COLLAGENS, KERATINS ET AL.
EVA CHOCHOLOVÁ
LABORATORY OF BIOLOGICAL AND MOLECULAR ANTHROPOLOGY
DEPARTMENT OF EXPERIMENTAL BIOLOGY
QUIZ
1. Write at least 3 examples of viruses studied in palaeogenomics:
2. How can we study past diseases in general?
3. What are the three types of plague?
4. Which types of viruses are hardest to study in palaeogenomics?
5. What is the difference between anthroponosis, zoonosis and sapronosis?
HBV, variola, influenza, bacteriphages…
RNA, ssDNA
Bubonic, septicemic, pneumonic
Human-human, human-animal, abiotic-living host
6. What are some examples of sedaDNA sources?
7. What can we study through sedaDNA?
Palaeoenvironment, climate change, biodiversity, domestication, ancient
hominins, parasites, …
QUIZ
8. What is DNA leaching and where was it observed so far?
10. What can we study from dental calculus?
11. How can we deal with the challenge of limited amount of dental calculus?
Movement of DNA vertically across sediment layers, observed in caves and
non-frozen soil
9. Domestication types and example:
…
COLLAGENS, KERATINS ET AL.
EVA CHOCHOLOVÁ
LABORATORY OF BIOLOGICAL AND MOLECULAR ANTHROPOLOGY
DEPARTMENT OF EXPERIMENTAL BIOLOGY
ANCIENT PROTEINS
Warinner et al., 2022 DOI: 10.1021/acs.chemrev.1c00703
• Most proteins preserved in mineralised matrix
• Degradation mainly through microbial activity
(slowed by cold and dry environment)
• Bottom-up approach
• Palaeoproteomics most often for taxonomy –
collagen and keratin – easier authentication
through modifications (glutamine deamidation)
COLLAGENS
• Aside from palaeoproteomics, collagens
are used in stable isotope analysis and
radiocarbon dating
• Collagen, type 1 (COL1)
• Most abundant protein in human
body (80 % of bone proteins)
• Stable – triple helix, hydrogen bonds
of hydroxyproline
• Tetrapods 2x COL1ɑ1 and 1x COL1ɑ2
• Teleost fish COL1ɑ1, COL1ɑ2, COL1ɑ3
• Chains consist of G-X-Y motif (G - glycine,
X and Y - various AA, often proline and
hydroxyproline)
• Taxonomically informative collagen
peptides - marker peptides.
Richter et al., 2022 DOI: 10.1073/pnas.2109323119
Overview of collagen structure and archaeological sources. Collagen can be retrieved from a wide range of animal
tissues. In most animals, the COL1 triple helix is composed of two ɑ1-chains and one ɑ2-chain. Five triple helices are
bundled into a microfibril. Bundles of microfibrils form a fibril, and bundles of fibrils form fibers. During the initial stages
of ZooMS, this structure is denatured, allowing the enzyme trypsin to cut the protein into peptides. Peptides differ in
sequence and mass across taxa, as shown for turkey ( M. gallopavo ), goat ( C. hircus ), and coho salmon ( O. kisutch ).
α-KERATINS AND CORNEOUS β-PROTEINS
Warinner et al., 2022 DOI: 10.1021/acs.chemrev.1c00703
• Less used since they‘re often contaminants (human and
sheep keratins)
• Most important structural proteins after collagens
• Skin, hair, claws, hooves, horns, feathers, beaks, turtle
shells, baleen
• Furs and wool
OTHER TARGETS FOR TAXONOMY
• Databases incomplete
• Best for single origin samples, complex samples studied by LC-MS/MS
• Fibroin in silk (not only from domesticated Bombyx mori, also wild
silkworms)
• Eggshells
• Mollusk shells
• Corals
• Insect exoskeleton
Richter et al., 2022 DOI: 10.1073/pnas.2109323119
ZooMS
Richter et al., 2022 DOI: 10.1073/pnas.2109323119
• Zooarchaelogy by mass spectrometry – collagen peptide mass
fingerprinting (PMF)
• Routinely MALDI-TOF (matrix-assisted laser desorption/ionisation-
time-of-flight)
• Cost-effective, fast, minimally invasive (low input)
• Applied to bone, ivory, antler, parchment and vellum, leather, fish
scales, dentine and cementum, horn core, animal glues, works of art
and various artefacts…
ZooMS
Richter et al., 2022 DOI: 10.1073/pnas.2109323119
Steps of MALDI-TOF and representative collagen spectra. Digested collagen peptides (pink) are embedded in the matrix (blue) and ionized with a laser. Charged peptides (+1) are then accelerated
through a TOF tube, where they separate by mass. The output of the detector is visualized as spectra, where time is converted to m/z based on calibration standards. Collagen spectra are shown
for turkey ( M. gallopavo ), goat ( C. hircus ), and coho salmon ( O. kisutch ). Authenticated collagen peptide peaks are indicated by pink circles ( Dataset S1 A ). Three taxonomically informative
marker peptides are annotated, with Insets indicating the collagen chain, position, m/z, and sequence; amino acids that differ across taxa are highlighted in green. Note that although isoleucine (I)
and leucine (L) differences are highlighted, they are not distinguishable by MALDI-TOF. Baseline correction, smoothing, and intensity normalization were performed in mMass ( 134 ).
a Location of the archaeological site “Salpetermosen Syd 10” on Zealand in Denmark in the Hillerød municipality 30 km north of Copenhagen. Map drawn in Mapbox
Studio using a custom style. b Cross section of an in situ wetland bone deposit. Scale bar is 50 cm. Four bones were radiocarbon dated between 1720 and 1570 BP.
Picture provided by the Museum of North Zealand. c Species identification results by SPIN (5 min method, library-based DIA) and by morphological assessment for 63
samples from the Salpetermosen site measured in technical duplicates and 3 blanks. Rows represent individual samples and have been ordered first by morphological
species assignment and then by decreasing mean site coverage. The upper left and lower right wedge of each cell represent results measured in two separate
experiments, one with higher (upper left, dark blue) and the other with lower (lower right, light blue) MS signal intensity. The first seven columns indicate SPIN species
by blue wedges and morphological species possibilities by pink boxes. Bovine species assignments are combined in column two. The eighth and ninth columns are
heatmaps showing the absolute number of covered amino acids and relative protease intensity, respectively. d Summary of SPIN species identifications from panel c in
the replicate with high MS intensity. Bovine identifications are separated into cow (Bos) and broader bovine identifications (Bos/Bison). Striped colors indicate samples
with insufficient sequence coverage to distinguish closely related taxa. Samples with insufficient sequence coverage for confident species identification are marked as
“signal too low” and correctly excluded blanks are marked in black. e Pseudo receiver operating characteristic (ROC) curves for comparing the sensitivity and success
rate of three different data acquisition and analysis strategies. Results of each dataset were sorted by decreasing number of identified sites. The y-axis shows the
cumulative number of correct species identifications in agreement with the morphology. The x-axis shows the cumulative number of false or missing identifications
below the relative protease intensity threshold. Color indicates data acquisition and analysis mode with pink for DDA, dark blue for library-based DIA, and sand color
for library-free DirectDIA. Experiments with lower MS intensity are shown by dashed and high intensity by solid lines.
Peters et al., 2022 DOI: 10.1016/j.isci.2022.104195
!
• ZooMS
• Clothing, silkworms
• Top-down, bottom-up
• Dust contamination
• Taxonomical resolution
• Whales
What is the main research question addressed in the paper?
What techniques and methods were used in the study to analyze ancient
protein samples?
• MS, radiocarbon dating – peptide markers in collagen for taxonomic identification
• ATR FT-IR spectroscopy (23 bones, 3 teeth) – decalcification Tris-EDTA buffer
• UHPLC-MS/MS – reconstruction PEAKS Studio Software
• COL1A1, COLA2
Were there any limitations or challenges associated with the methods
used?
What were the main findings of the study?
Were there any unexpected or surprising results?
What are the implications of the results for our understanding of ancient
human populations and their lifestyles?
• Populations exploited these domesticated animals mainly for their secondary products
(milk, wool, carrying) rather than meat
• Diffusion by gradual infiltration
Are there alternative interpretations of the data presented in the paper?
What additional evidence or analyses could strengthen the conclusions
drawn from the data?
How does this paper contribute to your understanding of palaeoproteomics
and its applications in archaeology and anthropology?
Student answers