Dataset Information

A Comparison of Proteomic, Genomic, and Osteological Methods of Archaeological Sex Estimation

ABSTRACT: Proteomic sex estimation of human remains via liquid chromatography tandem mass spectrometry (LC- MS/MS) of sexually dimorphic amelogenin peptides in tooth enamel. The human remains analyzed in this study were recovered from two archaeological sites, Síi Túupentak (CA-ALA-565; ca. 200-600 cal BP) and Rummey Ta Kuččuwiš Tiprectak (CA-ALA-704/H; ca. 180-2240 cal. BP), Late Holocene ancestral Ohlone villages in Central California. The X and Y chromosomes code for distinctive isoforms of amelogenin proteins, AMELX_HUMAN and AMELY_HUMAN. Fragments of these proteins can be detected using shotgun liquid chromatography mass spectrometry and assigned to the AMELX or AMELY isoform. We use nano-liquid chromatography coupled with orbitrap tandem mass spectrometry (LC-orbitrap-MS/MS). Signals of all peptides that can be unambiguously attributed to either AMELY or AMELX isoforms are summed and normalized for sample weight. Confident detection of AMELY specific peptides provides an unambiguous indicator of a male sex chromosome.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Tooth Enamel

SUBMITTER: Glendon Parker

LAB HEAD: Glendon J Parker

PROVIDER: PXD016076 | Pride | 2020-07-20

REPOSITORIES: Pride

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A comparison of proteomic, genomic, and osteological methods of archaeological sex estimation.

Buonasera Tammy T Eerkens Jelmer J de Flamingh Alida A Engbring Laurel L Yip Julia J Li Hongjie H Haas Randall R DiGiuseppe Diane D Grant Dave D Salemi Michelle M Nijmeh Charlene C Arellano Monica M Leventhal Alan A Phinney Brett B Byrd Brian F BF Malhi Ripan S RS Parker Glendon G

Scientific reports 20200717 1

Sex estimation of skeletons is fundamental to many archaeological studies. Currently, three approaches are available to estimate sex-osteology, genomics, or proteomics, but little is known about the relative reliability of these methods in applied settings. We present matching osteological, shotgun-genomic, and proteomic data to estimate the sex of 55 individuals, each with an independent radiocarbon date between 2,440 and 100 cal BP, from two ancestral Ohlone sites in Central California. Sex es ...[more]

PMID: 32681049

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Project description:In a systemic effort to survive environmental stress, organ systems fluctuate and adapt to overcome external pressures. The evolutionary drive back towards homeostasis, makes it difficult to determine if an organism experienced a toxic exposure to stress, especially in early prenatal and neonatal periods of development. Previous studies indicate that primary human teeth may provide historical records of experiences related to stressors during that early time window. To assess the molecular effects of early life adversity on enamel formation, we used a limited bedding and nesting (LBN) mouse model of early life adversity (ELA), to assess changes in enamel organ gene expression and enamel mineralization. Enamel of postnatal day 12 (P12) weight-matched ELA mice was more mineralized as compared control enamel. RNAseq showed 724 genes significantly upregulated in ELA enamel organs as compared to controls, and 574 significantly downregulated genes (DESeq2 p-value <= 0.05). Transcripts expressing the enamel matrix proteins amelogenin (Amelx) and enamelin (Enam) were among the top 4 most differentially expressed genes. The most significantly enriched GO and Reactome pathway was Extracellular Matrix Organization, while the most significantly enriched KEGG Pathways were Butanoate metabolism and Synthesis and Degradation of Ketone Bodies. qPCR analysis of weight-matched ELA and control enamel organs confirmed that expression of the ameloblast-specific proteins Amelx and Enam were highly upregulated in ELA mice. When evaluating molecular mechanisms for amelogenin upregulation, we found significantly increased expression of Dlx3, while transcripts for clock genes Per1 and Nrd1, which have also positively associated with amelogenin expression, were downregulated. These findings suggest that the developing enamel organs are sensitive to the pressures of early life adversity and produce structural biomarkers in tooth enamel mineral to reflect these changes. Together these results support the possibility that tooth enamel may contain biomarkers of cellular effects associated with systemic early life adversity.

Project description:The enamel proteome includes a range of proteins which are well-preserved in archaeological settings but have so far received less study than the sex-specific sequencing of enamel. We look beyond sex-specific sequencing of amelogenin to investigate the potential of several serum proteins, including immunoglobulin gamma (IgG), the major immunoglobulin found in blood serum, and C-reactive protein (CRP) which is associated with inflammatory response, to provide insight into the health and stresses experienced by individuals in the past. We apply this approach to enamel samples from Mission-Period ancestral Ohlone interred at Asistencia San Pedro y San Pablo (CA-SMA-71/H; n=11). For comparison, we also examine enamel from historic-period European-Americans interred in the City Cemetery in San Francisco, and extracted third molars from present-day military cadets. Results indicate that IgG is elevated among individuals at the asistencia relative to samples from present-day military cadets (n=8), or historic City Cemetery individuals (ANOVA with post-hoc Tukey Kramer tests, p < 0.02). Further, the inflammatory protein CRP, normally expressed at much lower levels than IgG, was present in 55% (6 of 11) of the asistencia samples, and in 17% (2 of 12) of the historic City Cemetery samples, but was not detected in enamel samples from contemporary military cadets. While more studies are needed, we argue that the difference in IgG could reflect higher levels of chronic diseases such as tuberculosis among Ohlone living within the Mission system, while the presence of measurable amounts of CRP could relate to high degrees of physical, social, and emotional stresses. To our knowledge, this is the first paleoproteomic study of immune proteins in tooth enamel. The ability to track immune responses during tooth formation could provide valuable and high-resolution information on ancient health and disease at the level of the individual over archaeological time-scales.

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.

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