High sensitivity LC-MS to understand the role of Proteomes in the rules of life for Plant scientists and N8 partners

高灵敏度 LC-MS 可帮助植物科学家和 N8 合作伙伴了解蛋白质组在生命规则中的作用

• 批准号: BB/W019825/1
• 负责人: Ari Sadanandom
• 金额: $ 53.8万
• 依托单位: Durham University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW019825%2F1
• 关键词: sensitivity; LC; MS; understand; role

It has become apparent that biological complexity is largely orchestrated not through the number of genes but through variation at the protein level. Post-translational modifications (PTMs) generate different forms of a protein called proteoforms. These are responsible for cell signaling in almost every biological process. The dynamic nature of PTMs allows organisms to respond to highly transient changes in the environment with precise fine-tuning. This is especially relevant to plants due to their sessile nature; their growth and development is dependent upon their ability to adapt to changes in the environment. The reliance upon PTMs for plant responses to environmental change is orchestrated by thousands of genes encoding components of PTM systems and expansion in genes encoding the PTM machinery in plants is more significant than in any other kingdom. Understanding how these proteins affect cellular responses to environmental stress is a critical component of plant breeding programmes. This will be central in addressing the global challenge of sustainably producing food in a changing climate.To date, a number of genetic variants which alter the PTM state of the corresponding protein have been found to be extremely valuable in agriculture. Examples are found in the DELLA genes, which result in a high-yielding variety of wheat that, due to the reduced height, is more resistant to damage from wind and rain. This valuable trait is controlled by a single mutant dwarfing DELLA, the rht-1 allele. The wheat rht1 allele produces a DELLA protein that is altered in its ubiquitinated state and acts as a dominant allele to increase resistance to adverse environmental stress, indicating that PTMs can have added value in expediting breeding programmes with dominant alleles. It is predicted that the critical difference between the flooding tolerant and intolerant SUB1A alleles in rice is the mutation of a phosphorylation site in the SUB1A protein, further highlighting the importance of PTMs in crop adaptation to the environment. Introgression of the flooding tolerant SUB1 allele is the single most significant advancement for generating flood-tolerant rice varieties in the last 30 years. PTMs can thus be exploited to generate novel alleles for boosting crop productivity. However, a systematic approach to exploit PTMs for plant improvement strategies has been limited by the lack of appropriate methodologies for target discovery, mass spectrometry machine access and training. With our track record in PTM analysis, especially in plants, we are proposing to bridge this gap by building a plant-cell-focused proteome research platform that will be used for method development and discovery of novel PTMs in crop species. This will add significantly to the huge tapestry of genomics data held by the UK and international model plant and crop communities and provide data for the design and implementation of future research and breeding programmes.The acquisition of a highly sensitive mass spectrometer, such as the Bruker timsTOF Pro 2, with capability to detect these often-ephemeral modifications (as detailed in the proposal), will be central to our goal. The spectrometer will identify PTMs associated with beneficial traits while other technologies will elucidate the cellular responses to them. Although the focus is on plants the new mass spectrometer will equally serve a range of animal and microbial scientists in Durham who investigate PTM mediated signalling to understand the rules of life in various organisms. In some aspects PTM analysis in animal and microbial fields is further advanced than in plants and cross fertilisation of knowledge between these fields brings added value to this proposal.

很明显，生物复杂性在很大程度上不是通过基因数量而是通过蛋白质水平的变异来协调的。翻译后修饰 (PTM) 会产生不同形式的蛋白质，称为蛋白质形式。它们负责几乎每个生物过程中的细胞信号传导。 PTM 的动态性质使生物体能够通过精确的微调来响应环境中的高度瞬态变化。由于植物的固着性质，这一点与植物尤其相关。他们的成长和发展取决于他们适应环境变化的能力。植物对环境变化的反应对 PTM 的依赖是由编码 PTM 系统组件的数千个基因精心策划的，并且植物中编码 PTM 机制的基因的扩展比任何其他领域都更为重要。了解这些蛋白质如何影响细胞对环境压力的反应是植物育种计划的重要组成部分。这对于应对气候变化下可持续生产粮食的全球挑战至关重要。迄今为止，已发现许多改变相应蛋白质 PTM 状态的遗传变异在农业中极具价值。 DELLA 基因就是一个例子，该基因产生高产小麦品种，由于高度降低，更能抵抗风雨的损害。这一宝贵的性状由单一突变矮化 DELLA（rht-1 等位基因）控制。小麦 rht1 等位基因产生 DELLA 蛋白，该蛋白在泛素化状态下发生改变，并作为显性等位基因增强对不利环境胁迫的抵抗力，这表明 PTM 在加快显性等位基因育种计划方面具有附加值。据预测，水稻耐水和不耐水的SUB1A等位基因之间的关键区别是SUB1A蛋白中磷酸化位点的突变，进一步凸显了PTM在作物适应环境中的重要性。耐洪 SUB1 等位基因的渗入是过去 30 年来培育耐洪水稻品种的最重大进展。因此，可以利用 PTM 来生成新的等位基因，以提高作物生产力。然而，由于缺乏适当的目标发现、质谱仪访问和培训方法，利用 PTM 进行植物改良策略的系统方法受到限制。凭借我们在 PTM 分析（尤其是植物中）方面的记录，我们建议通过建立一个以植物细胞为中心的蛋白质组研究平台来弥补这一差距，该平台将用于方法开发和发现作物物种中的新型 PTM。这将极大地丰富英国和国际模式植物和作物群落所持有的庞大基因组学数据，并为未来研究和育种计划的设计和实施提供数据。购买高灵敏度质谱仪，例如布鲁克timsTOF Pro 2 能够检测这些经常短暂的修改（如提案中详述），将成为我们目标的核心。光谱仪将识别与有益性状相关的 PTM，而其他技术将阐明细胞对它们的反应。尽管重点是植物，但新型质谱仪同样可以为达勒姆的一系列动物和微生物科学家服务，他们研究 PTM 介导的信号传导，以了解各种生物体的生命规则。在某些方面，动物和微生物领域的 PTM 分析比植物领域更先进，这些领域之间知识的交叉应用为该提案带来了附加值。