Structural Characterization of an Ancient Protein by MALD- MS: The Preservational State of Osteocalcin in Fossils

9805417 Ostrom这项赠款的主要目标是使用基质辅助激光解吸电离质谱仪(MALDI-MS)评估骨蛋白骨钙素(OC)的结构和保存潜力的成岩变化。我们的方法将是分析野牛和鹿的骨骼(最近到10万年代)的时间序列，以及模拟成岩作用的加热实验产生的样本。骨钙素的使用和MALDI-MS的应用为拟议的研究提供了独特的优势。骨钙素具有几个极其重要的属性，包括与骨的羟基磷灰石矿物相的密切结合，随时间的优先保存，仅在脊椎动物矿化组织中存在，不存在常见的污染物，包括微生物、无脊椎动物和植物，通过放射免疫分析(RIA)获得完整蛋白的免疫学特异性，以及存在广泛的骨钙素蛋白质序列数据库。此外，MALDI-MS技术可以识别非常少量的蛋白质(典型的蛋白质为8pmoL)，并且不会面临传统蛋白质测序方法面临的无数问题。我们的初步工作已经通过放射免疫分析证实了骨钙素在45万年前的骨骼中的存在。此外，我们还产生了其他证据，包括MALDI-MS数据，表明在800到53,000年前的骨骼中存在完整的OC。由于MALDI-MS具有多肽质量图谱和源后衰变分析提供蛋白质序列信息的能力，这种方法将使我们能够直接评估成岩过程中骨钙素一级结构的变化，并表征成岩过程中单个氨基酸的修饰。此外，这种方法将彻底改变我们从化石中明确识别本地成分的能力。

9805417OstromThe primary objectives of this grant are to evaluate diagenetic changes in structure and preservational potential of the bone protein, osteocalcin (OC), using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Our approach will be to analyze a time series of bison and deer bones (Recent to 100,000 yo) and samples resulting from a heating experiment to simulate diagenesis. The use of osteocalcin and the application of MALDI-MS provide a unique position of strength for the proposed research. Osteocalcin has several extremely important attributes which include intimate association with the hydroxyapatite mineral phase of bone, preferential preservation over time, exclusive occurrence in vertebrate mineralized tissues, absence in common contaminators including microbes, invertebrates and plants, immunological specificity of the intact protein via radioimmunoassay (RIA), and the existence of an extensive database of protein sequences for osteocalcin. Furthermore, the MALDI-MS technique can identify very small quantities of protein (8 pmol of protein is typical) and is not confronted by the myriad of problems that confront traditional methods of protein sequencing. Our preliminary work has confirmed the presence of osteocalcin via RIA in bones as old as 450,000 years. In addition, we have generated other lines of evidence, including MALDI-MS data that suggests the presence of intact OC in 800 to 53,000 year old bones. Owing to the ability of MALDI-MS with peptide mass mapping and post source decay analysis to provide protein sequence information, this approach will allow us to make a direct assessment of diagenetic changes in the primary structure of osteocalcin and to characterize modifications of individual amino acids throughout diagenesis. Furthermore, this approach will revolutionize our ability to unambiguously identify an indigenous component from a fossil.