Targeted Proteomics Core

靶向蛋白质组学核心：综述 靶向蛋白质组学核心设计并实施了越来越多使用的靶向分析 由中心研究人员快速定量和验证与他们的研究相关的潜在蛋白质生物标记物 神经信号与药物滥用的作用。通过利用我们内部有针对性的蛋白质组工作流程 对于MRM和数据独立(DIA)分析，我们建议与NIDA中心调查人员合作， 以及发现蛋白质组学、生物信息学和生物统计学核心，以准确地定量 在选定的感兴趣亚群中的蛋白质表达或其修饰。与总体情况完全一致 为了实现该中心的目标，将以创新的方式使用有针对性的蛋白质组方法，以系统地 通过设计MRM和DIA分析来解决单细胞类型分析的固有挑战 用于亚细胞细胞器和亚蛋白质组。还将使用有针对性的蛋白质组学方法来利用 来自选定脑区或神经细胞类型的RNA-SEQ数据，例如，在特定的定向分析中 在特定细胞类型中选择性表达或调节的蛋白质或相关蛋白质组。是这样的 有针对性的方法将是TPC独有的，并将对中心的能力产生重大影响 研究人员将使用最先进的方法进行创新研究，以研究药物对人体的作用 虐待。具体地说，我们将通过开发高度灵敏的MRM分析来利用我们优化的MRM工作流程 检查特定细胞器(如核、线粒体、突触小泡)、亚细胞、 从单一类型的神经细胞中分离或部分浓缩样品。我们还将扩大我们目前的 大鼠/小鼠脑靶向MRM PSD蛋白质组分析从112个蛋白质到200个蛋白质。此外，我们还将 将高分辨率DIA采集集成并扩展到我们的下一代大规模靶向蛋白质组中 针对特定类型神经元的亚蛋白质组的分析。其他项目包括实施 DIA检测尽可能多地定量已被确认为 人的肌醇磷脂结合蛋白质组。DIA分析有可能改变蛋白质组学 因为每个样本只需要运行一次，然后所有的肽片段都可以回溯 识别和量化。为了推进这些研究，我们将建立一个多肽生物标记物库，它将是 公开发布在我们的数据库中，可供中心的多名调查人员使用 其他人分析大脑中的基本信号转导机制，或研究 暴露在药物滥用的这些信号机制上。重要的是，我们将继续训练 神经蛋白质组学中心成员在质谱学技术方面的研究，包括实验设计、样品 制备(如组织、细胞)和处理、消化方案、MRM和DIA波谱的解释 他们可以以最佳方式利用该中心提供的先进技术。

Targeted Proteomics Core: Summary The Targeted Proteomics Core designs and implements targeted assays that have been used increasingly by Center investigators to rapidly quantitate and validate potential protein biomarkers related to their studies of neuronal signaling and the actions of drugs of abuse. By utilizing our in-house targeted proteome workflows for both MRM and data-independent (DIA) analysis, we propose to collaborate with NIDA Center investigators, and the Discovery Proteomics and Bioinformatics and Biostatistics Cores, to accurately quantitate either protein expression or their modifications in selected sub-groups of interest. Fully consistent with the overall goals of the Center, targeted proteomic approaches will be used in an innovative manner to systematically address the inherent challenges of single cell type analysis through the ability to design MRM and DIA assays for subcellular organelles and sub-proteomes. Targeted proteomic approaches will also be used to leverage RNA-seq data from selected brain regions or neuronal cell types, for example, in directed analysis of specific proteins or groups of related proteins that are selectively expressed or regulated in a specific cell type. Such targeted approaches will be unique to the TPC and will have a major impact on the ability of Center investigators to carry out innovative research using state-of-the-art approaches to study the action of drugs of abuse. Specifically, we will leverage our optimized MRM workflow by developing highly sensitive MRM assays to examine the proteomes of specific organelles (e.g., nuclei, mitochondria, synaptic vesicles), sub-cellular fractions or partially enriched samples from single types of neuronal cells. We will also expand our current rat/mouse brain targeted MRM PSD proteome assay from 112 proteins to 200 proteins. In addition, we will integrate and expand high resolution DIA acquisition into our next generation large scale targeted proteome assays directed at sub-proteomes from specific types of neurons. Other projects include implementation of a DIA assay to quantify as many as possible of the ~400 proteins that have been identified as being members of the human phosphoinositide-binding proteome. 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. To advance these studies, we will build a library of peptide biomarkers that will be posted publically on our YPED database and that can be used by multiple investigators in the Center and elsewhere who analyze basic signal transduction mechanisms in the brain, or who study the effects of exposure to drugs of abuse on these signaling mechanisms. Importantly, we will continue to train Neuroproteomics Center members in mass spectrometric techniques including experimental design, sample preparation (e.g. tissues, cells) and handling, digestion protocols, interpretation of MRM and DIA spectra so they can optimally utilize the advanced technologies available in the Center.