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，生物的复杂性在很大程度上不是通过基因的数量，而是通过蛋白质水平的变异来协调的。翻译后修饰（PTM）产生不同形式的蛋白质，称为蛋白质形式。它们负责几乎所有生物过程中的细胞信号传导。PTM的动态性质允许生物体通过精确的微调来响应环境中的高度瞬时变化。由于植物的固着性，这一点尤其重要;它们的生长和发育取决于它们适应环境变化的能力。植物对环境变化的响应依赖于PTM，这是由成千上万的编码PTM系统组分的基因协调的，并且在植物中编码PTM机制的基因的扩增比在任何其他王国中更重要。了解这些蛋白质如何影响细胞对环境胁迫的反应是植物育种计划的关键组成部分。这将是应对气候变化中可持续生产粮食这一全球挑战的核心。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基因就是一个很好的例子，它可以培育出高产的小麦品种，由于高度降低，这种小麦更能抵抗风雨的破坏。这种有价值的性状是由一个单一的突变体控制矮化DELLA，rht-1等位基因。小麦rht 1等位基因产生一种DELLA蛋白，该蛋白在其泛素化状态下发生改变，并作为显性等位基因增加对不利环境胁迫的抗性，这表明PTM可以在加快显性等位基因育种计划方面具有附加值。据预测，水稻耐淹和不耐淹SUB 1A等位基因之间的关键差异是SUB 1A蛋白磷酸化位点的突变，进一步突出了PTM在作物适应环境中的重要性。耐淹SUB1等位基因的渐渗是过去30年来产生耐淹水稻品种的最重要的进步。因此，可以利用PTM来产生新的等位基因以提高作物产量。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进行植物改进策略的系统方法受到限制。凭借我们在PTM分析方面的记录，特别是在植物中，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合这一差距，该平台将用于方法开发和发现作物物种中的新型PTM。这将大大增加英国和国际模式植物和作物社区所拥有的巨大基因组学数据，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，如布鲁克timsTOF Pro 2，能够检测这些通常短暂的修饰（如提案中所详述），将是我们目标的核心。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。虽然重点是植物，新的质谱仪将同样服务于一系列动物和微生物科学家在达勒姆谁调查PTM介导的信号，以了解生活在各种生物体的规则。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，这些领域之间的知识交叉融合为该提案带来了附加值。