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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=714107
• 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。