Discovery Proteomics Core

发现蛋白质组学核心

• 批准号: 9282574
• 负责人: TuKiet T Lam
• 金额: $ 62.85万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9282574
• 关键词: Amino Acids; Animals; Bioinformatics; Biological; Biological Assay; Biometry; Biophysics; Biostatistics Core; Brain; Brain region; Cell Culture Techniques; Cell Nucleus; Cells; Chemicals; Circular Dichroism; Collaborations; Complement; Complex; Coupling; Cytology; Cytometry; Data; Data Set; Databases; Digestion; Dissociation; Drug Addiction; Drug effect disorder; Electron Transport; Electrons; Experimental Designs; Exposure to; Field Flow Fractionation; Fluorescence; Heterogeneity; Histones; Immunologics; Individual; Information Dissemination; Kinetics; Label; Lasers; Libraries; Ligands; Lipids; Mammals; Mass Spectrum Analysis; Methodology; Methods; Microscopy; Mus; National Institute of Drug Abuse; Neuraxis; Neurologic; Neurons; Nucleic Acids; Organelles; Peptides; Phosphatidylinositols; Post-Translational Protein Processing; Preparation; Procedures; Protein Analysis; Proteins; Proteome; Proteomics; Psychotropic Drugs; Publications; RNA; Research; Research Personnel; Resources; Role; Sampling; Site; Software Tools; Source; Spottings; Stable Isotope Labeling; Surface Plasmon Resonance; System; Techniques; Technology; Thermodynamics; Tissues; Training; Transgenic Organisms; Trypsin; Ursidae Family; Viral; Work; addiction; base; biological systems; biophysical analysis; cell type; combinatorial; design; differential expression; drug of abuse; experimental study; holistic approach; improved; innovation; interest; light scattering; mass spectrometer; member; microcalorimetry; neuroproteomics; next generation; novel; polypeptide; potential biomarker; protein complex; protein expression; protein profiling; psychostimulant; relating to nervous system; repository; small molecule; success; tool; transcriptome sequencing; virtual

Discovery Proteomics Core: Summary The Discovery Proteomics Core (DPC) uses a full range of protein profiling technologies that are very positively leveraged by complementary biophysics and lipidomics analyses. Proteins whose expression or posttranslational modifications (PTMs) are altered by exposure to drugs of abuse in animal or cell-based systems will be identified by “bottom-up” MS-based technologies that will include iTRAQ, SILAC/SILAM, Label Free Quantitation (LFQ), Multi-dimensional Protein Identification Technology (MudPIT), and by coupling of LFQ to the data independent acquisition technology of SWATH. In addition, the Core will use Top-down/Middle- down proteomics methodologies to characterize intact proteoforms and their PTMs. Innovative sample preparation approaches will be used to reach deeper into the neural proteome with laser capture microscopy (LCM), fluorescence-based cytometry methods (FACS and FANS), and immuno-affinity methods in conjunction with transgenic and viral approaches that will enable analysis of cell type- and organelle-specific proteomes. Proteome expression will be validated by MRM/SWATH assays carried out in collaboration with the Targeted Proteomics Core (TPC). The availability of several biophysical technologies including isothermal microcalorimetry (ITC), static and dynamic light scattering, circular dichroism (CD), surface plasmon resonance (SPR), stopped-flow (SF), and asymmetric flow field-flow-fractionation (AFFF) will extend protein profiling analyses into the functional domain by quantitatively characterizing the thermodynamics and kinetics that underlie protein:protein and protein:ligand interactions of interest. Among the latter are phosphoinositides that have the ability to control virtually every aspect of neuronal function via their interactions with and modulation of the activities of neuronal proteins. Biophysical and phosphoinositide analyses, combined with proteome level analyses will provide an increasingly biological systems level approach. All protein profiling data will be stored in the Yale Protein Expression Database (YPED) and its Repository will be used to disseminate these data. Further, together with the Bioinformatics and Biostatistics Core (BBC), the DPC will use RNA-sequencing to construct experiment- and cell-type specific MS/MS protein reference databases that will be complemented by a PTMome database to increase peptide and protein identification rates. By taking a holistic approach, the DPC will provide Center investigators with the broad range of tools and training needed to identify, and then to understand why certain proteins and their phosphoinositide effectors are differentially expressed following exposure to psychostimulants and psychotropic drugs.

Discovery蛋白质组学核心：总结 Discovery Proteomics Core（DPC）使用全方位的蛋白质分析技术， 通过互补的生物物理学和脂质组学分析积极利用。蛋白质的表达或 翻译后修饰（PTM）在动物或细胞中通过暴露于滥用药物而改变， 系统将通过“自下而上”的基于MS的技术来识别，这些技术包括iTRAQ、SILAC/SILAM、标签 自由定量（LFQ），多维蛋白质鉴定技术（MudPIT），并通过耦合LFQ 小水线面双体船数据独立采集技术。此外，核心将使用自上而下/中间- 下蛋白质组学方法来表征完整的蛋白质型和它们的PTM。创新样本 制备方法将用于激光捕获显微镜深入神经蛋白质组 (LCM)基于荧光的细胞术方法（FACS和FANS）和免疫亲和方法结合 转基因和病毒的方法，这将使细胞类型和细胞器特异性蛋白质组的分析。 蛋白质组表达将通过MRM/SWATH测定法进行验证，该测定法与靶向的 蛋白质组学核心（TPC）。几种生物物理技术的可用性，包括等温 微量热法（ITC），静态和动态光散射，圆二色性（CD），表面等离子体共振 (SPR)停止流（SF）和不对称流场流分级（AFFF）将扩展蛋白质谱分析 通过定量表征热力学和动力学， 蛋白质：蛋白质和蛋白质：配体相互作用的基础。后者包括磷酸肌醇， 有能力通过与神经元的相互作用和调节来控制神经元功能的几乎每个方面。 神经元蛋白的活动。生物物理和磷酸肌醇分析，结合蛋白质组水平 分析将提供越来越多的生物系统水平的方法。所有蛋白质分析数据将被存储在 耶鲁大学蛋白质表达数据库（YPED）及其知识库中的数据将用于传播这些数据。 此外，DPC将与生物信息学和生物统计学核心（BBC）一起使用RNA测序， 构建实验和细胞类型特异性MS/MS蛋白质参考数据库， PTMome数据库，以提高肽和蛋白质鉴定率。通过采取全面的方法， DPC将为中心研究者提供识别所需的广泛工具和培训，然后 理解为什么某些蛋白质和它们的磷脂酰肌醇效应物在以下情况下差异表达 接触精神兴奋剂和精神药物。