A Label-Free Quantification Method for Assessing Sex from Modern and Ancient Bovine Tooth Enamel

A Label-Free Quantification Method for Assessing Sex from Modern and Ancient Bovine Tooth Enamel | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article A Label-Free Quantification Method for Assessing Sex from Modern and Ancient Bovine Tooth Enamel Paula Kotli This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4638090/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Identification of the sex of modern, fossil and archaeological animal remains offers many insights into their demography, mortality profiles and domestication pathways. However, due to manifold factors, sex determination of osteological remains is often problematic. To overcome this, we have developed an innovative protocol to determine an animal’s sex from tooth enamel, by applying label-free quantification (LFQ) of two unique AmelY peptides ‘ L R Y PYP’ (AmelY;[M+2] 2+ 404.7212 m/z) and ‘ L R Y PYPSY’ (AmelY;[M+2] 2+ 529.7689 m/z) that are only present in the enamel of males. We applied this method to eight modern cattle ( Bos taurus ) of known sex, and correctly assigned them to sex. We then applied the same protocol to twelve archaeological Bos teeth from the Neolithic site of Beisamoun, Israel (8 th –7 th millennium BC) and determined the sex of the archaeological samples. Since teeth are usually better preserved than bones, this innovative protocol has potential to facilitate sex determination in ancient and modern bovine remains, that currently cannot be sexed. Paleozoology Animal Science Archaeology Bioinformatics Paleoproteomics Bovine zooarchaeology peptidomics amelogenin sex determination enamelon enamel proteome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Sexing paleofaunal remains can provide important data on a broad spectrum of issues. These include the animal’s life history, extent of dimorphism, socio-ecological structure and behavior, as well as predator-prey relations and herd management strategies 1 – 7 . However, sex determination of fossil fauna is severely hampered by the often fragmentary nature of specimens, as it relies on the presence of sex-specific morphological features (e.g. on the pelvis, bacula, horns etc.) that are often missing or broken 8 , 9 . Additionally, to determine an animal’s sex, researchers have commonly relied on bone and tooth measurements (osteometries), and more recently, on geometric morphometrics 10 – 15 . However, these methods also rely on the presence of well-preserved remains, while there is often ambivalence in interpreting the mechanism/s responsible for size patterning, since this may be confounded by factors such as climate change, nutrition and domestication 1 , 16 . While, the advent of aDNA analyses has offered revolutionary possibilities for sexing ancient fauna remains 7 , 11 , 13 , 17 , in many situations its application is limited by DNA degradation and contamination 18 . Clearly, additional tools that will enable sexing of fossil (and fragmentary modern) animal remains will be invaluable to researchers in zoology, archaeology, archaeozoology and paleontology. To explore new methods of sexing mammals, researchers have turned to the analysis of amelogenin, an essential protein for tooth enamel development in mammals 19 – 22 . The enamel proteome is composed of around 90% of amelogenin (Amels) dimorphic proteins, comprising the X-linked AmelX that is present in both males and females and the Y-linked AmelY that is only present in males 23 – 25 . Compared to AmelX, AmelY carries multiple single nucleotide polymorphisms (SNPs) or DNA mutation (e.g.: Intron 3, 6 and Exon 5 regions), which translate into single amino acid variation (SAAVs). Thus, the identification of the unique AmelY peptide distinguishes males from females based on relative peptide abundance, and enables the unambiguous identification of males. Numerous studies have used this concept to determine the sex of recent animals by sampling their soft tissues and blood 26 , 27 , 27 , 28 , 28 , 29 , 29 – 35 . This approach also served as the foundation of groundbreaking sex determination in humans first undertaken by Stewart et al. 36 , 37 and Parker et al. 38 through the analysis of native peptides in the tooth enamel proteome using mass spectrometry techniques. Native peptides in enamel are the result of in-vivo enamel protease digestion, such as matrix-metalloproteinase 20 (Mmp20) and kallikrein-related peptidase 4 (Klk4) enzymes 22 , 39 , 40 . The extraction of the native amelogenin peptides facilitates the rapid analysis, without the need for enzymatic digestion that accompanies bottom-up proteomic pipelines that may contribute to sample loss in low abundance samples 41 . This approach has opened the door to sex identification of ancient remains by focusing on tooth enamel, the most resistant material in the skeleton, thereby overcoming the limitations imposed by poor bone preservation commonly encountered in aDNA and other standard sex determination methods outlined above. Not surprisingly, it has been extensively applied to sex ancient human populations including infants and ancient primates 42 – 44 , 44 – 54 . Far fewer studies have been undertaken on tooth enamel of fossil fauna. An exception is the study by Cappellini et al. 55 who successfully identified both the sex and species of fossil rhinoceros remains from the site of Dmanisi, dating to 1.77– 1.9 Mya. As a control, they used a male Medieval ovicaprine’s raw spectral data and the AmelY sequence from modern sheep as a matching database. The authors exploited single amino acid variances (SAAVs) in the AmelY sequence at position 171—where valine (V) replaces methionine (M), found in the corresponding position of the AmelX sequence. This key distinction enabled the sex identification of ancient faunal remains from this site. In the current study, we demonstrate that Label Free Quantification (LFQ) of native peptides extracted from teeth of modern bovine samples permits confident identification of sex. This method was then successfully applied to twelve archaeological samples from a Neolithic sited dated to the 8th − 7th millennium BC, using the same selected peptides. To our knowledge, this is the first application of a method to determine an animal’s sex using bovine tooth enamel. Results Sex Determination in Modern and Ancient Bovine Enamel Sex determination can be assessed through the identification and quantification of the unique peptides of AmelY (for a workflow pipeline, refer to Fig. 1 ). We selected peptides carrying SAAVs: Leu46, Tyr48. Namely ‘ L R Y PYP’ (AmelY;[M + 2] 2+ 404.7212 m/z) and ‘ L R Y PYPSY’ (AmelY;[M + 2] 2+ 529.7689 m/z). The sequence alignment of these unique peptides is illustrated in Fig. 2. In addition, to ensure the correct sex determination and quality of our acid enamel extraction, AmelX dimorphic unique peptides were also quantified. We selected peptides carrying SAAVs, Ser 44, Ile46 and His48. AmelX selected peptides were: ‘ S M(ox) I R H PYP’(AmelX;[M + 2] 2+ 508.7527 m/z) and ‘ I R H PYPSY’ (AmelX;[M + 2] 2+ 516.7667 m/z). A BLAST search for these sequences revealed that they exclusively exhibit homology with amelogenin proteins (reviewed sequence by Uniprot). The identifier (ID) for each of these peptides was validated manually (example in Fig. 3 b) and the isotopic envelope for the associated peak was correct (example in Fig. 3 a). Sex determination was performed by label-free quantification (LFQ) of native dimorphic peptides unique to AmelY. We found modern male samples had an intensity of AmelY unique peptides between XIC 6.47 10 7 to 1.34 10 8 for the short peptide version and between 2.76 10 7 to 8.97 10 8 for the long peptide version. For an example of the disparity between the sexes, in Fig. 4 we plotted sample WIS101 (a modern male) and sample WIS202 (a modern female). Figure 5 shows the intensities of modern male (blue dots) and female (red dots) samples. In addition, we calculated the average AmelX deamidation that was between 21% and 16.42% for Asp and Gln respectively. As proof of concept, we applied the above method to the analysis of archaeological bovine enamel obtained from twelve bovine teeth from the Neolithic (8th − 7th millennium BC) site of Beisamoun (for details on the sites see Materials section below). We were able to determine sex in all archaeological samples. AmelY unique peptides identified six males out of twelve archaeological samples from Beisamoun at XIC 2.76 10 7 to 5.33 10 9 (see Fig. 5 , violet dots). In addition, AmelX unique peptides were found across all archaeological samples at XIC between 1.10 10 8 to 6.87 10 9 (see Fig. 5 , red dots). Sex determination was possible due to the presence of AmelX unique peptides, and the presence or lack of AmelY. See also SI 1 and SI 2. Global deamidation was used as a tool to validate antiquity and study diagenesis in the archaeological samples 56 – 58 . We calculated Asp and Gln bulk deamidation of AmelX and compared the modern and archaeological samples. Archaeological samples showed 3 and 5 fold higher occupancy of deamidation (Asp and Gln, respectively) compared to modern samples. Validating the ancient origin of our fossil samples. Discussion We have presented here, a method for sex determination in bovine enamel based on LFQ of peptides unique to AmelY. We selected two peptides unique to AmelY (’ L R Y PYP’ and the ’ L R Y PYPSY’) that are found exclusively in male bovines. We also found two peptides unique to AmelX (‘ S Mox I R H PYP’ and ‘ I R H PYPSY’). All four peptides are found in both modern and archaeological samples in abundance, allowing reliable quantification and thereby robust sex determination. The use of peptidomics is advantageous, as it simplifies sample preparation and reduces the introduction of sample preparation artefacts, while still providing the desired methodological fidelity. Figure 1. Proteomic workflow for sex determination in modern and archaeological tooth samples.Teeth were cleaned and etched in acid. Acid-dissolved peptides are desalted and run on LC-MS instruments using the DDA method. The data search was undertaken using the Byonic search engine and quantified with Skyline. For greater details see Methods. Figure 2. To illustrate the unique sequence of the selected peptides, we aligned AmelY and AmelX sequences of cattle with our selected AmelY and AmelX peptide sequences (red, green lines respectively) as follow: ‘ L R Y PYP’ (AmelY;[M+2] 2+ 404.7212 m/z), ‘LRYPYPSY’ (AmelY;[M+2] 2+ 529.7689 m/z); AmelX peptide sequences (green lines): ‘ S M(ox) I R H PYP’ (AmelX;[M+2] 2+ 508.7527 m/z) and ‘ I R H PYPSY’ (AmelX;[M+2] 2+ 516.7667 m/z). SAAVs between the dimorphic proteins are show in light blue. Figure 3. XIC of the isotopic envelope precursor obtained for modern male WIS101 for two unique AmelY and two unique AmelX peptides. a.1)‘ L R Y PYP’ AmelY;[M+2] 2+ 404.7212 m/z a.2)‘ L R Y PYPSY’ AmelY;[M+2] 2+ 529.7689 m/z; AmelX unique peptides: a.3) ‘ S M(ox) I R H PYP’ (AmelX;[M+2] 2+ 508.7527 m/z and a.4) ‘ I R H PYPSY’ AmelX;[M+2] 2+ 516.7667 m/z. RT were as expected based on a previous Byonic search ID. Figures from b.1) to b.4) illustrated MS2 spectra of the peptide ID, below is the isotopic envelope of the precursor. Figure 4. XIC of the isotopic envelope precursor obtained using Skyline software for modern male WIS101 (a) and modern female WIS202 (b) for two unique AmelY and two unique AmelX peptides. First, ‘ L R Y PYP’ AmelY;[M+2] 2+ 404.7212 m/z (a.1 -male and b.1 -female), and ‘ L R Y PYPSY’ AmelY;[M+2] 2+ 529.7689 m/z (a.2 -male and b.2 -female). Second, AmelX unique peptides: ‘ S M(ox) I R H PYP’ (AmelX;[M+2] 2+ 508.7527 m/z (a.3 -male and b.3 -female) and ‘ I R H PYPSY’AmelX;[M+2] 2+ 516.7667 m/z (a.4 -male and b.4 -female) the RT were as expected based on previous Byonic search ID. AmelY peptides were exclusively found in male samples, while female samples lacked the unique AmelY peptides. However, both unique peptides of AmelX were found across both sexes. Conclusions Our method provides a simple and reliable method for sex determination in bovines using dental enamel of modern and archaeological samples, through the application of LFQ peptidomics. It will be of particular use for sexing poorly preserved osteological samples, those lacking diagnostic morphology, or assemblages where DNA methods cannot be applied due to poor preservation. Figure 5. Ion intensities of unique AmelY peptides vs AmelX unique peptide intensities, a.1) ‘ L R Y PYP’ (AmelY;[M+2]2+404.7212 m/z) vs ‘ S Mox I R H PYP’ (AmelX;[M+2] 2+ 508.7527 m/z) ; a.2) ‘ L R Y PYPSY’ (AmelY;[M+2] 2+ m/z 529.7689) vs ‘ S Mox I R H PYP’ (AmelX;[M+2] 2+ 508.7527 m/z); a.3) S M(ox) I R H PYP (AmelY;[M+2] 2+ 404.7212 m/z) vs ’ I R H PYPSY’ (AmelX;[M+2] 2+ 516.7667 m/z); c.4) ‘ L R Y PYPSY’ (AmelY;[M+2] 2+ m/z 529.7689) vs ‘ I R H PYPSY’ (AmelX;[M+2] 2+ 516.7667 m/z). Modern male: strong blue dots, modern female: bordeaux (strong red) dots; archaeological samples (Beisamoun) males (found by this method): violet dots and archaeological samples (Beisamoun) females: orange dots. Results are across eight modern samples and twelve archaeological samples. Ethics Statement The modern cattle samples used in this study derive from animals that were sold for slaughter to an authorized slaughterhouse (Beit Mitbahaim Tira Ltd.) and were not intentionally killed for this study. Lower jaws used in this study are routinely discarded by the slaughterhouse as they have no commercial value. The Neolithic site of Beisamoun was excavated under permit from the Israel Antiquities Authority (granted to co-authors FB & HK). For details on the site, its location and excavations, see section on Archaeozoological Samples below. Permission to sample bovine teeth was obtained from the excavators as well as from the archaeozoologist (LKH) working on this assemblage (all of whom are co-authors on this paper). The collection is currently held in the laboratory of the latter researcher, in the National Natural History Collections of the Hebrew University of Jerusalem. Methods Modern Samples A total of eight modern cattle lower jaws were donated by an Israeli meat manufacturer, Beit Mitbahaim Tira ltd.(BTL) (see Table 1 ). For samples pictures, see SI 3. Table 1 The table summarizes sample information including: Sample number, Proteomic Batch, Animal ID, Animal Species, Sex, Tooth type. Sample Number Proteomic Batch Animal ID Species Sex by docs Tooth Type WIS101 20701 Bostaurus10 Bos taurus Male M3 WIS201 20701 Bostaurus16 Bos taurus Female I3 WIS202 20701 Bostaurus14 Bos taurus Female M2 WIS203 20701 Bostaurus12 Bos taurus Male I2 WIS204 20701 Bostaurus11 Bos taurus Male M2 WIS208 20701 Bostaurus17 Bos taurus Female PM3 WIS209 20701 Bostaurus15 Bos taurus Female I3 WIS210 20701 Bostaurus13 Bos taurus Male M2 Archaeological Samples The twelve archaeological teeth samples analyzed derive from the Neolithic site of Beisamoun (see Table 2 , map ref NIG 25403–8/77682): The Neolithic site of Beisamoun is located in the Upper Jordan Valley, within the flood-plain of the now extinct Hula Lake, a region that is characterised by a Mediterranean climate 59 . The locality was first excavated in the 1960’s-1971 by Monique Lechevallier who reported finding remnants of a mid-Pre-Pottery Neolithic B village (PPNB, first half of the 8th millennium BC; Lechevallier 1978) 60 . Renewed excavations in 2007 through 2016, in an adjacent area were undertaken by a Franco–Israeli mission. They exposed in situ layers dating to the late PPNB (second half of the 8th millennium BC) and Pre-Pottery Neolithic C (PPNC, first half of the 7th millennium BC). The finds included architectural remains and built installations, with clearly identifiable successive phases of building and their associated lithic artefacts (e.g. arrowheads, sickle blades, axes, adzes, burins, scrapers, retouched blades etc.), groundstone artefacts (grinding slabs, mortars, hand stones, vessels etc.) predominantly made on basalt and limestone, bone tools, shell, stone and bone ornaments, as well as human burials and faunal remains (Bocquentin et al. 2014, 2020) 61 , 62 . The PPNC sediment is composed of a sandy-clay sediment with fine-grained inclusions of charcoal, clay, and ochre (Bocquentin et al. 2014) 61 . For samples pictures, see SI 3. Samples Preparation and Cleaning Teeth (modern and archaeological) were first cleaned mechanically using soft brushes to exclude any external material (e.g. sediments) was performed with bistoury and tweezers under a microscope binocular (Nikon SMZ800N, see picture SI 6). A clean enamel piece (10mm x 15 mm) was extracted. A final examination was carried out using a Nikon SMZ800N binocular microscope. Finally, etching was performed under a chemical hood (see subsection, Sample Preparation for MS). Sample Preparation for MS The extracted and clean piece of bovine enamel was immersed for 30 seconds in 3% H 2 O 2 , rinsed with DDW and the solution discarded. Then, the enamel piece was etched for 2 minutes in freshly prepared 5% HCl. This solution was discarded. A second etching step in 1000 µl 5% HCl for 60 minutes was performed at room temperature, and the solution was retained in a new Eppendorf on ice. The section was then allowed to completely dissolve in 1000 µl 5% HCl (aprox. 90 min). Then, both solutions were then combined to a final volume of 2000 µl (about 100 mg dry weight). The samples were then frozen and stored in -80C for desalting. Mass Spectrometry Dissolved samples were desalted using Oasis HLB 96 well plate (Waters), using the manufacturer’s instructions; in short samples were loaded onto the wells using vacuum pull, followed by 3 washes of 300ul 0.1% TFA. the peptides were then eluted by passing 50ul 50% Acetonitrile, 0.1% FA. The resulting peptides were loaded onto a reversed-phase Symmetry C18 trapping column (20*0.18 mm, 5 µm particle, Water) and resolved on an analytical HSS T3 column (250*0.07 5mm, 1.8 µm particles, Water), mounted on a nanoAquity (Waters) nanoLC instrument. Peptides were eluted from the column using a 50-minute gradient from 4–30%B (99.9% acetonitrile, 0.1% formic acid) at 350 nl/min flow. The peptides were eluted into a Q-Exactive HF mass spectrometer (Thermo Fisher) using a FlexIon nano-ESI source, through a 20 µm ID emitter (Fossil IonTech, Madrid), at 2.8kV. Blank injections interspaced sample to allow washing off peptide carryover. Data were acquired in data-dependent acquisition (DDA) mode using Top10 method. MS1 resolution was set to 120,000@ 400 m/z, with a mass range of 350–1650 m/z, using a maximum injection time of 60 ms. Precursors selected for MS2 were limited to charge states of 2–8, intensity threshold of 3.3 4 and dynamic exclusion of 20s. Precursors were isolated using a window of 1.7 m/z, AGC target was set to 10 5 or a maximum of 60ms injection time, fragmented using HCD at 27 normalized collision energy (NCE). Scans were performed using a first mass setting of 100 m/z at 15,000 resolution (@200 m/z). Table 2 The table summarizes sample information including: Sample Number, Sample Locus and Square in the archaeological site of Beisamoun, Animal Species, Sex by Amelogenin (this research study), Tooth type, Period. Samples Number Proteomic Batch Loc. Sq. (Location Square) Species Sex by Amels Tooth Type Period WIS116 20701 BN14, Area E, Loc.334, sq. T7b/T8a/U7c/U8d, Numb. 3316 Bos Male LM1-right PPNC WIS115 20701 BN14, Area E, sq: T7c/d, Numb. 3291 Bos Male UPM3- left PPNC WIS164 20701 BN13, Area E, Loc. 371, sq. S7a / T7d, Numb. 3174 Bos Female UM3 right PPNC WIS111 20701 BN12, Area E, sq:R9d; Loc. 339, Numb. 2877 Bos Male LM2 PPNC WIS100 20701 BN12, Area E, Loc.333, sq: S10d/R10b, Numb. 2727 Bos Male PM4-right PPNC WIS114 20701 BN14, Area E, Loc.352, Numb. 3391, sq: U7a-d Bos Female LM1 left PPNC WIS102 20701 BN10,AreaF,Sq.W25a; Numb.1827 Bos Female LM PPNB-PPNC WIS110 20701 BN 11, Area F, Loc. 228, sq: V24c; Numb. 5034 Bos Female UM-1/2 PPNB-PPNC WIS165 20701 BN16, Area E, sq. S7 a/d, Numb. 3907 Bos Female UM1 right PPNB-PPNC WIS166 20701 BN11, Area F, Numb.5045, sq. T21b, Loc 243/246 Bos Female LM1 right PPNB-PPNC WIS163 20701 BN10, Area F, q.U25d, Numb. 1833 Bos Male UM(?) PPNB-PPNC WIS109 20701 BN 11, Area F, Numb.5021 sq. U26c Bos Male UM-1/2- left PPNB-PPNC Data analysis Data was searched using Byonic search engine (Protein Metrics Inc) using a database that was tailored for modern and ancient samples against the relevant sequences (P02817 AMELX BOVIN Amelogenin, X isoform, Bos taurus and Q99004 AMELY BOVIN Amelogenin, Y isoform Bos taurus , Uniprot). Fixed and variable modifications were adjusted as follows: common2: Oxidation (F, H, K, M, P, W, Y), Deamidated (N, Q, R); rare1: Acetyl (NTerm), Gln to pyro-Glu (NTerm Q), Glu to pyro-Glu (NTerm E) and Phosphorilation (S); rare2: Di-oxidation (F, M, P, W, Y). maximum of 2 common and 2 rare modifications were allowed. MS1 tolerance was set to 10ppm while MS2 was set to 20ppm. Data was filtered using Byonic’s target-decoy method set to 1% percent FDR. We used a post-search cut-off of LogProb > 3 (FDR 200. Identified unique AmelY peptides were examined manually to avoid false positive identifications. Quantification was performed using Skyline software (version 23.1.0.455). Byonic mzID files were imported into Skyline along with the RAW files and used to identify the correct peak for quantification positively. Quantification was performed by XIC extraction of the MS1 signal. Quantification of AmelY base on peptide1: ‘LRYPYP’ (AmelY;[M + 2] 2+ 404.7212 m/z); peptode2:‘LRYPYPSY’ (AmelY;[M + 2] 2+ 529.7689 m/z). AmelX: peptide3:‘SMoxIRHPYP’ (AmelX;[M + 2] 2+ 508.7527 m/z); peptide4:‘IRHPYPSY’ (AmelX;[M + 2] 2+ 516.7667 m/z). Ion fragment validation was performed manually. We performed sequence similarity searches using protein-protein BLAST program ( www.uniprot.org/blast ). Bulk modification calculation were perform by in-house RStudio scrips. Declarations Data Availability The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 63 partner repository with following dataset identifiers:1) PXD053146, 10.6019/PXD053146; 2) PXD053148, 10.6019/PXD053148. LFQ files at PXD053225. ProteomeXchange via the PRIDE database - Password and User Names for Reviewers The data was divided into 2 projects; see all the details below. a) Project Name: Label-Free Quantification Method for Assessing Sex from Modern and Ancient Bovine Tooth Enamel. Project accession: PXD053146 Project DOI: 10.6019/PXD053146 Reviewer account details: – Username: [email protected] b) Project Name: Label-Free Quantification Method for Assessing Sex from Modern and Ancient Bovine Tooth Enamel PART II. Project accession: PXD0531486 • Project DOI: 10.6019/PXD053148 Reviewer account details: – Username: [email protected] c) Project Name: Label-Free Quantification Method for Assessing Sex from Modern and Ancient Bovine Tooth Enamel. • Project accession: PXD053225 Token: dU4QhIY3tPti Reviewer account details: – Username: [email protected] Acknowledgements P.K. was supported by the Helen and Martin Kimmel Center for Archaeological Science. We wish to thank the George Schwartzman Fund for funding laboratory work and sample analyses. Author contributions statement P.K. E.B. and L.K.H. conceived the idea; P.K. and E.B. designed the study; P.K. performed all laboratory work and analyses; L.K.H., F.B. and H.K. provided the archaeological samples; P.K., D.M. and E.B. analyzed and interpreted the data; P.K. wrote the manuscript with the contribution of E.B., .D.M. and L.K.H. References Horwitz, L., Cope, C. & Tchernov, E. 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Analysis of 5000 year-old human teeth using optimized large-scale and targeted proteomics approaches for detection of sex-specific peptides. J. Proteomics 211, 103548 (2020). Rebay-Salisbury, K. et al. Child murder in the Early Bronze Age: Proteomic sex identification of a cold case from Schleinbach, Austria. Archaeol. Anthropol. Sci. 12, 1–13 (2020). Granja, R. et al. Unbalanced sex-ratio in the Neolithic individuals from the Escoural Cave (Montemor-o-Novo, Portugal) revealed by peptide analysis. Sci. Reports 13, 19902 (2023). Gasparini, A. et al. Biological sex vs. archaeological gender: Enamel peptide analysis of the horsemen of the Early Middle age necropolises of Campochiaro (Molise, Italy). J. Archaeol. Sci. Reports 41, 103337 (2022). Koenig, C. et al. Automated high-throughput biological sex identification from archaeological human dental enamel using targeted proteomics. BioRxiv 2024–02 (2024). Lugli, F. et al. Enamel peptides reveal the sex of the Late Antique ‘Lovers of Modena’. Sci. Reports 9, 13130 (2019). Lugli, F. et al. Sex-related morbidity and mortality in non-adult individuals from the Early Medieval site of Valdaro (Italy): the contribution of dental enamel peptide analysis. J. Archaeol. Sci. Reports 34, 102625 (2020). Welker, F. et al. The dental proteome of Homo antecessor . Nature 580, 235–238 (2020). Gowland, R. et al. Sex estimation of teeth at different developmental stages using dimorphic enamel peptide analysis. Am. J. Phys. Anthropol. 174, 859–869 (2021). Demeter, F. et al. A Middle Pleistocene Denisovan molar from the Annamite chain of northern Laos. Nat. Comm. 13, 2557 (2022). Mikšík, I., Morvan, M. & Bru˚žek, J. Peptide analysis of tooth enamel–a sex estimation tool for archaeological, anthropological, or forensic research. J. Sep. Sci. 46, 2300183 (2023). Gamble, J. A. et al. Advancing sex estimation from amelogenin: Applications to archaeological, deciduous, and fragmentary dental enamel. J. Archaeol. Sci. Reports 54, 104430 (2024). Welker, F. et al. Enamel proteome shows that Gigantopithecus was an early diverging Pongine. Nature 576, 262–265 (2019). Cappellini, E. et al. Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny. Nature 574, 103–107 (2019). Hendy, J. et al. A guide to ancient protein studies. Nat. Ecol. & Evol. 2, 791–799 (2018). Coutu, A. N. et al. Palaeoproteomics confirm earliest domesticated sheep in Southern Africa ca. 2000 bp. Sci. Reports 11, 6631 (2021). Cucina, A. et al. Meta-proteomic analysis of two mammoth’s trunks by EVA technology and high-resolution mass spectrometry for an indirect picture of their habitat and the characterization of the Collagen Type I, Alpha-1 and Alpha-2 sequence. Amino Acids 54, 935–954 (2022). Danin, A. Flora and vegetation of israel and adjacent areas. In TheZoogeography of Israel , vol. 30, 251–276 (Dr. W. Junk Publishers Dordrecht, The Netherlands, 1988). Lechevallier, M. (ed.) Abou Gosh et Beisamoun. Deux gisements du VII millénaire avant l’ère Chrétienne en Israël (Mém et Trav du Centre de Recherches Préhistoriques Français de Jérusalem 2, 1978). Bocquentin, F. et al. Renewed excavations at Beisamoun: investigating the 7th millennium cal. BC of the Southern Levant. J. Isr. Prehist. Soc. 44, 5–100 (2014). Bocquentin, F. et al. Between two worlds: The PPNB–PPNC transition in the central Levant as seen through discoveries at Beisamoun. In The Mega-project at Motza (Moza): The Neolithic and Later Occupations up to the 20th Century. New Studies in the Archaeology of Jerusalem and its Region. , 163–199 (Israel Antiquities Authority, Jerusalem, 2020). Perez-Riverol, Y. et al. The pride database resources in 2022: A hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 50, D543–D552 (2022). Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryInformationKotlietal.2024.pdf Supplementary Information See files: "Supplementary Information Kotli et al. 2024.pdf" and "20701 Samples order in the MS.xlsx". Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4638090","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":318973628,"identity":"4d48886d-447b-45b7-bef7-7dc6b4128950","order_by":0,"name":"Paula Kotli","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIie3QsUrDQBzH8d9xkCyHrgdKfALhoHA49V6lJVCXiwguAYtECskSnH0N3+BKIC6RrgWXdnHKoIs4lOJZClLppavDfdf/ffj/OcDn+4epDMQAAyAk9wuA/46CcD8R9v2GMEomYpfQgwQB3525yHMjzBdGkaI0v2XphTovXuoFxn0cOYgqtZiW0D17WP7KGk5lc3UpUMfuw6CFYUiH5Q9Jch5IoyVHYNzkuBXT1ZbcJGvO5Ky1ZN1BuBYVg94QmmScy7ndQvIu8nZdnYpRj1VkcrKquZBzu2X4EDP3YfHTsk3jKCyK5cfj+E7Jmd3y/tmPzopsv9l+wp8GAOt47/P5fL4DfQPkI0/FAOr8mAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0007-1238-0150","institution":"Weizmann Institute of Sciencec","correspondingAuthor":true,"prefix":"","firstName":"Paula","middleName":"","lastName":"Kotli","suffix":""}],"badges":[],"createdAt":"2024-06-25 17:31:02","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-4638090/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4638090/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59087362,"identity":"dd068388-a985-47ee-99db-21eb9ab43860","added_by":"auto","created_at":"2024-06-26 08:11:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":126311,"visible":true,"origin":"","legend":"\u003cp\u003eProteomic workflow for sex determination in modern and archaeological tooth samples.Teeth were cleaned and etched in acid. Acid-dissolved peptides are desalted and run on LC-MS instruments using the DDA method. The data search was undertaken using the Byonic search engine and quantified with Skyline. For greater details see Methods.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4638090/v1/790768b803349b445d4c8d76.png"},{"id":59087360,"identity":"9370a6d7-a886-4747-84a6-ac52874d957e","added_by":"auto","created_at":"2024-06-26 08:11:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":104563,"visible":true,"origin":"","legend":"\u003cp\u003eTo illustrate the unique sequence of the selected peptides, we aligned AmelY and AmelX sequences of cattle with our selected AmelY and AmelX peptide sequences (red, green lines respectively) as follow: ‘\u003cu\u003eL\u003c/u\u003eR\u003cu\u003eY\u003c/u\u003ePYP’ (AmelY;[M+2]\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e404.7212 m/z), ‘LRYPYPSY’ (AmelY;[M+2]\u003csup\u003e2+ \u003c/sup\u003e529.7689 m/z); AmelX peptide sequences (green lines): ‘\u003cu\u003eS\u003c/u\u003eM(ox)\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYP’ (AmelX;[M+2]\u003csup\u003e2+ \u003c/sup\u003e508.7527 m/z) and ‘\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYPSY’ (AmelX;[M+2]\u003csup\u003e2+ \u003c/sup\u003e516.7667 m/z). SAAVs between the dimorphic proteins are show in light blue.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4638090/v1/f9871020b874d32ccda62e8a.png"},{"id":59087365,"identity":"3487ba82-a75f-4e97-8f59-3f2ae294fcf2","added_by":"auto","created_at":"2024-06-26 08:11:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":177581,"visible":true,"origin":"","legend":"\u003cp\u003eXIC of the isotopic envelope precursor obtained for modern male WIS101 for two unique AmelY and two unique AmelX peptides. a.1)‘\u003cu\u003eL\u003c/u\u003eR\u003cu\u003eY\u003c/u\u003ePYP’ AmelY;[M+2]\u003csup\u003e2+ \u003c/sup\u003e404.7212 m/z a.2)‘\u003cu\u003eL\u003c/u\u003eR\u003cu\u003eY\u003c/u\u003ePYPSY’ AmelY;[M+2]\u003csup\u003e2+ \u003c/sup\u003e529.7689 m/z; AmelX unique peptides: a.3) ‘\u003cu\u003eS\u003c/u\u003eM(ox)\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYP’ (AmelX;[M+2]\u003csup\u003e2+ \u003c/sup\u003e508.7527 m/z and a.4) ‘\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYPSY’ AmelX;[M+2]\u003csup\u003e2+ \u003c/sup\u003e516.7667 m/z. RT were as expected based on a previous Byonic search ID. Figures from b.1) to b.4) illustrated MS2 spectra of the peptide ID, below is the isotopic envelope of the precursor.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4638090/v1/a4417efd53efaa6825542d40.png"},{"id":59087366,"identity":"f1be2dbe-a168-4b25-bf66-d0123d6e1403","added_by":"auto","created_at":"2024-06-26 08:11:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":88636,"visible":true,"origin":"","legend":"\u003cp\u003eXIC of the isotopic envelope precursor obtained using Skyline software for modern male WIS101 (a) and modern female WIS202 (b) for two unique AmelY and two unique AmelX peptides. First, ‘\u003cu\u003eL\u003c/u\u003eR\u003cu\u003eY\u003c/u\u003ePYP’ AmelY;[M+2]\u003csup\u003e2+ \u003c/sup\u003e404.7212 m/z (a.1 -male and b.1 -female), and ‘\u003cu\u003eL\u003c/u\u003eR\u003cu\u003eY\u003c/u\u003ePYPSY’ AmelY;[M+2]\u003csup\u003e2+ \u003c/sup\u003e529.7689 m/z (a.2 -male and b.2 -female). Second, AmelX unique peptides: ‘\u003cu\u003eS\u003c/u\u003eM(ox)\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYP’ (AmelX;[M+2]\u003csup\u003e2+ \u003c/sup\u003e508.7527 m/z (a.3 -male and b.3 -female) and ‘\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYPSY’ AmelX;[M+2]\u003csup\u003e2+ \u003c/sup\u003e516.7667 m/z (a.4 -male and b.4 -female) the RT were as expected based on previous Byonic search ID. AmelY peptides were exclusively found in male samples, while female samples lacked the unique AmelY peptides. However, both unique peptides of AmelX were found across both sexes.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4638090/v1/0aff0329cce445fabac41741.png"},{"id":59087896,"identity":"e64e8c83-d980-42fb-a29c-70d155554d83","added_by":"auto","created_at":"2024-06-26 08:19:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":55438,"visible":true,"origin":"","legend":"\u003cp\u003eIon intensities of unique AmelY peptides vs AmelX unique peptide intensities, a.1) ‘LRYPYP’ (AmelY;[M+2]2+ 404.7212 m/z) vs ‘SMoxIRHPYP’ (AmelX;[M+2]2+ 508.7527 m/z) ; a.2) ‘LRYPYPSY’ (AmelY;[M+2]2+ m/z 529.7689) vs ‘SMoxIRHPYP’ (AmelX;[M+2]2+ 508.7527 m/z); a.3) SM(ox)IRHPYP (AmelY;[M+2]2+ 404.7212 m/z) vs ’IRHPYPSY’ (AmelX;[M+2]2+ 516.7667 m/z); c.4) ‘LRYPYPSY’ (AmelY;[M+2]2+ m/z 529.7689) vs ‘IRHPYPSY’ (AmelX;[M+2]2+ 516.7667 m/z). Modern male: strong blue dots, modern female: bordeaux (strong red) dots; archaeological samples (Beisamoun) males (found by this method): violet dots and archaeological samples (Beisamoun) females: orange dots. Results are across eight modern samples and twelve archaeological samples.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4638090/v1/16bf039e265a68e59b799328.png"},{"id":59088705,"identity":"7f6aeda9-368e-40b9-aaed-01eebc3bb595","added_by":"auto","created_at":"2024-06-26 08:27:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1098425,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4638090/v1/a7946da4-ab52-408d-9c71-b7bbff0dd84e.pdf"},{"id":59087361,"identity":"986758eb-345d-4a29-a8f8-0786b3d0bedf","added_by":"auto","created_at":"2024-06-26 08:11:57","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2193417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSee files: \"Supplementary Information Kotli et al. 2024.pdf\" and \"20701 Samples order in the MS.xlsx\".\u003c/p\u003e","description":"","filename":"SupplementaryInformationKotlietal.2024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4638090/v1/49023ae0ffc2a223ca2778fd.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eA Label-Free Quantification Method for Assessing Sex from Modern and Ancient Bovine Tooth Enamel\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSexing paleofaunal remains can provide important data on a broad spectrum of issues. These include the animal\u0026rsquo;s life history, extent of dimorphism, socio-ecological structure and behavior, as well as predator-prey relations and herd management strategies\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, sex determination of fossil fauna is severely hampered by the often fragmentary nature of specimens, as it relies on the presence of sex-specific morphological features (e.g. on the pelvis, bacula, horns etc.) that are often missing or broken\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Additionally, to determine an animal\u0026rsquo;s sex, researchers have commonly relied on bone and tooth measurements (osteometries), and more recently, on geometric morphometrics\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, these methods also rely on the presence of well-preserved remains, while there is often ambivalence in interpreting the mechanism/s responsible for size patterning, since this may be confounded by factors such as climate change, nutrition and domestication\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. While, the advent of aDNA analyses has offered revolutionary possibilities for sexing ancient fauna remains\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, in many situations its application is limited by DNA degradation and contamination\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Clearly, additional tools that will enable sexing of fossil (and fragmentary modern) animal remains will be invaluable to researchers in zoology, archaeology, archaeozoology and paleontology.\u003c/p\u003e \u003cp\u003eTo explore new methods of sexing mammals, researchers have turned to the analysis of amelogenin, an essential protein for tooth enamel development in mammals\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The enamel proteome is composed of around 90% of amelogenin (Amels) dimorphic proteins, comprising the X-linked AmelX that is present in both males and females and the Y-linked AmelY that is only present in males\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Compared to AmelX, AmelY carries multiple single nucleotide polymorphisms (SNPs) or DNA mutation (e.g.: Intron 3, 6 and Exon 5 regions), which translate into single amino acid variation (SAAVs). Thus, the identification of the unique AmelY peptide distinguishes males from females based on relative peptide abundance, and enables the unambiguous identification of males. Numerous studies have used this concept to determine the sex of recent animals by sampling their soft tissues and blood\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan additionalcitationids=\"CR30 CR31 CR32 CR33 CR34\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This approach also served as the foundation of groundbreaking sex determination in humans first undertaken by Stewart et al.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and Parker et al.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e through the analysis of native peptides in the tooth enamel proteome using mass spectrometry techniques. Native peptides in enamel are the result of in-vivo enamel protease digestion, such as matrix-metalloproteinase 20 (Mmp20) and kallikrein-related peptidase 4 (Klk4) enzymes\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe extraction of the native amelogenin peptides facilitates the rapid analysis, without the need for enzymatic digestion that accompanies bottom-up proteomic pipelines that may contribute to sample loss in low abundance samples\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis approach has opened the door to sex identification of ancient remains by focusing on tooth enamel, the most resistant material in the skeleton, thereby overcoming the limitations imposed by poor bone preservation commonly encountered in aDNA and other standard sex determination methods outlined above. Not surprisingly, it has been extensively applied to sex ancient human populations including infants and ancient primates\u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan additionalcitationids=\"CR45 CR46 CR47 CR48 CR49 CR50 CR51 CR52 CR53\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFar fewer studies have been undertaken on tooth enamel of fossil fauna. An exception is the study by Cappellini et al.\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e who successfully identified both the sex and species of fossil rhinoceros remains from the site of Dmanisi, dating to 1.77\u0026ndash; 1.9 Mya. As a control, they used a male Medieval ovicaprine\u0026rsquo;s raw spectral data and the AmelY sequence from modern sheep as a matching database. The authors exploited single amino acid variances (SAAVs) in the AmelY sequence at position 171\u0026mdash;where valine (V) replaces methionine (M), found in the corresponding position of the AmelX sequence. This key distinction enabled the sex identification of ancient faunal remains from this site.\u003c/p\u003e \u003cp\u003eIn the current study, we demonstrate that Label Free Quantification (LFQ) of native peptides extracted from teeth of modern bovine samples permits confident identification of sex. This method was then successfully applied to twelve archaeological samples from a Neolithic sited dated to the 8th \u0026minus;\u0026thinsp;7th millennium BC, using the same selected peptides. To our knowledge, this is the first application of a method to determine an animal\u0026rsquo;s sex using bovine tooth enamel.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSex Determination in Modern and Ancient Bovine Enamel\u003c/h2\u003e \u003cp\u003eSex determination can be assessed through the identification and quantification of the unique peptides of AmelY (for a workflow pipeline, refer to Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We selected peptides carrying SAAVs: Leu46, Tyr48. Namely \u0026lsquo;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eL\u003c/span\u003eR\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eY\u003c/span\u003ePYP\u0026rsquo; (AmelY;[M\u0026thinsp;+\u0026thinsp;2]\u003csup\u003e2+\u003c/sup\u003e 404.7212 m/z) and \u0026lsquo;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eL\u003c/span\u003eR\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eY\u003c/span\u003ePYPSY\u0026rsquo; (AmelY;[M\u0026thinsp;+\u0026thinsp;2]\u003csup\u003e2+\u003c/sup\u003e 529.7689 m/z). The sequence alignment of these unique peptides is illustrated in Fig.\u0026nbsp;2. In addition, to ensure the correct sex determination and quality of our acid enamel extraction, AmelX dimorphic unique peptides were also quantified. We selected peptides carrying SAAVs, Ser 44, Ile46 and His48. AmelX selected peptides were:\u003c/p\u003e \u003cp\u003e\u0026lsquo;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS\u003c/span\u003eM(ox)\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eI\u003c/span\u003eR\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003ePYP\u0026rsquo;(AmelX;[M\u0026thinsp;+\u0026thinsp;2]\u003csup\u003e2+\u003c/sup\u003e 508.7527 m/z) and \u0026lsquo;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eI\u003c/span\u003eR\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003ePYPSY\u0026rsquo; (AmelX;[M\u0026thinsp;+\u0026thinsp;2]\u003csup\u003e2+\u003c/sup\u003e 516.7667 m/z). A BLAST search for these sequences revealed that they exclusively exhibit homology with amelogenin proteins (reviewed sequence by Uniprot). The identifier (ID) for each of these peptides was validated manually (example in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) and the isotopic envelope for the associated peak was correct (example in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Sex determination was performed by label-free quantification (LFQ) of native dimorphic peptides unique to AmelY. We found modern male samples had an intensity of AmelY unique peptides between XIC 6.47 10\u003csup\u003e7\u003c/sup\u003e to 1.34 10\u003csup\u003e8\u003c/sup\u003e for the short peptide version and between 2.76 10\u003csup\u003e7\u003c/sup\u003e to 8.97 10\u003csup\u003e8\u003c/sup\u003e for the long peptide version. For an example of the disparity between the sexes, in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e we plotted sample WIS101 (a modern male) and sample WIS202 (a modern female). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the intensities of modern male (blue dots) and female (red dots) samples. In addition, we calculated the average AmelX deamidation that was between 21% and 16.42% for Asp and Gln respectively.\u003c/p\u003e \u003cp\u003eAs proof of concept, we applied the above method to the analysis of archaeological bovine enamel obtained from twelve bovine teeth from the Neolithic (8th \u0026minus;\u0026thinsp;7th millennium BC) site of Beisamoun (for details on the sites see Materials section below). We were able to determine sex in all archaeological samples. AmelY unique peptides identified six males out of twelve archaeological samples from Beisamoun at XIC 2.76 10\u003csup\u003e7\u003c/sup\u003e to 5.33 10\u003csup\u003e9\u003c/sup\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e, violet dots). In addition, AmelX unique peptides were found across all archaeological samples at XIC between 1.10 10\u003csup\u003e8\u003c/sup\u003e to 6.87 10\u003csup\u003e9\u003c/sup\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e, red dots). Sex determination was possible due to the presence of AmelX unique peptides, and the presence or lack of AmelY. See also SI 1 and SI 2.\u003c/p\u003e \u003cp\u003eGlobal deamidation was used as a tool to validate antiquity and study diagenesis in the archaeological samples\u003csup\u003e\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. We calculated Asp and Gln bulk deamidation of AmelX and compared the modern and archaeological samples. Archaeological samples showed 3 and 5 fold higher occupancy of deamidation (Asp and Gln, respectively) compared to modern samples. Validating the ancient origin of our fossil samples.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe have presented here, a method for sex determination in bovine enamel based on LFQ of peptides unique to AmelY. We selected two peptides unique to AmelY (\u0026rsquo;\u003cu\u003eL\u003c/u\u003eR\u003cu\u003eY\u003c/u\u003ePYP\u0026rsquo; and the \u0026rsquo;\u003cu\u003eL\u003c/u\u003eR\u003cu\u003eY\u003c/u\u003ePYPSY\u0026rsquo;) that are found exclusively in male bovines. We also found two peptides unique to AmelX (\u0026lsquo;\u003cu\u003eS\u003c/u\u003eMox\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYP\u0026rsquo; and \u0026lsquo;\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYPSY\u0026rsquo;). All four peptides are found in both modern and archaeological samples in abundance, allowing reliable quantification and thereby robust sex determination. The use of peptidomics is advantageous, as it simplifies sample preparation and reduces the introduction of sample preparation artefacts, while still providing the desired methodological fidelity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 1.\u0026nbsp;\u003c/strong\u003eProteomic workflow for sex determination in modern and archaeological tooth samples.Teeth were cleaned and etched in acid. Acid-dissolved peptides are desalted and run on LC-MS instruments using the DDA method. The data search was undertaken using the Byonic search engine and quantified with Skyline. For greater details see Methods.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 2.\u0026nbsp;\u003c/strong\u003eTo illustrate the unique sequence of the selected peptides, we aligned AmelY and AmelX sequences of cattle with our selected AmelY and AmelX peptide sequences (red, green lines respectively) as follow: \u0026lsquo;\u003cu\u003eL\u003c/u\u003eR\u003cu\u003eY\u003c/u\u003ePYP\u0026rsquo; (AmelY;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e404.7212 m/z), \u0026lsquo;LRYPYPSY\u0026rsquo; (AmelY;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e529.7689 m/z); AmelX peptide sequences (green lines): \u0026lsquo;\u003cu\u003eS\u003c/u\u003eM(ox)\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYP\u0026rsquo; (AmelX;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e508.7527 m/z) and \u0026lsquo;\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYPSY\u0026rsquo; (AmelX;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e516.7667 m/z). SAAVs between the dimorphic proteins are show in light blue.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 3.\u0026nbsp;\u003c/strong\u003eXIC of the isotopic envelope precursor obtained for modern male WIS101 for two unique AmelY and two unique AmelX peptides. a.1)\u0026lsquo;\u003cu\u003eL\u003c/u\u003eR\u003cu\u003eY\u003c/u\u003ePYP\u0026rsquo; AmelY;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e404.7212 m/z a.2)\u0026lsquo;\u003cu\u003eL\u003c/u\u003eR\u003cu\u003eY\u003c/u\u003ePYPSY\u0026rsquo; AmelY;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e529.7689 m/z; AmelX unique peptides: a.3) \u0026lsquo;\u003cu\u003eS\u003c/u\u003eM(ox)\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYP\u0026rsquo; (AmelX;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e508.7527 m/z and a.4) \u0026lsquo;\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYPSY\u0026rsquo; AmelX;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e516.7667 m/z. RT were as expected based on a previous Byonic search ID. Figures from b.1) to b.4) illustrated MS2 spectra of the peptide ID, below is the isotopic envelope of the precursor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 4.\u0026nbsp;\u003c/strong\u003eXIC of the isotopic envelope precursor obtained using Skyline software for modern male WIS101 (a) and modern female WIS202 (b) for two unique AmelY and two unique AmelX peptides. First, \u0026lsquo;\u003cu\u003eL\u003c/u\u003eR\u003cu\u003eY\u003c/u\u003ePYP\u0026rsquo; AmelY;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e404.7212 m/z (a.1 -male and b.1 -female), and \u0026lsquo;\u003cu\u003eL\u003c/u\u003eR\u003cu\u003eY\u003c/u\u003ePYPSY\u0026rsquo; AmelY;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e529.7689 m/z (a.2 -male and b.2 -female). Second, AmelX unique peptides: \u0026lsquo;\u003cu\u003eS\u003c/u\u003eM(ox)\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYP\u0026rsquo; (AmelX;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e508.7527 m/z (a.3 -male and b.3 -female) and \u0026lsquo;\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYPSY\u0026rsquo;AmelX;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e516.7667 m/z (a.4 -male and b.4 -female) the RT were as expected based on previous Byonic search ID. AmelY peptides were exclusively found in male samples, while female samples lacked the unique AmelY peptides. However, both unique peptides of AmelX were found across both sexes.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur method provides a simple and reliable method for sex determination in bovines using dental enamel of modern and archaeological samples, through the application of LFQ peptidomics. It will be of particular use for sexing poorly preserved osteological samples, those lacking diagnostic morphology, or assemblages where DNA methods cannot be applied due to poor preservation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 5.\u0026nbsp;\u003c/strong\u003eIon intensities of unique AmelY peptides vs AmelX unique peptide intensities, a.1) \u0026lsquo;\u003cu\u003eL\u003c/u\u003eR\u003cu\u003eY\u003c/u\u003ePYP\u0026rsquo; (AmelY;[M+2]2+404.7212 m/z) vs \u0026lsquo;\u003cu\u003eS\u003c/u\u003eMox\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYP\u0026rsquo; (AmelX;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e508.7527 m/z) ; a.2) \u0026lsquo;\u003cu\u003eL\u003c/u\u003eR\u003cu\u003eY\u003c/u\u003ePYPSY\u0026rsquo; (AmelY;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003em/z 529.7689) vs\u003c/p\u003e\n\u003cp\u003e\u0026lsquo;\u003cu\u003eS\u003c/u\u003eMox\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYP\u0026rsquo; (AmelX;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e508.7527 m/z); a.3) \u003cu\u003eS\u003c/u\u003eM(ox)\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYP (AmelY;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e404.7212 m/z) vs \u0026rsquo;\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYPSY\u0026rsquo;\u003c/p\u003e\n\u003cp\u003e(AmelX;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e516.7667 m/z); c.4) \u0026lsquo;\u003cu\u003eL\u003c/u\u003eR\u003cu\u003eY\u003c/u\u003ePYPSY\u0026rsquo; (AmelY;[M+2]\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003em/z 529.7689) vs \u0026lsquo;\u003cu\u003eI\u003c/u\u003eR\u003cu\u003eH\u003c/u\u003ePYPSY\u0026rsquo; (AmelX;[M+2]\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e516.7667 m/z). Modern male: strong blue dots, modern female: bordeaux (strong red) dots; archaeological samples (Beisamoun) males (found by this method): violet dots and archaeological samples (Beisamoun) females: orange dots. Results are across eight modern samples and twelve archaeological samples.\u003c/p\u003e\n\u003cp\u003eEthics Statement\u003c/p\u003e\n\u003cp\u003eThe modern cattle samples used in this study derive from animals that were sold for slaughter to an authorized slaughterhouse\u003c/p\u003e\n\u003cp\u003e(Beit Mitbahaim Tira Ltd.) and were not intentionally killed for this study. Lower jaws used in this study are routinely discarded by the slaughterhouse as they have no commercial value. The Neolithic site of Beisamoun was excavated under permit from the Israel Antiquities Authority (granted to co-authors FB \u0026amp; HK). For details on the site, its location and excavations, see section on Archaeozoological Samples below. Permission to sample bovine teeth was obtained from the excavators as well as from the archaeozoologist (LKH) working on this assemblage (all of whom are co-authors on this paper). The collection is currently held in the laboratory of the latter researcher, in the National Natural History Collections of the Hebrew University of Jerusalem.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003cp\u003eModern Samples\u003c/p\u003e\n \u003cp\u003eA total of eight modern cattle lower jaws were donated by an Israeli meat manufacturer, Beit Mitbahaim Tira ltd.(BTL) (see Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). For samples pictures, see SI 3.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe table summarizes sample information including: Sample number, Proteomic Batch, Animal ID, Animal Species, Sex, Tooth type.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample Number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProteomic Batch\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAnimal ID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSex by docs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTooth Type\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS101\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBostaurus10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBos taurus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS201\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBostaurus16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBos taurus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS202\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBostaurus14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBos taurus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS203\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBostaurus12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBos taurus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS204\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBostaurus11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBos taurus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBostaurus17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBos taurus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePM3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS209\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBostaurus15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBos taurus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS210\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBostaurus13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBos taurus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003cp\u003eArchaeological Samples\u003c/p\u003e\n \u003cp\u003eThe twelve archaeological teeth samples analyzed derive from the Neolithic site of Beisamoun (see Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, map ref NIG 25403\u0026ndash;8/77682): The Neolithic site of Beisamoun is located in the Upper Jordan Valley, within the flood-plain of the now extinct Hula Lake, a region that is characterised by a Mediterranean climate\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. The locality was first excavated in the 1960\u0026rsquo;s-1971 by Monique Lechevallier who reported finding remnants of a mid-Pre-Pottery Neolithic B village (PPNB, first half of the 8th millennium BC; Lechevallier 1978)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Renewed excavations in 2007 through 2016, in an adjacent area were undertaken by a Franco\u0026ndash;Israeli mission. They exposed in situ layers dating to the late PPNB (second half of the 8th millennium BC) and Pre-Pottery Neolithic C (PPNC, first half of the 7th millennium BC). The finds included architectural remains and built installations, with clearly identifiable successive phases of building and their associated lithic artefacts (e.g. arrowheads, sickle blades, axes, adzes, burins, scrapers, retouched blades etc.), groundstone artefacts (grinding slabs, mortars, hand stones, vessels etc.) predominantly made on basalt and limestone, bone tools, shell, stone and bone ornaments, as well as human burials and faunal remains (Bocquentin et al. 2014, 2020)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. The PPNC sediment is composed of a sandy-clay sediment with fine-grained inclusions of charcoal, clay, and ochre (Bocquentin et al. 2014)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. For samples pictures, see SI 3.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003cp\u003eSamples Preparation and Cleaning\u003c/p\u003e\n \u003cp\u003eTeeth (modern and archaeological) were first cleaned mechanically using soft brushes to exclude any external material (e.g. sediments) was performed with bistoury and tweezers under a microscope binocular (Nikon SMZ800N, see picture SI 6). A clean enamel piece (10mm x 15 mm) was extracted. A final examination was carried out using a Nikon SMZ800N binocular microscope. Finally, etching was performed under a chemical hood (see subsection, Sample Preparation for MS).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003cp\u003eSample Preparation for MS\u003c/p\u003e\n \u003cp\u003eThe extracted and clean piece of bovine enamel was immersed for 30 seconds in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, rinsed with DDW and the solution discarded. Then, the enamel piece was etched for 2 minutes in freshly prepared 5% HCl. This solution was discarded. A second etching step in 1000 \u0026micro;l 5% HCl for 60 minutes was performed at room temperature, and the solution was retained in a new Eppendorf on ice. The section was then allowed to completely dissolve in 1000 \u0026micro;l 5% HCl (aprox. 90 min). Then, both solutions were then combined to a final volume of 2000 \u0026micro;l (about 100 mg dry weight). The samples were then frozen and stored in -80C for desalting.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003cp\u003eMass Spectrometry\u003c/p\u003e\n \u003cp\u003eDissolved samples were desalted using Oasis HLB 96 well plate (Waters), using the manufacturer\u0026rsquo;s instructions; in short samples were loaded onto the wells using vacuum pull, followed by 3 washes of 300ul 0.1% TFA. the peptides were then eluted by passing 50ul 50% Acetonitrile, 0.1% FA. The resulting peptides were loaded onto a reversed-phase Symmetry C18 trapping column (20*0.18 mm, 5 \u0026micro;m particle, Water) and resolved on an analytical HSS T3 column (250*0.07 5mm, 1.8 \u0026micro;m particles, Water), mounted on a nanoAquity (Waters) nanoLC instrument. Peptides were eluted from the column using a 50-minute gradient from 4\u0026ndash;30%B (99.9% acetonitrile, 0.1% formic acid) at 350 nl/min flow. The peptides were eluted into a Q-Exactive HF mass spectrometer (Thermo Fisher) using a FlexIon nano-ESI source, through a 20 \u0026micro;m ID emitter (Fossil IonTech, Madrid), at 2.8kV. Blank injections interspaced sample to allow washing off peptide carryover. Data were acquired in data-dependent acquisition (DDA) mode using Top10 method. MS1 resolution was set to 120,000@ 400 m/z, with a mass range of 350\u0026ndash;1650 m/z, using a maximum injection time of 60 ms. Precursors selected for MS2 were limited to charge states of 2\u0026ndash;8, intensity threshold of 3.3\u003csup\u003e4\u003c/sup\u003e and dynamic exclusion of 20s. Precursors were isolated using a window of 1.7 m/z, AGC target was set to 10\u003csup\u003e5\u003c/sup\u003e or a maximum of 60ms injection time, fragmented using HCD at 27 normalized collision energy (NCE). Scans were performed using a first mass setting of 100 m/z at 15,000 resolution (@200 m/z).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe table summarizes sample information including: Sample Number, Sample Locus and Square in the archaeological site of Beisamoun, Animal Species, Sex by Amelogenin (this research study), Tooth type, Period.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSamples\u003c/p\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProteomic\u003c/p\u003e\n \u003cp\u003eBatch\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLoc. Sq.\u003c/p\u003e\n \u003cp\u003e(Location Square)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSex by\u003c/p\u003e\n \u003cp\u003eAmels\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTooth\u003c/p\u003e\n \u003cp\u003eType\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeriod\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBN14, Area E, Loc.334, sq.\u003c/p\u003e\n \u003cp\u003eT7b/T8a/U7c/U8d, Numb. 3316\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBos\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLM1-right\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPNC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBN14, Area E, sq: T7c/d, Numb.\u003c/p\u003e\n \u003cp\u003e3291\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBos\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUPM3-\u003c/p\u003e\n \u003cp\u003eleft\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPNC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBN13, Area E, Loc. 371, sq. S7a /\u003c/p\u003e\n \u003cp\u003eT7d, Numb. 3174\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBos\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUM3 right\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPNC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBN12, Area E, sq:R9d; Loc. 339,\u003c/p\u003e\n \u003cp\u003eNumb. 2877\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBos\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPNC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBN12, Area E, Loc.333, sq:\u003c/p\u003e\n \u003cp\u003eS10d/R10b, Numb. 2727\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBos\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePM4-right\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPNC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBN14, Area E, Loc.352, Numb.\u003c/p\u003e\n \u003cp\u003e3391, sq: U7a-d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBos\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLM1 left\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPNC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBN10,AreaF,Sq.W25a;\u003c/p\u003e\n \u003cp\u003eNumb.1827\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBos\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPNB-PPNC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBN 11, Area F, Loc. 228, sq: V24c;\u003c/p\u003e\n \u003cp\u003eNumb. 5034\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBos\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUM-1/2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPNB-PPNC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBN16, Area E, sq. S7 a/d, Numb.\u003c/p\u003e\n \u003cp\u003e3907\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBos\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUM1 right\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPNB-PPNC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS166\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBN11, Area F, Numb.5045, sq.\u003c/p\u003e\n \u003cp\u003eT21b, Loc 243/246\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBos\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLM1 right\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPNB-PPNC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS163\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBN10, Area F, q.U25d, Numb.\u003c/p\u003e\n \u003cp\u003e1833\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBos\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUM(?)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPNB-PPNC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWIS109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBN 11, Area F, Numb.5021 sq.\u003c/p\u003e\n \u003cp\u003eU26c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBos\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUM-1/2-\u003c/p\u003e\n \u003cp\u003eleft\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPNB-PPNC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eData analysis Data was searched using Byonic search engine (Protein Metrics Inc) using a database that was tailored for modern and ancient samples against the relevant sequences (P02817 AMELX BOVIN Amelogenin, X isoform, \u003cem\u003eBos taurus\u003c/em\u003e and Q99004 AMELY BOVIN Amelogenin, Y isoform \u003cem\u003eBos taurus\u003c/em\u003e, Uniprot). Fixed and variable modifications were adjusted as follows: common2: Oxidation (F, H, K, M, P, W, Y), Deamidated (N, Q, R); rare1: Acetyl (NTerm), Gln to pyro-Glu (NTerm Q), Glu to pyro-Glu (NTerm E) and Phosphorilation (S); rare2: Di-oxidation (F, M, P, W, Y). maximum of 2 common and 2 rare modifications were allowed. MS1 tolerance was set to 10ppm while MS2 was set to 20ppm. Data was filtered using\u003c/p\u003e\n \u003cp\u003eByonic\u0026rsquo;s target-decoy method set to 1% percent FDR. We used a post-search cut-off of LogProb\u0026thinsp;\u0026gt;\u0026thinsp;3 (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and score \u0026gt;\u0026thinsp;200. Identified unique AmelY peptides were examined manually to avoid false positive identifications. Quantification was performed using Skyline software (version 23.1.0.455). Byonic mzID files were imported into Skyline along with the RAW files and used to identify the correct peak for quantification positively. Quantification was performed by XIC extraction of the MS1 signal. Quantification of AmelY base on peptide1: \u0026lsquo;LRYPYP\u0026rsquo; (AmelY;[M\u0026thinsp;+\u0026thinsp;2]\u003csup\u003e2+\u003c/sup\u003e 404.7212 m/z); peptode2:\u0026lsquo;LRYPYPSY\u0026rsquo; (AmelY;[M\u0026thinsp;+\u0026thinsp;2]\u003csup\u003e2+\u003c/sup\u003e 529.7689 m/z). AmelX: peptide3:\u0026lsquo;SMoxIRHPYP\u0026rsquo; (AmelX;[M\u0026thinsp;+\u0026thinsp;2]\u003csup\u003e2+\u003c/sup\u003e 508.7527 m/z); peptide4:\u0026lsquo;IRHPYPSY\u0026rsquo; (AmelX;[M\u0026thinsp;+\u0026thinsp;2]\u003csup\u003e2+\u003c/sup\u003e 516.7667 m/z). Ion fragment validation was performed manually. We performed sequence similarity searches using protein-protein BLAST program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.uniprot.org/blast\u003c/span\u003e\u003c/span\u003e). Bulk modification calculation were perform by in-house RStudio scrips.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eData Availability\u003c/p\u003e\n\u003cp\u003eThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE\u003csup\u003e63 \u003c/sup\u003epartner repository with following dataset identifiers:1) PXD053146, 10.6019/PXD053146; 2) PXD053148, 10.6019/PXD053148. LFQ files at PXD053225.\u003c/p\u003e\n\u003cp\u003eProteomeXchange via the PRIDE database - Password and User Names for Reviewers The data was divided into 2 projects; see all the details below.\u003c/p\u003e\n\u003cp\u003ea) Project Name: Label-Free Quantification Method for Assessing Sex from Modern and Ancient Bovine Tooth Enamel.\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003eProject accession: PXD053146\u003c/li\u003e\n\u003cli\u003eProject DOI: 10.6019/PXD053146\u003c/li\u003e\n\u003cli\u003eReviewer account details:\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u0026ndash; Username: [email protected]\u003c/p\u003e\n\u003cp\u003eb) Project Name: Label-Free Quantification Method for Assessing Sex from Modern and Ancient Bovine Tooth Enamel PART II.\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003eProject accession: PXD0531486 \u0026bull; Project DOI: 10.6019/PXD053148\u003c/li\u003e\n\u003cli\u003eReviewer account details:\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u0026ndash; Username: [email protected]\u003c/p\u003e\n\u003cp\u003ec) Project Name: Label-Free Quantification Method for Assessing Sex from Modern and Ancient Bovine Tooth Enamel. \u0026bull; Project accession: PXD053225\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003eToken: dU4QhIY3tPti\u003c/li\u003e\n\u003cli\u003eReviewer account details:\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u0026ndash; Username: [email protected]\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eP.K. was supported by the Helen and Martin Kimmel Center for Archaeological Science. We wish to thank the George Schwartzman Fund for funding laboratory work and sample analyses.\u003c/p\u003e\n\u003cp\u003eAuthor contributions statement\u003c/p\u003e\n\u003cp\u003eP.K. E.B. and L.K.H. conceived the idea; P.K. and E.B. designed the study; P.K. performed all laboratory work and analyses; L.K.H., F.B. and H.K. provided the archaeological samples; P.K., D.M. and E.B. analyzed and interpreted the data; P.K. wrote the manuscript with the contribution of E.B., .D.M. and L.K.H.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHorwitz, L., Cope, C. \u0026amp; Tchernov, E. Sexing the bones of mountain-gazelle (\u003cem\u003eGazella gazella\u003c/em\u003e) from prehistoric sites in the Southern Levant. \u003cem\u003ePal\u0026eacute;orient\u0026nbsp;\u003c/em\u003e1\u0026ndash;12 (1990).\u003c/li\u003e\n \u003cli\u003eBarden, H. E. \u0026amp; Maidment, S. C. 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New Studies in the Archaeology of Jerusalem and its Region.\u003c/em\u003e, 163\u0026ndash;199 (Israel Antiquities Authority, Jerusalem, 2020).\u003c/li\u003e\n \u003cli\u003ePerez-Riverol, Y. \u003cem\u003eet al.\u0026nbsp;\u003c/em\u003eThe pride database resources in 2022: A hub for mass spectrometry-based proteomics evidences. \u003cem\u003eNucleic Acids Res.\u0026nbsp;\u003c/em\u003e50, D543\u0026ndash;D552 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Paleoproteomics, Bovine, zooarchaeology, peptidomics, amelogenin, sex determination, enamelon, enamel proteome","lastPublishedDoi":"10.21203/rs.3.rs-4638090/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4638090/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIdentification of the sex of modern, fossil and archaeological animal remains offers many insights into their demography, mortality profiles and domestication pathways. However, due to manifold factors, sex determination of osteological remains is often problematic. To overcome this, we have developed an innovative protocol to determine an animal’s sex from tooth enamel, by applying label-free quantification (LFQ) of two unique AmelY peptides ‘\u003cu\u003e\u003cstrong\u003eL\u003c/strong\u003e\u003c/u\u003eR\u003cu\u003e\u003cstrong\u003eY\u003c/strong\u003e\u003c/u\u003ePYP’ (AmelY;[M+2]\u003csup\u003e2+ \u003c/sup\u003e404.7212 m/z) and ‘\u003cu\u003e\u003cstrong\u003eL\u003c/strong\u003e\u003c/u\u003eR\u003cu\u003e\u003cstrong\u003eY\u003c/strong\u003e\u003c/u\u003ePYPSY’ (AmelY;[M+2]\u003csup\u003e2+ \u003c/sup\u003e529.7689 m/z) that are only present in the enamel of males. We applied this method to eight modern cattle (\u003cem\u003eBos taurus\u003c/em\u003e) of known sex, and correctly assigned them to sex. We then applied the same protocol to twelve archaeological \u003cem\u003eBos \u003c/em\u003eteeth from the Neolithic site of Beisamoun, Israel (8\u003csup\u003eth\u003c/sup\u003e–7\u003csup\u003eth \u003c/sup\u003emillennium BC) and determined the sex of the archaeological samples. Since teeth are usually better preserved than bones, this innovative protocol has potential to facilitate sex determination in ancient and modern bovine remains, that currently cannot be sexed.\u003c/p\u003e","manuscriptTitle":"A Label-Free Quantification Method for Assessing Sex from Modern and Ancient Bovine Tooth Enamel","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-26 08:11:52","doi":"10.21203/rs.3.rs-4638090/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c7efc927-83cc-4d67-9f60-c4f16561177d","owner":[],"postedDate":"June 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33730203,"name":"Paleozoology"},{"id":33730204,"name":"Animal Science"},{"id":33730205,"name":"Archaeology"},{"id":33730206,"name":"Bioinformatics"}],"tags":[],"updatedAt":"2024-06-26T08:11:52+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-26 08:11:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4638090","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4638090","identity":"rs-4638090","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}