SILAC proteomics for quantitation of protein isoforms from alternative splicing in Arabidopsis seedlings

遗传变异是生物多样性和表型变异的重要基础。植物的生长和生产力以及植物对外界刺激（如病原体/害虫或逆境条件）的反应取决于植物物种的基因含量和基因表达的调控。基因在许多不同的水平上受到调控。一个重要的水平是基因的开启或关闭，或上升或下降，称为转录控制。第二个水平发生在基因转录或复制成RNA之后，称为转录后控制。有许多不同的转录后控制机制，选择性剪接（AS）是最重要的机制之一。选择性剪接是指基因转录物的不同部分以不同的组合连接在一起，从而从一个基因中产生多个信使RNA （mRNA）。由此产生的mrna可以被翻译成具有不同功能的蛋白质，或者可以作为降解的目标。因此，AS增加了生物体蛋白质组的复杂性，并可以调节mRNA的水平。选择性剪接影响植物发育、生存能力、对外部条件的适应性、代谢和生理的许多方面，最近的估计表明，植物中至少60%的内含子基因经历了AS。越来越多的例子被描述为AS调节表达或功能蛋白多样性。随着我们对这一重要调控水平的全面了解的增加，有必要在蛋白质水平上研究As的影响和动态变化。在鉴定和定量由AS产生的不同蛋白质同种异构体衍生的肽方面存在特殊的挑战（例如，区分特定同种异构体与共同同种异构体的肽的丰度）在各种基于质谱（MS）的定量蛋白质组学方法中，SILAC在这种特定应用中具有许多优势：常规检测成千上万种蛋白质，通常很大一部分蛋白质具有多个多肽，蛋白质的定量相对简单，并且可以使用良好的质谱分析软件。此外，SILAC能够量化低丰度肽和磷酸肽，并且已经开发出分析人类细胞中蛋白质同种异构体的分析方法。SILAC在植物（细胞培养）中的应用非常有限，因为植物是自养的，用含有稳定同位素的氨基酸进行标记效率低下。我们已经开发出一种方法来获得高水平的SILAC标记（>90%），更重要的是，在拟南芥幼苗中（与细胞培养相反）。这使得这项前沿技术首次适用于整个植物系统，这意味着它可以应用于植物生物学的广泛领域，从发育到对生物和非生物胁迫的反应，并有助于在蛋白质水平上对突变体进行比较。在本提案中，我们将使用这种SILAC技术的新修改，使其能够应用于植物幼苗，以研究蛋白质水平上的选择性剪接。我们将分析三组遗传系，其中剪接因子或NMD因子过度表达或突变，并且我们从转录分析中得知许多基因的AS显着改变。这些细胞系将最大限度地提高鉴定异构体特异性肽的机会。目前关于植物中蛋白质异构体变异的数据很少，利用SILAC和使用特定的遗传系将使我们能够更好地了解AS在蛋白质水平上的影响。最后，我们希望我们的植物SILAC系统能够更广泛地应用于其他非模式植物物种，并为研究翻译后修饰、蛋白质转换、microRNA对蛋白质水平的影响以及蛋白质相互作用网络等生化问题提供一种新的方法。

Genetic variation is an important basis for biodiversity and phenotypic variation. Plant growth and productivity and how plants respond to external stimuli such as pathogens/pests or stress conditions depend on the gene content of the plant species and the regulation of expression of the genes. Genes are regulated at many different levels. One important level is where genes are turned on or off or up or down - called transcriptional control. A second level occurs after the gene is transcribed or copied into RNA - called post-transcriptional control. There are many different mechanisms of post-transcriptional control and alternative splicing (AS) is one of the most important. Alternative splicing is where different portions of a gene transcript are joined in different combinations to generate more than one messenger RNA (mRNA) from a gene. The resultant mRNAs can be translated into proteins with different functions or can be targeted for degradation. Thus, AS increases the proteome complexity of an organism and can regulate mRNA levels. Alternative splicing affects many aspects of plant development, viability, adaptability to external conditions, metabolism and physiology and the most recent estimate suggests that at least 60% of intron-containing genes in plants undergo AS. More and more examples are being described where AS regulates expression or functional protein diversity. As our overall knowledge of this important regulatory level increases, it becomes necessary to be able to investigate the impact and dynamic changes of AS at the protein level. There are particular challenges in identifying and quantifying peptides deriving from different protein isoforms generated by AS (e.g. the abundance of peptides that distinguish specific isoforms from peptides common to isoforms)Of various mass spectrometry (MS)-based quantitative proteomics methods, SILAC has a number of advantages for this specific application: many thousands of proteins are routinely detected, usually a significant fraction of proteins have multiple peptides, quantitation of proteins is relatively straightforward and good MS analysis software is available. In addition, SILAC,is able to quantify low abundance peptides and phosphopeptides, and analysis methods have been developed for analysis of protein isoforms in human cells. SILAC has had very limited use in plants (cell culture) due to inefficient labelling with stable-isotope-containing amino acids as plants are autotrophic. We have developed a method to obtain high levels of SILAC labelling (>90%) and, more significantly, in Arabidopsis seedlings (as opposed to cell cultures). This makes this frontline technology amenable to whole plant systems for the very first time and means that it can be applied to a wide range of areas of plant biology from development to responses to biotic and abiotic stresses, and facilitates the comparison of mutants at the protein level.In this proposal we will use this new modification of SILAC technology which allows it to be applied to plant seedlings to investigate alternative splicing at the protein level. We will analyse three sets of genetic lines where splicing factors or NMD factors are over-expressed or mutated and where we know from transcript analysis that the AS of many genes is significantly altered. These lines will maximise the opportunity for identifying isoform-specific peptides. There is currently very little data on protein isoform variants in plants and exploiting SILAC and using specific genetic lines will allow us to better understand the impact of AS at the protein level. Finally, we expect our plant SILAC system to be applied more widely to other, non-model plant species and to provide a new method to investigate biochemical questions of post-translational modification, protein turnover, microRNA effects on protein levels, and protein interaction networks.