Developing new technology and novel strategies to enhance the quality of quantitative proteomic analyses

开发新技术和新策略以提高定量蛋白质组分析的质量

• 批准号: 371929-2009
• 负责人: Smith, Jeffrey
• 金额: $ 2.19万
• 依托单位: Carleton University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2010
• 资助国家: 加拿大
• 起止时间: 2010-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=461431
• 关键词: Developing; new; technology; novel; strategies

For over two decades, mass spectrometry (MS) has proven to be particularly well suited to analyze proteins. Initial efforts in this field successfully identified dozens of proteins in a single experiment and it is now possible to identify thousands of proteins from complicated biological extracts with relative ease. MS-based protein characterization (proteomics) studies have become increasingly prevalent and attention has begun to shift from merely identifying the presence of proteins in a given sample to characterizing the dynamics of proteins in the context of a changing biological system. Although a number of strategies to quantify protein dynamics using MS are available, many are cost prohibitive or negatively affect the analytical sensitivity. Our ongoing research program will strive to develop technology and strategies to improve the reproducibility and overall quality of quantitative proteomic analyses; this work will concurrently develop improved protein identification methods as well. To begin, we will investigate the dynamics of protein phosphorylation, an important protein modification that is employed by cells to control protein function. To date, relatively few predicted phosphorylation sites have been experimentally confirmed despite the fact that many different disease states are associated with faulty phosphorylation mechanics. Current methods to chemically modify and enrich phosphorylated peptides involve multiple transfer steps to containers with relatively large volumes, resulting in sample loss and depletion of analytical sensitivity. To overcome this, microliter-scale columns packed with peptide-binding resin will be used to concentrate and process complicated biological samples so that phosphorylated peptides may be isolated with minimal transfer steps and user intervention. Furthermore, the ability to chemically modify peptides in distinct ways will be available which will allow peptide/phosphopeptide concentration dynamics to be measured over the course of an external stimulus (e.g. determining the proteomic response of cells exposed to a drug). The application of this technology to probe specific biological questions will be carried out in parallel with its development.

二十多年来，质谱（MS）已被证明特别适合于分析蛋白质。在这一领域的初步努力成功地在一次实验中鉴定了几十种蛋白质，现在有可能相对容易地从复杂的生物提取物中鉴定数千种蛋白质。基于质谱的蛋白质表征（蛋白质组学）研究已经变得越来越普遍，人们的注意力已经开始从仅仅识别给定样品中蛋白质的存在转向在不断变化的生物系统背景下表征蛋白质的动力学。虽然有许多策略可以使用质谱来量化蛋白质动力学，但许多策略成本过高或对分析灵敏度产生负面影响。我们正在进行的研究项目将努力开发技术和策略，以提高定量蛋白质组学分析的可重复性和整体质量；这项工作将同时开发改进的蛋白质鉴定方法。首先，我们将研究蛋白质磷酸化的动力学，这是细胞用来控制蛋白质功能的一种重要的蛋白质修饰。迄今为止，尽管许多不同的疾病状态都与错误的磷酸化机制有关，但实验证实的预测磷酸化位点相对较少。目前化学修饰和富集磷酸化肽的方法涉及多个转移步骤到相对大容量的容器中，导致样品损失和分析灵敏度的降低。为了克服这一点，将使用挤满肽结合树脂的微升级色谱柱来浓缩和处理复杂的生物样品，以便磷酸化肽可以通过最小的转移步骤和用户干预来分离。此外，以不同方式化学修饰肽的能力将是可用的，这将允许在外部刺激过程中测量肽/磷酸肽浓度动态（例如确定暴露于药物的细胞的蛋白质组反应）。这项技术在探索特定生物学问题上的应用将与它的发展同步进行。