its origins predate the characterization of DNA, it
was only with the advent of soft ionization mass spectrometry that the
study of ancient proteins became truly feasible. Technological gains over
the past 20 years have allowed increasing opportunities to better
understand preservation, degradation, and recovery of the rich
bioarchive of ancient proteins found in the archaeological and
paleontological records. Growing from a handful of studies in the 1990s
on individual highly abundant ancient proteins, paleoproteomics today is
an expanding field with diverse applications ranging from the taxonomic
identification of highly fragmented bones and shells and the
phylogenetic resolution of extinct species to the exploration of past
cuisines from dental calculus and pottery food crusts and the
characterization of past diseases. More broadly, these studies have
opened new doors in understanding past human–animal interactions,
the reconstruction of past environments and environmental changes, the
expansion of the hominin fossil record through large scale screening of
nondiagnostic bone fragments, and the phylogenetic resolution of the
vertebrate fossil record. Even with these advances, much of the ancient
proteomic record still remains unexplored. Here we provide an overview
of the history of the field, a summary of the major methods and
applications currently in use, and a critical evaluation of current
challenges. We conclude by looking to the future, for which innovative
solutions and emerging technology will play an important role in
enabling us to access the still unexplored “dark” proteome, allowing for a
fuller understanding of the role ancient proteins can play in the
interpretation of the past.
Proteins are long-lived biomolecules capable of surviving over millions

of years.9,10 They routinely outlast even the oldest surviving DNA,11−14 and
their full longevity has yet to be determined.15−17 Although proteins do not
persist into deep time as long as lipids,18 their sequence diversity makes
them more informative, and consequently proteins represent one of our
most valuable bioarchives of the past.
The longevity and biological utility of proteins derive in large part from
their structure. Proteins are large biomolecules built from linear
sequences of amino acids folded into complex three-dimensional forms.
The 20 standard amino acids, each formed around a central carbon,
contain a carboxyl group and an amino group, which form the peptide
bonds linking the amino acids together into proteins, and an R group,
which varies between amino acids and imparts distinct chemical
properties. R groups are chemically diverse, consisting of positively
charged, negatively charged, polar, and nonpolar groups that can be
small, large, or structurally constrained. The sequence of amino acids
making up the primary protein structure is encoded by DNA, which is
then transcribed to RNA and translated into proteins using trinucleotide
codon sequences for each amino acid. Because proteins are derived from
the genetic code, individual proteins preserve part of the heritable
genetic signal of an organism, and therefore, protein sequences can be
used to make taxonomic identifications and reconstruct phylogenies. 19−21
Digging deeper into the Denisovans

Anthropologists are particularly keen to learn more about the Denisovans, a
mysterious tribe of humans who lived at least 200,000 to 50,000 years ago. So far,
their genetic material has only been recovered from one site: the Denisova Cave in
the foothills of Siberia’s Altai Mountains.
But there’s evidence that they were much more widespread. People alive today in
Asia, Australia and Papua New Guinea have Denisovan DNA in their genetic code.
“Almost every [question we have] about the Denisovans is unanswered,” says Dr

Frido Welker at the University of Copenhagen in Denmark who’s a leader in the field
of ancient proteins. “It’s not only that we need to know where they lived, we also
don’t know what kinds of stone tools they made, we don’t know their hunting
behaviour.”
The most complete remains of a Denisovan found so far is half a lower jawbone
with two teeth attached that was discovered by a monk in Baishiya Karst Cave high
on the Tibetan Plateau in China.
The DNA inside the jaw, thought to be at least 160,000 years old, was too degraded
to analyse. But in 2019, a team that included Welker managed to analyse the
collagen protein in the teeth and it was a match for the Denisovans found at
Denisova Cave. It was the first time an ancient human had been identified only
from its proteins.
Protein analysis is now being used to comb through thousands of bone fragments
dug up from archaeological sites across Europe and Asia to identify which belonged
to ancient humans, and which belonged to animals such as hyenas and
mammoths.
By doing this, the FINDER research project at the Max Planck Institute for the
Science of Human History in Jena, Germany, is increasing the number of bone
samples known to be from ancient humans. This will provide a much larger range
of ancient human bones for analysis.
Understanding ancient diets

As well as using DNA, archaeologists have studied the shapes and dimensions of
bones to identify species and consider where they might fit into our evolutionary
past.
But much of what they find is just small shards that can’t be identified. “Back in the
1950s, or even earlier, [archaeologists] would chuck these bone fragments away
because they’d be of no value to them,” says Dr Katerina Douka, who is leading
FINDER.
Douka is using a technique called Zooarchaeology by Mass Spectrometry (ZooMS)
to identify the bone shards. In ZooMS, collagen protein is extracted from the bones
and broken down with trypsin, an enzyme that helps to digest protein in our
stomachs.
Trypsin chops the collagen into peptides (chains of amino acids), which are then
placed in a mass spectrometer so their masses can be measured. The peptides are
present in different ratios in human remains compared to those of animals,
allowing human bones to be identified.
So far 11,000 bone fragments from the Denisova Cave have been analysed using
ZooMS, and 10 human bones identified. Some of them are almost 250,000 years
old, so likely beyond genetic analysis. After all, DNA has only been sequenced from
three hominin groups; Neanderthals, Denisovans and Homo sapiens – and mostly
from the past 100,000 years.
DNA’s tendency to break down over time is a problem Welker is familiar with from
his days as an undergraduate. He was trying to sequence genetic material trapped
within the fossilised dung of an extinct mountain goat (Myotragus balearicus) to find
out what it ate by identifying the genes of any plants it had digested.
“It didn’t work because [the DNA] was absolutely knackered in terms of
preservation,” he says. “The next best thing seemed to be proteins.”
In December 2020, Welker started work on a major new research project

sequencing the proteins in hominin fossils from the past million years, that had
been collected across Africa, Europe and Asia.
He has received €1.5m (£1.35m approx) from the European Research Council to
analyse bone and teeth samples from museums and universities. “Between 700,000
and 200,000 years ago is an exciting period for understanding where we, as a
species, originate and what the hominin species at the time were doing,
behaviourally speaking,” says Welker.
It’s the period when Homo heidelbergensis, the species that we, Homo sapiens, are
thought to have descended from, first came about, having evolved from Homo
erectus.
“There are several species designations out there, like Homo heidelbergensis where

either people disagree on how it should be placed in relation to us, or whether it
exists or not,” says Welker. “The good thing is that, for that time period and Homo
heidelbergensis specifically, proteins can be very informative in the coming years to
resolve some of those questions.”
The shotgun proteomics technique Welker will be using starts by drilling into bone
or tooth to create a breadcrumb-sized amount of powder. Typically, the powder is
placed in hydrochloric acid to release the proteins, which are then sliced into
peptides using trypsin.
Just as in ZooMS, the peptides’ masses are measured in a mass spectrometer. But
shotgun proteomics differs from ZooMS in that the data from the mass
spectrometer also allows researchers to determine the sequence of amino acids
within the peptides – and it does this for all the proteins in the sample, rather than
just one.
So while ZooMS can tell whether a bone came from an ancient human or
something else, a protein sequence from shotgun proteomics can be compared
with those already known to occur in hominin species to identify the specific
species.
Furthermore, as the amino acid sequence of a protein is determined by the

genome, variation in the sequence that might exist between species tells
researchers something about the evolutionary relationship between the fossil
being studied and other hominin species.
The future of proteomics

Over the next few years, it’s expected that shotgun proteomics will allow
researchers to identify the species present at more archaeological sites, providing a
clearer picture of where the likes of the Denisovans lived.
Also, by identifying the species present at a location, archaeologists can use the
artefacts there to piece together more information about them, such as the prey
they hunted and whether or not they used fire.
But it won’t be easy. Many of the shards of bone that researchers work with only
have a limited number of proteins in them – they are, after all, a tiny fragment from
one part of the skeleton. So they contain much less information than an entire
genetic code.
Proteins are also ‘evolutionarily conserved’, meaning they often don’t change a
great deal between species because they’re doing the same job. This limits the
extent to which they can be used to link a bone sample to a specific species.
But proteins have already shown themselves to be much more resilient than DNA,
allowing us to peer further back in time than ever before. Right now, we just don’t
know how far back proteomics will allow us to see.
“It’s exciting to have been part of what has happened in the field in the past couple
of years,” says Welker. “Even now, I still don’t know what its limits are. That’s only a
good thing, because it means we still have lots of things to explore.”

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Beyond DNA: How proteins let us get up

close and personal to our ancient relatives

Palaeoproteomics, a new technology that studies the proteins of ancient

It means we’re on the cusp of a ‘palaeoproteomics’ revolution that promises to

Paleoproteomics, the study of ancient proteins, is a rapidly growing field

Proteins are long-lived biomolecules capable of surviving over millions

Digging deeper into the Denisovans

“Almost every [question we have] about the Denisovans is unanswered,” says Dr

Understanding ancient diets

In December 2020, Welker started work on a major new research project

“There are several species designations out there, like Homo heidelbergensis where

Furthermore, as the amino acid sequence of a protein is determined by the

The future of proteomics

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