Multiplexing Protein Quantification Core

摘要 多重蛋白质定量核心采用LC-Tandem MS和选择性反应 监测(SRM)以测量蛋白质丰度。SRM在众多产品中独树一帜 因为，就像蛋白质印迹一样，蛋白质是非常小心的 选择并确定量化的目标。通过设计SRM方法来实现目标 测量蛋白质在消化时所特有的几种多肽的丰度。 胰酶。然后在复杂的样本中检测到这些多肽，例如整个组织 匀浆，基于它们由碰撞-驱动的序列特异的碎裂反应- 诱导解离(CID)。这些多肽被鉴定为色谱峰 特征保留时间和丰度的计算基于 那些山峰。SRM的两个关键特性使其成为老年科学的强大新工具 研究。首先，SRM是一种高通量方法，一次可以测量大约30个蛋白质 单项检测。核心已经开发了一系列蛋白质分析小组，使研究人员能够 询问整个途径，如抗氧化蛋白，β氧化，克雷布斯循环，糖酵解， TCA-Cycle，以及其他正在不断开发的新的分析面板。第二，一个新的 可以在几个小时内设计和验证来自任何动物的任何蛋白质的分析。《核心》 已经开发出一种有条不紊的设计过程，使用 对多肽层析和气相离子化学有基本的了解，以及 公共数据库中的信息，以快速选择、测试和验证每个多肽的最佳多肽 蛋白。这种快速有效的化验设计/验证过程对于 使用抗体可获得性严重有限的动物模型进行衰老研究。 尽管SRM比以免疫化学为基础的检测方法(如免疫印迹)有许多优势 和ELISA，SRM确实需要一台主要设备(先进的、高灵敏度的LC- 串联质谱分析系统)，操作该仪器的专业知识，以及 用于测量每种蛋白质的多肽的开发和验证。系统会这样做 有能力每年运行多达6000到8000个样品，这使得一个核心 实验室是最大限度地利用这些强大工具的绝佳方式。 目标蛋白质定量核心的具体目标是： 目的1.提供高通量的多组蛋白质定量 小鼠和大鼠的组织/细胞的多种途径。 目标2.为无脊椎动物和其他动物模型开发新的分析方法和板，例如， 长寿物种，以及个别研究人员所要求的蛋白质。 目的3.发展蛋白质翻译后修饰的检测技术。

ABSTRACT The Multiplexed Protein Quantification Core uses LC-tandem MS with selected reaction monitoring (SRM) to measure protein abundance. SRM is unique among the various quantitative mass spectrometry methods because, like Western blots, proteins are carefully selected and targeted for quantification. Targeting is achieved by designing the SRM method to measure the abundance of several peptides, which are unique to the protein when digested with trypsin. These peptides are then detected in complex samples, such as whole tissue homogenates, based on their sequence-specific fragmentation reactions driven by collision- induced dissociation (CID). The peptides are identified as chromatographic peaks at characteristic retention times and the abundance calculated based on the integrated area of those peaks. Two key features of SRM make this a powerful new tool for Geroscience research. First, SRM is a high throughput method where one can measure ~30 proteins in a single assay. The Core has developed panels of protein assays that allow investigators to interrogate entire pathways such as antioxidant proteins, beta oxidation, Krebs cycle, glycolysis, TCA-cycle, and others with new assay panels continuously being developed. Second, a new assay can be designed and validated for any protein from any animal in a few hours. The Core has developed a methodical design process that evaluates the protein sequence using a fundamental understanding of peptide chromatography and gas phase ion chemistry, along with information in public databases, to rapidly select, test, and validate the best peptides for each protein. This rapid and effective assay design/validation process is especially important for aging research using animal models where the availability of antibodies is severely limited. Although SRM has many advantages over immunochemical-based assays like Western blot and ELISA, SRM does require a major piece of equipment (an advanced, high sensitivity LC- tandem mass spectrometry system), expertise to operate that instrument, and expertise for the development and validation of the peptides used to measure each protein. The systems do have the capacity to run as many as 6000 to 8000 samples per year, which makes a core laboratory an excellent way to maximize the benefit of these powerful tools. The Specific Aims for the Targeted Protein Quantitation Core are: Aim 1. Provide high throughput multiplexed protein quantification of panels of proteins in multiple pathways for tissues/cells from mice and rats. Aim 2. Develop new assays and panels for invertebrates and other animal models, e.g., long-lived species, and for proteins requested by individual investigators. Aim 3. Develop the technology to measure the post-translational modifications of proteins.