Proteomes of proteoforms - improving routine top-down proteomic analyses

蛋白质驱动细胞过程--它们是生命的分子主力。蛋白质大规模分析(蛋白质组学)是支持一系列研究的基石，例如与环境、农业、健康和工业研究有关的研究。一种特定的蛋白质以多个分子物种(蛋白质形式)的形式存在，这是因为特定的化学变化称为翻译后修饰(PTM)，这种变化调整了它的行为。为了分析生物样本(蛋白质组)中的蛋白质，我们必须有效地评估蛋白质形式；然而，常用的蛋白质组分析并不常规地做到这一点，而是使用随机氨基酸测序来对可能存在的蛋白质进行分类。我们正在进一步开发通过双向凝胶电泳法(2DE)进行的自上而下的分析，以在单一凝胶中解析数千种蛋白质形式，然后通过使用质谱仪(MS)进行氨基酸测序来识别这些蛋白质形式。我们为定量蛋白质组学的2DE进行了系统的优化，包括改进了用于蛋白质检测的高灵敏度、高成本效益的染色(CCBB)。随着对蛋白质形式的关注，我们的计划在未来五年集中于以下目标：1.通过将CCBB与其他菌株相结合来建立CCBB的卓越检测灵敏度，并合成和测试CCBB的化学变体，部分是通过定义另一种敏感菌株如何以不同的方式与蛋白质结合并相应地针对CCBB进行修饰。2.为了鉴定蛋白质形式，从2D凝胶中切下它们，用特定的酶处理，得到的多肽由MS测序。最常见的方法会导致信息丢失。我们将系统地改进程序，以确保最佳的MS数据，从而确保蛋白质鉴定。3.在2DE之后引入常规的第三步分离，以确保最好的分辨率，从而通过后续的多肽测序来鉴定蛋白质形式。4.建立新的成像仪器原型，以最高的灵敏度对已分解的蛋白质进行凝胶内检测，从而能够在每次分析中提取最多的信息，并识别那些对所研究的生物学至关重要的蛋白质。5.为了适应行业标准材料，在超薄(~0.2 mm)凝胶中分解蛋白质组，显著减少每次分析的总蛋白质并提高检测灵敏度，我们的研究计划将主要通过高质量、低成本的改进来提高蛋白质形式的分辨率、检测和鉴定，从而使高口径蛋白质组分析得以广泛使用。我们将为研究、医疗和工业领域的下一代领导者提供尖端培训和职业发展。这些成果将在基础研究和应用研究中引起全球关注并从中受益。多个研究领域和行业将受到影响。这是具有最广泛潜在影响的基础研究的概念。

Proteins drive cellular processes-they 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.