School of Natural Sciences, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
Institute for Prehistoric and Protohistoric Archaeology, Kiel University, Johanna-Mestorf Strasse 2-6, D-24118 Kiel, Germany.
J Proteome Res. 2021 Mar 5;20(3):1689-1704. doi: 10.1021/acs.jproteome.0c00862. Epub 2021 Feb 17.
Over the past two decades, proteomic analysis has greatly developed in application to the field of biomolecular archaeology, coinciding with advancements in LC-MS/MS instrumentation sensitivity and improvements in sample preparation methods. Recently, human dental calculus has received much attention for its well-preserved proteomes locked in mineralized dental plaque which stores information on human diets and the oral microbiome otherwise invisible to other biomolecular approaches. Maximizing proteome recovery in ancient dental calculus, available only in minute quantities and irreplaceable after destructive analysis, is of paramount importance. Here, we compare the more traditional ultrafiltration-based and acetone precipitation approaches with the newer paramagnetic bead approach in order to test the influence of demineralization acid on recovered proteome complexity obtained from specimens as well as the sequence coverages matched for significant proteins. We found that a protocol utilizing EDTA combined with paramagnetic beads increased proteome complexity, in some cases doubling the number of unique peptides and number of proteins matched, compared to protocols involving the use of HCl and either acetone precipitation or ultrafiltration. Although the increase in the number of proteins was almost exclusively of bacterial origin, a development that has implications for the study of diseases within these ancient populations, an increase in the peptide number for the dairy proteins β-lactoglobulin and casein was also observed reflecting an increase in sequence coverage for these dietary proteins of interest. We also consider structural explanations for the discrepancies observed between these two key dietary proteins preserved in archaeological dental calculus.
在过去的二十年中，蛋白质组学分析在生物分子考古学领域的应用得到了极大的发展，这与 LC-MS/MS 仪器灵敏度的提高和样品制备方法的改进是一致的。最近，人类牙垢因其在矿化牙菌斑中保存完好的蛋白质组而受到广泛关注，这些蛋白质组储存了关于人类饮食和口腔微生物组的信息，而这些信息是其他生物分子方法无法获得的。最大限度地从古代牙垢中回收蛋白质组，对于仅可用少量且在破坏性分析后不可替代的牙垢来说至关重要。在这里，我们比较了基于超滤和丙酮沉淀的更传统方法与较新的顺磁珠方法，以测试脱矿酸对从标本中获得的回收蛋白质组复杂性以及与显著蛋白质匹配的序列覆盖率的影响。我们发现，与使用 HCl 结合丙酮沉淀或超滤的方案相比，使用 EDTA 结合顺磁珠的方案增加了蛋白质组的复杂性，在某些情况下，独特肽和匹配蛋白质的数量增加了一倍。尽管增加的蛋白质几乎完全来自细菌，但这一发展对研究这些古代人群中的疾病具有重要意义，还观察到乳制品蛋白β-乳球蛋白和酪蛋白的肽数量增加，反映了这些感兴趣的饮食蛋白的序列覆盖率增加。我们还考虑了在考古牙垢中保存的这两种关键膳食蛋白之间观察到的差异的结构解释。
