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Expanding the Proteomics Toolbox for Sex Determination in Human Remains
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Este estudo apresenta uma análise proteómica abrangente de amostras dentárias antigas, investigando diferenças de expressão proteica com base no sexo e fornecendo informações sobre os hábitos alimentares de populações passadas. Conseguimos extrair o peptidoma e proteoma do esmalte dentário usando um método não destrutivo. O peptidoma, enriquecido por purificação em colunas HLB, permitiu a deteção de várias sequências de AMELX e AMELY, possibilitando a determinação do sexo. Especificamente, identificámos péptidos chave da AMEL: m/z = 540,2796 com carga +2, correspondente ao ião AMELX (sequência: SIRPPYPSY), e m/z = 440,2233 com carga +2, correspondente ao ião AMELY (sequência: SM(ox)IRPPY). Sequências modificadas, como LRPLPPILPDLHLEAWPATDKTKQ(+0,98)EEVD e SYGYEPMGGW, aumentaram a fiabilidade na diferenciação do sexo. Comparações revelaram menor expressão combinada de AMELX e AMELY nos homens, com uma cópia de cada gene, comparado com as mulheres, que possuem dois genes AMELX funcionais.
A análise proteómica, realizada com o método FASP (Filter-Aided Sample Preparation) e LC-MS/MS, identificou 196 proteínas humanas e 47 de outras espécies, como Gallus gallus (galinha), Sus scrofa (porco) e Ovis aries (ovelha). As proteínas humanas eram principalmente colagénio, refletindo a sua estabilidade estrutural e abundância nos tecidos dentários. Vias relacionadas com colagénio, como trimerização, biossíntese e montagem de fibrilas, estavam significativamente representadas, com os tipos COL1A1, COL1A2 e COL4A1 sendo críticos para a integridade da dentina e esmalte. 52 proteínas apresentaram deamidação, uma modificação pós-traducional associada a envelhecimento ou degradação proteica. A deteção de proteínas não-humanas de Gallus gallus, Sus scrofa e Ovis aries sugere uma dieta rica em aves, porco e leite de ovelha. A presença de caseínas de Ovis aries apoia a hipótese de consumo de laticínios, nomeadamente leite de ovelha. Esta análise proteómica oferece informações promissoras sobre práticas alimentares antigas e diferenças na expressão proteica baseada no sexo.
This study presents a comprehensive proteomic analysis of ancient dental samples aimed at investigating sex-based differences in protein expression and providing insights into the dietary habits of past populations. We successfully extracted the peptidome and proteome from tooth enamel using a non-destructive method. The peptidome, enriched via HLB purification, enabled the detection of several AMELX and AMELY sequences, allowing for sex determination. Specifically, we identified key AMEL protein peptides: m/z = 540.2796 with a +2 charge corresponding to the AMELX ion (sequence: SIRPPYPSY) and m/z = 440.2233 with a +2 charge corresponding to the AMELY ion (sequence: SM(ox)IRPPY). Additionally, modified sequences, such as LRPLPPILPDLHLEAWPATDKTKQ(+0.98)EEVD and SYGYEPMGGW, were detected, further enhancing the reliability of sex differentiation. Additionally, sex-based comparisons revealed lower combined expression of AMELX and AMELY in males, who possess only one copy of each gene, compared to females with two functional AMELX genes. These findings were corroborated by peptide intensity analyses. Proteomic analysis, performed using the Filter-Aided Sample Preparation (FASP) method and LC-MS/MS, identified 196 human proteins and 47 from other species, including Gallus gallus (chicken), Sus scrofa (pig), and Ovis aries (sheep). The human proteins were predominantly collagens, reflecting their structural stability and abundance in dental tissues. Key collagen-related pathways, such as collagen trimerisation, biosynthesis, and fibril assembly, were significantly represented, with collagen types COL1A1, COL1A2, and COL4A1 being critical for the structural integrity of dentin and enamel. Notably, 52 proteins exhibited deamidation at asparagine and/or glutamine residues, a post-translational modification associated with protein ageing or degradation. Detecting non-human proteins from Gallus gallus, Sus scrofa, and Ovis aries provides preliminary evidence for the dietary habits of these individuals, suggesting a diet rich in poultry, pork, and sheep’s milk. The presence of caseins from Ovis aries supports the hypothesis of dairy consumption, specifically sheep’s milk. While further validation is needed, this proteomic analysis offers promising insights into ancient dietary practices and sex-based protein expression differences.
This study presents a comprehensive proteomic analysis of ancient dental samples aimed at investigating sex-based differences in protein expression and providing insights into the dietary habits of past populations. We successfully extracted the peptidome and proteome from tooth enamel using a non-destructive method. The peptidome, enriched via HLB purification, enabled the detection of several AMELX and AMELY sequences, allowing for sex determination. Specifically, we identified key AMEL protein peptides: m/z = 540.2796 with a +2 charge corresponding to the AMELX ion (sequence: SIRPPYPSY) and m/z = 440.2233 with a +2 charge corresponding to the AMELY ion (sequence: SM(ox)IRPPY). Additionally, modified sequences, such as LRPLPPILPDLHLEAWPATDKTKQ(+0.98)EEVD and SYGYEPMGGW, were detected, further enhancing the reliability of sex differentiation. Additionally, sex-based comparisons revealed lower combined expression of AMELX and AMELY in males, who possess only one copy of each gene, compared to females with two functional AMELX genes. These findings were corroborated by peptide intensity analyses. Proteomic analysis, performed using the Filter-Aided Sample Preparation (FASP) method and LC-MS/MS, identified 196 human proteins and 47 from other species, including Gallus gallus (chicken), Sus scrofa (pig), and Ovis aries (sheep). The human proteins were predominantly collagens, reflecting their structural stability and abundance in dental tissues. Key collagen-related pathways, such as collagen trimerisation, biosynthesis, and fibril assembly, were significantly represented, with collagen types COL1A1, COL1A2, and COL4A1 being critical for the structural integrity of dentin and enamel. Notably, 52 proteins exhibited deamidation at asparagine and/or glutamine residues, a post-translational modification associated with protein ageing or degradation. Detecting non-human proteins from Gallus gallus, Sus scrofa, and Ovis aries provides preliminary evidence for the dietary habits of these individuals, suggesting a diet rich in poultry, pork, and sheep’s milk. The presence of caseins from Ovis aries supports the hypothesis of dairy consumption, specifically sheep’s milk. While further validation is needed, this proteomic analysis offers promising insights into ancient dietary practices and sex-based protein expression differences.
Descrição
Palavras-chave
Human teeth Sex-determination Peptidome Proteome Mass Spectrometry