Targeted Proteomics Core

靶向蛋白质组学核心设计和实施靶向测定， 中心研究者对最初从蛋白质中鉴定的潜在蛋白质生物标志物进行定量和验证 由发现蛋白质组学核心（DPC）进行的分析，基因组研究，包括使用 神经元细胞类型特异性转录和翻译谱分析或文献。这些分析将 包括靶蛋白水平或其翻译后改变相对和绝对定量 在模型和其他生物体中涉及的修饰，作为发生在 对滥用药物的反应。为了解决中枢神经系统细胞和亚细胞异质性的巨大水平， 神经系统，另一个目标是开发分析亚细胞所需的目标工作流程 来自单一类型神经元的细胞器和亚蛋白质组，它们定义了构成动作基础的回路 以及滥用药物的成瘾性。在目标1中，我们将集成高分辨率数据独立 采用分级数据依赖采集（DIA）技术，将其应用于我们的大规模靶向蛋白质组分析 (DDA)由DPC从小鼠、大鼠和人脑区域产生的肽文库。DIA分析显示， 改变蛋白质组学格局的潜力，因为每个样品只需要运行一次，然后所有 可以回顾性地鉴定和定量肽片段。在目标2中，我们将制定有针对性的，高度 检测特定神经元亚型及其细胞器的蛋白质组（例如， 细胞核，突触囊泡），亚细胞级分或部分富集的样品（例如，PSD），然后使用并行 反应监测（PRM）以验证最显著差异表达的蛋白质。为支持这一 目的是，我们将开发改进的方法，用于蛋白质谱的有限数量的蛋白质， 从LCM和基于FACS的方法获得，需要分离神经元亚型。自从真核生物 蛋白质几乎总是在体内组装成多蛋白复合物，Aim 3将利用来自 我们通过实施改进的近距离生物素化技术来鉴定蛋白质， 我们的靶向测定发现这些蛋白质的表达差异最大。到 为了进一步增强对目标蛋白质组分析数据的解释，我们将在目标4中实现 用于翻译组谱分析的嘌呤霉素相关新生链蛋白质组学（PUNCH-P）测定。因为根据 稳态条件下蛋白质水平主要由转录物浓度决定， 相当大的兴趣，比较近似稳态蛋白质水平确定我们的DIA分析 通过我们的PUNCH-P分析确定了暴露于以下药物之前和之后的新生蛋白水平： 虐待在目标5中，我们将继续培训神经蛋白质组学中心的成员， 技术，包括实验设计、样品制备和处理、消化方案、解释 PRM和DIA光谱，以便他们能够最佳地利用中心提供的先进技术。

The Targeted Proteomics Core designs and implements targeted assays that have been used increasingly by Center investigators to quantitate and validate potential protein biomarkers initially identified from the protein profiling analyses carried out by the Discovery Proteomics Core (DPC), genomic studies, including the use of neuronal cell type-specific transcriptional and translational profiling, or the literature. These analyses will include relative and absolute quantitation of targeted protein levels or alterations in their post-translational modifications that have been implicated in model and other organisms as adaptive changes that occur in response to drugs of abuse. To address the huge level of cellular and sub-cellular heterogeneity in the central nervous system, another objective is to develop the targeted workflows needed to analyze subcellular organelles and sub-proteomes from the single types of neurons that define the circuits that underlie the actions and addictive properties of drugs of abuse. In Aim 1 we will integrate high resolution Data-Independent Acquisition (DIA) into our large scale targeted proteome assays using fractionated Data-Dependent Acquisition (DDA) peptide libraries generated by the DPC from mouse, rat, and human brain regions. DIA analysis has the potential to change the proteomic landscape since each sample only needs to be run once, and then all peptide fragments can retrospectively be identified and quantified. In Aim 2 we will develop targeted, highly sensitive DIA assays to examine the proteomes of specific neuronal sub-types and their organelles (e.g., nuclei, synaptic vesicles), sub-cellular fractions or partially enriched samples (e.g., PSD) and then use Parallel Reaction Monitoring (PRM) to validate the most significantly differentially expressed proteins. In support of this Aim, we will develop improved methodologies for protein profiling of the limiting amounts of protein that can be obtained from the LCM and FACS-based approaches needed to isolate neuronal sub-types. Since eukaryotic proteins almost always are assembled into multiprotein complexes in vivo, Aim 3 will leverage the data from our targeted assays by implementing improved proximity biotinylation technologies for identifying protein interactomes for those proteins that are found to be most differentially expressed by our targeted assays. To further enhance the interpretation of the data from our targeted proteome analyses, in Aim 4 we will implement a Puromycin-associated Nascent CHain Proteomics (PUNCH-P) assay for translatome profiling. Since under steady state conditions protein levels are largely determined by transcript concentrations, it will be of considerable interest to compare the approximately steady state protein levels determined by our DIA analyses with the nascent protein levels determined by our PUNCH-P analyses before and after exposure to drugs of abuse. In Aim 5, we will continue to train Neuroproteomics Center members in targeted mass spectrometric techniques including experimental design, sample preparation and handling, digestion protocols, interpretation of PRM and DIA spectra so they can optimally utilize the advanced technologies available in the Center.