PROTEOMICS AND BIOMINERALIZATION OF DENTIN

描述（由申请人提供）：该独立科学家奖（K02）申请旨在支持蛋白质辅助生物矿化研究的职业发展：细胞外基质蛋白（ECM）翻译后修饰的作用及其对矿物质形成的影响。该专业知识将应用于申请人资助的R01“牙本质生物矿化”，该项目涉及使用质谱分析重组产生的磷酸化蛋白（PP）以及从稳定转染的非矿化细胞（NIH3T3）和具有矿化能力的细胞（MC3T3-E1和MDPC-23）中纯化的PP，以确定牙本质基质蛋白3的磷酸化模式和外显子5位点的差异。此外，我们将评估这种翻译后修饰如何影响矿物形成。职业发展技能将应用于与Co-PI和顾问的合作研究。蛋白质组学的新职业发展活动将包括匹兹堡大学提供的蛋白质组学课程、密集讲习班和在合作伙伴和核心设施实验室进行的动手实验。这将包括样品制备，在MALDI-TOF/TOF-MS/MS上运行样品和样品分析。矿物表征的职业发展将包括必要设备（x射线衍射，FTIR， SEM， TEM和EDAX）的教程，卡内基梅隆大学的材料科学和工程课程，重点是陶瓷，研讨会，合作实验室的样品分析。在5年的提案期限内，候选人将投入至少75%的时间进行研究，并计划参加一系列高级课程，包括科学中的负责任行为、职业发展和拨款写作、生物伦理学、数据管理、写作和演讲技巧。该奖项的支持将使申请人有时间学习质谱和矿物表征的基础知识，并建立一个专注于生理/病理生物矿化的强大研究计划。

DESCRIPTION (provided by applicant): This Independent Scientist Award (K02) application is designed to support career development in the studies of protein-assisted biomineralization: The role of post-translational modifications of extracellular matrix proteins (ECM) and its impact on mineral formation. This expertise will be applied to the applicant's funded R01 "Dentin Biomineralization" which involves the determination of the difference in phosphorylation patterns and sites of exon 5 of Dentin Matrix Protein 3 using mass spectrometry by analysing recombinantly produced phosphophoryn (PP) as well as purified PP from stably transfected non-mineralizing cells (NIH3T3) and cells that have the capability to mineralize (MC3T3-E1 and MDPC-23). In addition, we will assess how this post- translational modification impacts mineral formation. Career development skills will be applied to collaborative research with Co-PI and consultant. New career development activities in proteomics will include proteomics courses offered at the University of Pittsburgh, intensive workshops and hands on experiments in collaborator and core facility laboratories. This will involve sample preparation, running sample on MALDI-TOF/TOF-MS/MS and sample analysis. Career development in mineral characterization will include tutorials of the necessary equipment (x-ray diffraction, FTIR, SEM, TEM and EDAX), courses in Materials Sciences and Engineering focused on ceramics at Carnegie Mellon University, workshops, sample analysis in collaborator laboratory. The candidate will devote at least 75% of his time to research during the 5-year proposal duration and plans to attend a series of advanced courses, which include responsible conduct in science, career development and grant writing, bioethics, data management, writing and presentation skills. Support of this award will allow the applicant protected time to learn the fundamentals of mass spectrometry and mineral characterization as well as establish a strong research program focused on physiological/pathological biomineralization.