SGER: Phylogenetic and Indigeneity Implications of MALDI-MS Determined Osteocalcin Protein Sequences

9614235 Ostrom一种新的方法，即基质辅助激光解吸电离质谱仪(MALDIMS)，用于测定古代生物及其最近的现代祖先骨骼中骨钙素蛋白质的氨基酸序列，以建立初步的进化关系并评估土著起源。骨钙素的使用，可用的特殊样品，以及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)保持免疫学特异性，以及存在广泛的骨钙素蛋白质序列数据库。在我们的特殊情况下，先前的工作已经通过放射免疫分析证实了所有古代样本中骨钙素的存在。MALDI-MS技术不受传统蛋白质测序方法所面临的无数问题的影响。虽然这种方法从未在古代蛋白质上尝试过，但我们打算证明它在描绘古代蛋白质序列方面的潜力，从而识别本地有机物质。鉴于骨钙素在脊椎动物中的独有但普遍存在，该项目开发的技术将直接适用于任何数量的其他进化关系模糊的脊椎动物谱系的分子研究。

9614235 Ostrom A new approach, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), to determine the amino acid sequence of the protein osteocalcin in bones of ancient organisms and their closest modern ancestors to establish preliminary evolutionary relationships and assess indigeneity. The use of osteocalcin, the particular samples available, 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, retention of immunological specificity via radioimmunoassay (RIA), and the existence of an extensive database of protein sequences for osteocalcin. In our particular case, prior work has confirmed the presence of osteocalcin via RIA for all ancient samples. The MALDI-MS technique is not subject to the myriad of problems that confront traditional methods for protein sequencing. Although this approach has never been attempted on ancient proteins, we intend to demonstrate its potential to delineate ancient protein sequences and, thus, identify indigenous organic matter. Given the exclusive yet ubiquitous occurrence of osteocalcin among vertebrates, the techniques developed in this project will be directly applicable for molecular studies on any number of other vertebrate lineages with obscured evolutionary relationships.