Dataset Information

Human enamel peptides LC-MS/MS.

ABSTRACT: Recently, enamel peptides revealed their critical role in sex classification of old skeletal remains. In particular, the presence of protein AMELY (amelogenin isoform Y) within the enamel dental tissue is restricted to the male sex only, while AMELX (isoform X) can be found in both sexes. AMELY can be easily detected through peptide SM(ox)IRPPY by means of LC-MS/MS and retrieved from ion chromatograms (exact mass [M+2H]+2 440.2233 m/z). In this paper, we exploited the dimorphic features of the amelogenin protein to unravel the sex of the so-called ‘Lovers of Modena’, two Late Antique individuals whose skeletons were intentionally buried hand-in-hand.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Tooth Enamel

SUBMITTER: Federico Lugli

LAB HEAD: Giulia Di Rocco

PROVIDER: PXD012587 | Pride | 2019-11-05

REPOSITORIES: Pride

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Publications

Enamel peptides reveal the sex of the Late Antique 'Lovers of Modena'.

Lugli Federico F Di Rocco Giulia G Vazzana Antonino A Genovese Filippo F Pinetti Diego D Cilli Elisabetta E Carile Maria Cristina MC Silvestrini Sara S Gabanini Gaia G Arrighi Simona S Buti Laura L Bortolini Eugenio E Cipriani Anna A Figus Carla C Marciani Giulia G Oxilia Gregorio G Romandini Matteo M Sorrentino Rita R Sola Marco M Benazzi Stefano S

Scientific reports 20190911 1

Recent work has disclosed the critical role played by enamel peptides in sex classification of old skeletal remains. In particular, protein AMELY (amelogenin isoform Y) is present in the enamel dental tissue of male individuals only, while AMELX (isoform X) can be found in both sexes. AMELY can be easily detected by LC-MS/MS in the ion extracted chromatograms of the SM<sub>(ox)</sub>IRPPY peptide (monoisotopic [M + 2 H]<sup>+2</sup> mass = 440.2233 m/z). In this paper, we exploited the dimorphic ...[more]

PMID: 31511583

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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: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.

Dataset Information

Human enamel peptides LC-MS/MS.

Publications

Enamel peptides reveal the sex of the Late Antique 'Lovers of Modena'.

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