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

很明显，生物的复杂性在很大程度上不是由基因的数量决定的，而是通过蛋白质水平上的变异来决定的。翻译后修饰(PTM)产生一种不同形式的蛋白质，称为蛋白质形式。这些细胞在几乎每个生物过程中都负责细胞信号传递。PTMS的动态性质使生物体能够通过精确的微调对环境中的高度瞬变变化做出反应。由于植物的固位性，这一点与植物特别相关；它们的生长和发育取决于它们适应环境变化的能力。植物对环境变化的反应依赖于PTM，这是由数千个编码PTM系统组成部分的基因调控的，而编码PTM机制的基因在植物中的扩张比任何其他王国都更显着。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。这将是应对在不断变化的气候中可持续生产粮食的全球挑战的核心。到目前为止，一些改变相应蛋白质的PTM状态的遗传变异在农业中被发现非常有价值。在della基因中发现了这样的例子，这种基因导致了一种高产品种的小麦，由于高度降低，更能抵抗风雨的破坏。这一有价值的性状由一个突变的矮化della控制，即RHT-1等位基因。小麦rht1等位基因产生一种在其泛素化状态下发生改变的della蛋白，并作为显性等位基因来增强对不利环境胁迫的抗性，这表明PTMS在加快具有显性等位基因的育种计划方面具有附加值。据预测，水稻耐淹和不耐涝等位基因的关键区别在于SUB1A蛋白中一个磷酸化位点的突变，这进一步突显了PTMS在作物适应环境中的重要性。在过去的30年里，耐涝等位基因SUB1的导入是培育耐涝水稻品种的最大进展。因此，可以利用光敏核不育系产生新的等位基因来提高作物产量。然而，由于缺乏适当的目标发现、质谱机接入和培训方法，利用PTMS进行植物改良战略的系统方法一直受到限制。凭借我们在PTM分析方面的记录，特别是在植物方面，我们建议通过建立一个以植物细胞为重点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。这将显著增加英国和国际模式植物和作物社区拥有的庞大的基因组数据，并为未来研究和育种计划的设计和实施提供数据。获得高灵敏度的质谱仪，如Bruker timsTOF Pro 2，具有检测这些经常短暂的修改的能力(如提案中详细说明的那样)，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。虽然重点放在植物上，但新的质谱仪将同样服务于达勒姆的一系列动物和微生物科学家，他们研究PTM介导的信号传递，以了解各种有机体的生命规则。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交流为这一提议带来了附加值。

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.