Analysis of the dynamic sulfotyrosine proteome.

生物体的生存取决于不同类型的细胞在正确的时间、正确的地点组装正确的蛋白质复合物，从而相互沟通的能力。实现这一目标的一种方法是使用一种叫做酪氨酸蛋白硫转移酶（TPSTs）的化学和生物催化剂（酶）来改变聚合物（如蛋白质）的生物特性。TPSTs通过添加少量带电化学物质作为快速（通常）可逆调节的手段来驱动这一过程。这些事件，更准确地称为“翻译后修饰”，或PTMs，作为改变信息流的开关，并决定不同生物结果的类型，如细胞运动、生长、生存或死亡。我们的提议旨在利用新的工具和蛋白质组学技术（使用质谱法，以公正的方式观察细胞中的所有蛋白质）来评估向蛋白质中添加一种称为硫酸盐的特定化学基团。目前，对硫酸化的分析缺乏重点，吸引的战略资金很少，难以进行整体操作或研究，对其全球意义的研究具有挑战性。由于它支撑了如此多的基础生物学，但关于这种调节机制的机制的详细信息仍然缺乏，硫酸化研究需要协调一致的策略来开发分析，编译和分发有关蛋白质硫酸化如何调节细胞和非细胞生物学的大量生物学数据的方法。事实上，基于技术的方法分析蛋白质上的不同化学基团，磷酸盐，导致了我们对细胞如何沟通的理解的革命，并且对于40多年来在结构生物学，细胞信号和通信以及药物设计领域工作的生物学家至关重要，对世界各地的生物技术，制药工业和临床干预产生了连锁反应。为了快速推进我们对细胞蛋白酪氨酸硫酸化（sTyr）的理解，我们的目标将通过相关但不同的工作包来实现。其中包括：WP1：多种多肽和蛋白质中sTyr（和sThr/sSer）位点特异性的生化分析；WP2：从复杂的细胞衍生混合物中分离sTyr和基于质谱的定量sTyr分析；wp3：优化基于细胞的方法来操纵TPST1/2和干扰哺乳动物细胞中的sTyr含量；swp4：质谱数据的计算分析和酪氨酸硫酸化网络的解剖，用于公共分布和生物学推断；分析与Tyr硫酸化相关的新细胞作用的工作流程

The survival of an organism depends upon the ability of different cell types to communicate with each other by assembling the correct complexes of proteins at the correct time in the correct place. One way this is achieved is to use the tricks of chemistry and biological catalysts (enzymes) called tyrosyl protein sulfotransferases, or TPSTs, to change the biological properties of polymers, such as proteins. TPSTs drive this process by adding small charged chemicals as a means of rapid (usually) reversible regulation. These events, more accurately called 'post-translational modifications', or PTMs, act as switches to change information flow and dictate the types of different biological outcomes elicited, such as cell movement, growth, survival or death. Our proposal aims to exploit new tools and proteomics technology (using mass spectrometry, which looks at all proteins in cells in an unbiased way) to evaluate the addition of a specific chemical group, called sulfate, to proteins. Currently, the analysis of sulfation is unfocused, it attracts little strategic funding, and it is difficult to manipulate or study in a holistic manner, making efforts to study its global significance challenging. Since it underpins so much basic biology, yet detailed information as to the mechanism of this regulatory mechanism is still lacking, sulfation research requires concerted strategies to develop ways of analysing, compiling and distributing, the large amounts of biological data pertaining to how protein sulphation regulates cellular and acellular biology. Indeed, technology-based approaches for the analysis of a different chemical group on proteins, phosphate, led to a revolution in our understanding of how cells communicate, and has been critically important for biologists working in the areas of structural biology, cell signalling and communication and drug design for over 40 years, with knock-on effects on biotechnology, pharmaceutical industries and clinical intervention across the world. To rapidly advance our understanding of cellular protein tyrosine sulfation (sTyr), our aims will be achieved through related, but distinct, work packages. These are:WP1: Biochemical analysis of sTyr (and sThr/sSer) site-specificity in a variety of peptides and proteins WP2: sTyr isolation and mass spectrometry-based quantitative sTyr analysis from complex cell-derived mixturesWP3: Optimise cell-based approaches to manipulate TPST1/2 and perturb sTyr content in mammalian cellsWP4: Computational analysis of mass spectrometry data and dissection of tyrosine sulfation networks for public distribution and biological inferenceWP5: A workflow for analysis of new cellular roles associated with Tyr sulphation