Proteomes of proteoforms - improving routine top-down proteomic analyses

蛋白质形式的蛋白质组 - 改进常规自上而下的蛋白质组分析

• 批准号: RGPIN-2019-04324
• 负责人: Coorssen, Jens
• 金额: $ 3.06万
• 依托单位: Brock University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=688534
• 关键词: Proteomes; proteoforms; improving; routine; top

Proteins drive cellular processesthey are the molecular workhorses of life. The large-scale analysis of proteins (Proteomics) is a cornerstone of research supporting a range of studies related, for example, to environmental, agricultural, health and industrial research. ***A given protein exists as multiple molecular species (proteoforms) because of specific chemical alterations called post-translational modifications (PTM) that tune its actions. To analyze the proteins in a biological sample (the proteome), we must assess proteoforms effectively; yet, commonly applied proteome analyses do not routinely do this, but use random amino acid sequencing to catalog proteins possibly present. We are further developing top-down' analyses by two-dimensional gel electrophoresis (2DE) to resolve, in a single gel, thousands of proteoforms that can then be identified by amino acid sequencing using mass spectrometry (MS). We are recognized for the systematic optimization of 2DE for quantitative proteomics, including refinement of a highly sensitive, cost-effective stain (cCBB) for protein detection.***With attention to proteoforms, our program centers on the following objectives over the next five years: ***1. To build on the superior detection sensitivity of cCBB by combining it with other stains, and synthesize and test chemical variants of cCBB, in part by defining how another sensitive stain binds differently to proteins and targeting modifications to cCBB accordingly.***2. To identify proteoforms they are cut from 2D gels, treated with specific enzymes, and the resulting peptides sequenced by MS. The most common protocol results in lost information. We will systematically refine the procedure to ensure the best MS data and thus proteoform identifications. ***3. To introduce a routine third separation step after 2DE, ensuring the best possible resolution and thus proteoform identifications by subsequent peptide sequencing. ***4. To prototype new imaging instrumentation for the highest sensitivity in-gel detection of resolved proteoforms, enabling extraction of the most information per analysis and identification of those proteoforms critical to the biology under study.***5. To resolve proteomes in ultrathin (~0.2mm) 2D gels adapting industry standard materials, markedly reducing total protein per analysis and improving detection sensitivity, ***Overall, our research program will improve proteoform resolution, detection, and identification, largely by high quality, low-cost refinements that enable the widespread use of high-calibre proteomic analyses. We will provide cutting-edge training and career development for the next generations of leaders in research, healthcare, and industry. The results will be of global interest and benefit across fundamental and applied research. Multiple research areas and industry will be impacted. This is the conception of fundamental research having the broadest potential ramifications. **

蛋白质驱动细胞过程，它们是生命的分子主力。蛋白质的大规模分析（蛋白质组学）是支持一系列相关研究（例如环境、农业、卫生和工业研究）的研究基石。***由于翻译后修饰（PTM）的特定化学变化，特定蛋白质以多种分子物种（蛋白质形态）的形式存在，从而调整其行为。为了分析生物样品中的蛋白质（蛋白质组），我们必须有效地评估蛋白质形态；然而，通常应用的蛋白质组分析并不常规地这样做，而是使用随机氨基酸测序来编目可能存在的蛋白质。我们正在进一步发展自上而下的二维凝胶电泳（2DE）分析，以在单个凝胶中解析数千种蛋白质，然后通过质谱（MS）进行氨基酸测序来鉴定。我们被公认为定量蛋白质组学的2DE系统优化，包括用于蛋白质检测的高灵敏度，高成本效益的染色（cCBB）的改进。***关注变形，我们的计划在未来五年内集中在以下目标：***1。通过将cCBB与其他染色剂结合，建立cCBB优越的检测灵敏度，合成和测试cCBB的化学变体，部分方法是通过定义另一种敏感染色剂如何与蛋白质结合并相应地靶向cCBB修饰。***2。为了鉴定蛋白质形态，他们从2D凝胶中切割，用特定的酶处理，然后用质谱测序得到的肽。最常见的方法导致信息丢失。我们将系统地完善程序，以确保最佳的质谱数据，从而鉴定蛋白质形态。* * * 3。在2DE后引入常规的第三个分离步骤，确保最佳的分辨率，从而通过随后的肽测序确定蛋白质形态。* * * 4。*** 4 .开发新型成像仪器的原型，用于最高灵敏度的凝胶内分解蛋白检测，从而能够在每次分析中提取最多的信息，并识别对所研究的生物学至关重要的蛋白。为了在超薄（~0.2mm） 2D凝胶中分析蛋白质组，显著减少每次分析的总蛋白，提高检测灵敏度，***总的来说，我们的研究项目将提高蛋白质的分辨率、检测和鉴定，主要是通过高质量、低成本的改进，使高质量的蛋白质组学分析得到广泛应用。我们将为研究、医疗和工业领域的下一代领导者提供尖端的培训和职业发展。其结果将在基础研究和应用研究领域引起全球的兴趣和利益。多个研究领域和行业将受到影响。这是基础研究的概念，具有最广泛的潜在影响。**