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

9805417 Ostrom该基金的主要目的是使用基质辅助激光解吸电离质谱（MALDI-MS）评估骨蛋白骨钙素（OC）结构和分解潜力的成岩变化。 我们的方法将是分析野牛和鹿骨的时间序列（最近到100，000年）和加热实验产生的样品，以模拟成岩作用。 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地位。 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紧密结合，随时间优先保存，仅存在于脊椎动物矿化组织中，不存在包括微生物、无脊椎动物和植物在内的常见污染物，通过放射免疫测定（RIA）的完整蛋白质的免疫特异性，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（典型的是8 pmol的蛋白质），并且不会遇到传统蛋白质测序方法所面临的无数问题。 我们的初步工作已经通过RIA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，这些数据表明在800至53，000年前的骨骼中存在完整的OC。 由于MALDI-MS与肽质量映射和后源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变化的一级结构的骨钙素和表征整个成岩过程中的个别氨基酸的修饰。 此外，这种方法将彻底改变我们从化石中明确识别土著成分的能力。

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.