Project description:Tooth enamel forms in an ephemeral protein matrix where changes in protein abundance, composition and post-translational modifications are critical to achieve healthy enamel properties. Amelogenin (AMELX) with its splice variants is the most abundant enamel matrix protein, with only one known phosphorylation site at serine 16 shown in vitro to be critical for regulating mineralization. The phosphorylated form of AMELX stabilizes amorphous calcium phosphate, while crystalline hydroxyapatite forms in the presence of the unphosphorylated protein. While it is necessary to our understanding of how AMELX regulates mineral transitions over space and time, it is unknown whether and when un-phosphorylated amleogenin occurs during enamel mineralization. This study aims to reveal the spatiotemporal distribution of the most abundant AMLEX splice variants including the full length P173, the shorter leucine-rich amelogenin protein (LRAP), and the exon 4-containing P190 in forming enamel, all within the context of the changing enamel matrix proteome during mineralization. We microsampled permanent pig molars, capturing known stages of enamel formation from both crown surface and inner enamel. Nano-LC-MS/MS proteomic analyses after tryptic digestion rendered more than 500 unique protein identifications in enamel, dentin, and bone. We mapped collagens, keratins, and proteolytic enzymes (CTSL, MMP2, MMP10) and determined distributions of P173, LRAP and P190, the enamel proteins enamelin (ENAM) and ameloblastin (AMBN), and matrix-metalloprotease-20 (MMP20) and kallikrein-4 (KLK4). All enamel proteins and KLK4 were near-exclusive to enamel and in excellent agreement with published expression levels. Phosphorylated P173 and LRAP decreased in abundance from recently deposited matrix towards older enamel, mirrored by increasing abundances of testicular acid phosphatase (ACPT). Our results showed that hierarchical clustering analysis of secretory enamel links closely matching distributions of unphosphorylated P173 and LRAP with ACPT and nontraditional amelogenesis proteins, many associated with enamel defects. We report higher protein diversity than previously published and Gene Ontology (GO)-defined protein functions related to the regulation of mineral formation in secretory enamel (e.g. casein α-S1, CSN1S1), immune response in erupted enamel (e.g. peptidoglycan recognition protein, PGRP), and phosphorylation. This study presents a novel approach to characterize and study functional relationships through spatiotemporal mapping of the ephemeral extracellular matrix proteome.
Project description:Genetic, linguistic, and archaeological studies have demonstrated the existence of strong links between eastern and southern Africa over the past millennia, including the diffusion of the first domesticated sheep and goats. However, the proportions at which they were introduced into past human subsistence strategies in Africa is difficult to assess archaeologically, as caprines share skeletal features with a number of wild bovids. Palaeoproteomics has proven effective at retrieving biological information from archaeological remains in African arid contexts. Using published collagen sequences and generated de novo ones of wild bovids, we present the molecular (re-)attribution of remains morphologically identified as sheep/goat or unidentifiable bovids from seventeen archaeological sites distributed between eastern and southern Africa and spanning seven millennia. More than 70% of the remains were identified and the direct radiocarbon dating of domesticates specimens allowed the chronological refinement of the arrival of caprines in both African regions. Our results further substantiate a predominance of sheep in the assemblages along with a similar arrival chronology. Beyond adding substantial biological data to the field of (palaeo-)proteomics, it is the first large-scale palaeoproteomics investigation to include both eastern and southern African sites, opening promising future applications of the method on the continent.
Project description:We describe the first dental proteomic profiles of Iron Age individuals (c2000-1000 years B.P), collected from the site of Long Long Rak rock shelter (LLR) in northwest Thailand. A bias toward the preservation of small, acidic and hydrophobic amino acids is observed. It is evident that the 212 proteins identified (2 peptide, FDR <1%) comprise a palimpsest of alterations that occurred both ante-mortem and post-mortem. Conservation of particular amino acids has contributed to the identification of amelogenin peptides. A novel MRM method for sexing individuals using the amelogenin protein is described, with four teeth indicating male origin. Stable isotope analysis using carbon and oxygen isotopes highlights the strongly C3 based (~80%) diet of the Long Long Rak cemetery people, which probably comprised rice combined with protein from freshwater fish among other food items. The combination of pathway and isotopic analysis adds weight to the relatively simple C3 based diets having contributed to the enrichment of pathways associated with metabolic conditions and shows capacity for harboring these conditions prior to death. The combination of proteomics and stable isotope analysis provides a complementary strategy for assessing the demography, diet, lifestyle and possible diseases experienced by ancient populations.
Project description:The project aimed to characterize the collagen type I (COL1) sequences from various modern, Holocene and Pleistocene bone, antler and skin samples for phylogenetic purposes. All extractions were performed at BioArCh, University of York (UK) or the Department of Human Evolution, MPI-EVA (Germany). Analyses took place on Q-Exactive Hybrid Quadrupole-Orbitrap MS.
Project description:Biological sex determination of human remains by means of a minimally destructive surface acid etch of tooth enamel and subsequent identification of sex chromosome linked isoforms of Amelogenin– an enamel-forming protein - by nano-liquid chromatography mass spectrometry(nanoLC-MS).
Project description:Embryologically the tooth is derived from both the ectoderm and neural crest (ectomesenchyme). It is often used as a model to study how epithelialmesenchymal interactions can control differentiation and morphogenesis. During early development organs of ectodermal origin share both a set of signalling molecules and exhibit common morphological features, subsequently proceeding along separate developmental programs.<br><br>Tooth development is a continuous process that can be divided into the initiation -, bud -, cap -, and bell-stages. In mice, tooth development begins at embryonic day 11.5 (E11.5), by thickening of the dental epithelium, while mineralization of enamel and dentin in first molar starts at postnatal day 0 (P0) (5). A multistep and complex process of the gene expression are involved in the early stage of tooth development. So far expression of more than 1300 genes and/or proteins have been detected during tooth germ development by microarrays/immunocytochemistry/in situ hybridization. Studies with mutant mice have identified a number of genes that regulate tooth development and morphology. For example, deficiency of Lef-1 or P63 arrests tooth development at early stages. Deficiency of Msx1 or Pax9 results in arrest of tooth development at the bud stage , while deficiency of Runx2/Cbfa1 or Sp3 inhibits cyto-differentiation of ameloblasts and/or odontoblasts. Shh is required for normal growth and morphogenesis, but is not essential for cyto-differentiation of the ameloblast and odontoblast populations. Ameloblastin and amelogenin knock-out mice develop severe enamel hypoplasia with abnormal ameloblast differentiation. <br><br>Recently, new connections between retinoid metabolism and PPAR responses have been identified. It has also been shown that endogenous retinoic acid is necessary for the initiation of odontogenesis , and that some of the genes that catalyze the oxidation of retinaldehyde into retinoic acid, exhibit distinct patterns of expression in developing murine teeth. Little is known about functions of PPAR-a as regards tooth germs or mature teeth. It is, however, likely that mitochondrial oxidative metabolism well as fatty acid metabolism is enhanced in late odontogenesis. These are metabolic activities which in other tissues are stimulated by PPAR-a agonists.<br><br>For this reason it was of interest to carry out comparative gene expression profiling of the first molar tooth germs of PPAR-a knock-out mouse and of the corresponding wild-type mice. The results suggest marked differences in gene expression, parts of which may be associated with an observed hypomineralization of enamel in the mature PPAR-a knock-out murine tooth.
Project description:This study tests the application of an innovative new method of sex determination utilizing enamel peptides on a sample of incompletely developed deciduous teeth, including those of perinates.
Project description:Tooth is a remarkable source of information for paleoanthropologists. Investigation of ancient dental proteomes is able to provide biomolecular data helping at understanding past life. In particular, the enamel protein amelogenin (AMEL) offers the possibility to determine the sex of the specimen. Here, we have analysed eleven 5000 year-old human teeth from a French Neolithic site using a dedicated paleoproteomics strategy for phenotypic characterization of ancient samples. We have set up a shotgun proteomics analysis based on an optimized 3-step iterative search strategy. This allowed the identification of a total number of 1496 proteins including the main proteins characteristic of dental tissues. Most of the proteins were identified by the no enzyme database search mode included in the iterative bioinformatics data analysis pipeline. This demonstrates that a number of peptides issued from randomly degraded proteins may be missed when using the conventional semi-tryptic database search mode. Based on the identification of the sex-specific peptides TALVLTPLK, IALVLTPLK and WYQSIRPPYP of amelogenin, a targeted MS approach using a parallel reaction monitoring (PRM) mode was performed to maximize the sensitivity and the reproducibility of detecting these unique peptides for sex estimation. This led to confirm the sex of individualsin all the samples.
Project description:Sex of individuals, estimated on human remains, is important information in anthropological, archaeological and forensic researches. In forensic anthropology, this is one of the key points for identifying a person. In archeology and paleoanthropology, information about sex is important for reconstructing the social and biological structure of a group, lifestyle, and for modeling demographic and growth processes in the past. Sex estimation of adult individuals is possible according to morphological criteria when metric and descriptive features are used. In case of poor preservation of the adult skeletons, as well as on children's remains, sex estimation by morphological criteria has some limitations. Therefore regular attempts are made to attract alternative methods for sexing human remains. As one of such methods a liquid chromatography-mass spectrometry analysis of tooth enamel peptides can be used. The result of this analysis is the identification of certain fragments of amelogenins - the main structural proteins of the organic matrix of tooth enamel, which make up more than 90% of its protein content. Amelogenin genes are located on both the X- and Y-chromosomes and the products of these genes have structural differences that allow them to be differentiated from each other. In the process of maturation of tooth enamel, its protein matrix undergoes proteolytic degradation, as a result enamel already contains proteins in the form of their peptide fragments. Reliable and reproducible detection of unique peptide fragments of amelogenins encoded on the X- and Y-chromosomes in human enamel will make it possible to estimate sex of human remains. The work presents a peptidomic analysis of tooth enamel using chromatography-mass spectrometry. Experiments were carried out on a series of teeth of various archaeological data, tafonomic condition of crown and different biological generations of teeth (deciduous and permanent ones).