PROTEINS, PROTEOMS, METAPROTEOMS EVA CHOCHOLOVÁ LABORATORY OF BIOLOGICAL AND MOLECULAR ANTHROPOLOGY DEPARTMENT OF EXPERIMENTAL BIOLOGY PROTEINS • Collagens • Keratins and corneous β-proteins • Fibroin • Amelogenin Warinner et al., 2022 DOI: 10.1021/acs.chemrev.1c00703 Previous presentation • Mostly taxonomy, identification SEX DETERMINATION • Amelogenin cleaved and incorporated during enamel maturation • Gene AMEL • Non-mammals – autosomal (no difference) • Mammals– AMELX, AMELY • Most species only AMELX funtional or even complete loss of AMELY • Some species AMELX = AMELY in sequence • Sequence difference enables sex determination in e.g., Homo, Bovis, Capra, Ovis, Equus… • Ideal in cases of lower preservation (subadults, incomplete skeletal material, low DNA preservation, very old samples) • Cost-effective SEX DETERMINATION • acid etching of tooth enamel – minimally destructive • nanoflow liquid chromatography mass spectrometry (nanoLC-MS) SEX DETERMINATION • Changing preconceptions • Possible even in poor DNA preservation • Suggest additional unique peptides beside 440.2233 (SM(ox)IRPPY), e.g. 432.2258 (SMIRPPY) and 396.7073 (M(ox)IRPPY) • Most probably war comrades or relatives - unknown Ion chromatograms representing selected peptides of the ‘Lovers’ enamel proteome20. Chromatograms search was performed using Xcalibur software (Thermo Scientific) with a mass tolerance of 5 ppm. Peptide sequences, protein names and retention times are reported in the graphs. The presence of peptide SM (ox)IRPPY (AMELY; [M+ 2 H]+2 440.2233 m/z) in both the specimens suggests that the two individuals were males. SEX DETERMINATION preprint, 2023 • 2 million years old, Paranthropus robustus • Oldest African hominin DNA about 0.018 Ma • May help elucidate taxonomic placement SEX DETERMINATION preprint, 2024 Left: Spectral counting-based coverage of AmelY protein in peptidomics data from eight modern samples - fourmales and four females, y-axis presents the number of ID spectra per amino acid position in the protein in log base 10 scale.Note, the difference between male and female samples in this region. Right: Human and cattle Amelogenin sequencealignments using Jalview (version 2.11.2.6). The close up are alignment and consensus sequences: Left, shows the uniqueAmelY peptide identified in human and its alignment with our unique cattle AmelY sequence. Right, cattle AmelY and AmelXalignments showing SAAVs obtained in this research that successfully determine sex in cattle. • Sex determination in cattle PROTEOMS • Bone and dentine • Enamel • Eggshells • Mollusc shells • Mummified remains • Plants Warinner et al., 2022 DOI: 10.1021/acs.chemrev.1c00703 SKIN • Improved taxonomy, beyond taxonomic assignment • Identification of proteins with expression varying in age groups – estimation of age of death from calf skin SKIN • Improved taxonomy, beyond taxonomic assignment • Identification of proteins with expression varying in age groups – estimation of age of death from calf skin Tissue-thin parchment made it possible to produce the first pocket Bibles: Thousands were made in the 13th century. The source of this parchment, often called “uterine vellum,” has been a long-standing controversy in codicology. Use of the Latin term abortivum in many sources has led some scholars to suggest that the skin of fetal calves or sheep was used. Others have argued that it would not be possible to sustain herds if so many pocket Bibles were produced from fetal skins, arguing instead for unexpected alternatives, such as rabbit. Here, we report a simple and objective technique using standard conservation treatments to identify the animal origin of parchment. The noninvasive method is a variant on zooarchaeology by mass spectrometry (ZooMS) peptide mass fingerprinting but extracts protein from the parchment surface by using an electrostatic charge generated by gentle rubbing of a PVC eraser on the membrane surface. Using this method, we analyzed 72 pocket Bibles originating in France, England, and Italy and 293 additional parchment samples that bracket this period. We found no evidence for the use of unexpected animals; however, we did identify the use of more than one mammal species in a single manuscript, consistent with the local availability of hides. These results suggest that ultrafine vellum does not necessarily derive from the use of abortive or newborn animals with ultrathin hides, but could equally well reflect a production process that allowed the skins of maturing animals of several species to be rendered into vellum of equal quality and fineness. Representative examples of ancient proteomes. Well-preserved ancient proteomes contain distinctive groups of proteins that reflect the protein composition of the original tissue or material, such as human bone364 (A), human dental calculus445 (B), artist materials137 (C), and pottery crusts99 (D). As such, the composition of an ancient proteome can aid in its authentication. Data were searched against the SwissProt database using Mascot using the parameters described in ref 102. Protein identifications were established at <5.0% protein FDR and <1.0% peptide FDR in Scaffold v.5 (Proteome Software), and proteins with a minimum of 97% protein identification probability and at least two unique peptides were accepted. The top 15 proteins (by number of PSMs) per sample source were visualized as a treemap and labeled by their corresponding gene name; trypsin, keratins, serum albumin, and microbial proteins were excluded from the analysis. *Ovostatin; **riboflavin-binding protein; ***B3-hordein. Warinner et al., 2022 DOI: 10.1021/acs.chemrev.1c00703 DINOSAUR FEATHERS • „Modern bird feathers more stiff compared to dinosaur“ • Keratins (formerly α-keratins) – softer • Corneous β-proteins (formerly β-keratins) – stiff • Modern bird feather rich in β-keratins – stronger for flight • Further research shows the differences were probably due to taphonomy – change observed in heated feathers (β » α) • „you can’t read the fossil record literally“ Slater et al., 2023 DOI: 10.1038/s41559-023-02177-8 METAPROTEOMS • Microbiomes • Food remains • Pathogens and disease • Cultural heritage • Missing taxa in databases – often palaeoproteomic studies add both modern and ancient proteomes to databases Warinner et al., 2022 DOI: 10.1021/acs.chemrev.1c00703 DIET AND SUBSISTENCE PRACTICES • ZooMS • Food remains, crusts … • Fermentation • Calculus • Plant microremains DIET AND SUBSISTENCE PRACTICES • ZooMS • Food remains, crusts … • Mostly ceramics • Calcified and charred food • Ingredients as well as processing • Fermentation • Calculus • Plant microremains • Sourdough bread made from millet and barley • Fermenting with baker's yeast and lactic acid bacteria • Broom corn (but not foxtail) millet DIET AND SUBSISTENCE PRACTICES • DNA + carbohydrates + proteins from medieval Vitis vinifera • ZooMS • Food remains, crusts … • Fermentation • Calculus • Plant microremains • Usually waterlogged, mineralised, or charred seeds • Arid and/or cold areas • Low protein recovery compared to carbohydrates and lipids in seeds DIET AND SUBSISTENCE PRACTICES HEALTH AND DISEASE • Pathogens • Better phylogenetic resolution with DNA • Enables research of pathophysiology • Often impossible to discern from other bacteria (e.g. Mycobacterium) • Best in combination with palaeogenomics (Mycobacterium leprae in calculus) • Palaeopathology • Palaeogenomic analysis conclusive • Only 4 proteins assigned to Mycobacterium genus HEALTH AND DISEASE • Pathogens • Better phylogenetic resolution with DNA • Enables research of pathophysiology • Often impossible to discern from other bacteria (e.g. Mycobacterium) • Best in combination with palaeogenomics (Mycobacterium leprae in calculus) HEALTH AND DISEASE • Pathogens • Better phylogenetic resolution with DNA • Enables research of pathophysiology • Often impossible to discern from other bacteria (e.g. Mycobacterium) • Best in combination with palaeogenomics (Mycobacterium leprae in calculus) • Immunity HEALTH AND DISEASE • Pathogens • Better phylogenetic resolution with DNA • Enables research of pathophysiology • Often impossible to discern from other bacteria (e.g. Mycobacterium) • Best in combination with palaeogenomics (Mycobacterium leprae in calculus) • Immunity • Medicinal practise • Evidence for the use of honey, cereals, ovicaprine milk and legumes • Cervico-vaginal fluid • Opens up research of stains • „stable isotope analysis from bone proteins and investigations on dental calculus using DNA analysis, light microscopy, and gas chromatography coupled with mass spectrometry“ • „knowledge of ethnopharmacological tradition and the application of medicinal plants (e.g. Punica granatum L., Ephedra sp. L.) were also identified“ • Artemisinin CULTURAL HERITAGE Extensive review – voluntary reading • Paint • Glue, binders CULTURAL HERITAGE • Paint • Glue, binders • Mortars CULTURAL HERITAGE • Paint • Glue, binders • Mortars • Coffins, clothing, tools, toys, religious objects, kayaks, paintings … • Tissue type CULTURAL HERITAGE • Paint • Glue, binders • Mortars • Coffins, clothing, tools, toys, religious objects, kayaks, paintings … • Tissue type • Biocodicology CULTURAL HERITAGE • Paint • Glue, binders • Mortars • Coffins, clothing, tools, toys, religious objects, kayaks, paintings … • Tissue type • Biocodicology • Preservation of proteins is higher • Different information than DNA • Lower taxonomical resolution, tissue specifity, active processes • No latex gloves, wool, silk, leather or exposed skin • Ancient biomolecules laboratories • Feasibility of a few samples • Blanks, injections of blanks between samples in LC-MS/MS • Inject oldest and most precious samples first • Diagenesis (mostly deamidation) • Contaminating taxa in search (cRAP - common Repository of Adventitious Proteins) • Share data in public repositories • Combine with other methods! ! • Dinosaurs, beta and aplha keratins, taphonomy • Birthing girdles, analysis of stains • Kayaks from multiple species • Grape and all seeds • Sex determination CASE STUDIES • Groups of 2 or 3 • Pick a case • Individual proposal • Brainstorming together only AFTER individual work • Project proposal is only a suggestion, you can think of very different approach! • Try what you personally would like to or could do • Scaleable – try showing cost-effective, minimal scenario, as well as wild research fantasy without funding limits • Project proposal (max 1 page, no minimum, bullet points allowed) BEFORE May 15th • 10 min presentation of the project on May 15th • PowerPoint / no props / science slam style / shark tank / (almost) anything goes EXAM • Suggestion for final exam: everyone together with coffee and cookies, defense afterwards • Wednesday, 29th May, 2pm ?