Analysis of the PKC-δ Signaling Pathway by Proteomics with Embryonic and Genetic Engeneering

基因组序列数据的积累促进了在mRNA(转录组)水平上系统描述基因表达谱的新方法的建立。然而，这种策略不一定提供关于蛋白质表达(蛋白质组)的直接信息，因为给定的mRNA的丰度不一定与编码的蛋白质的丰度相关。此外，蛋白质的许多特性，包括它们的亚细胞定位、与其他分子的相互作用、稳定性和翻译后修饰，都只能在蛋白质水平上进行研究。基于MS的蛋白质鉴定方法和蛋白质或DNA序列数据库的搜索的最新进展使蛋白质组的高通量分析成为可能。翻译后修饰，包括磷酸化，通过影响蛋白质与其他分子的相互作用、酶活性或其亚单位…来调节蛋白质的功能。更多的细胞定位，是控制许多细胞过程的关键。然而，这种修饰很难用标准的蛋白质组学方法来识别，因为修饰的蛋白质通常只占所有蛋白质分子的一小部分。因此，有必要首先浓缩这种修饰的蛋白质，并防止被高度丰富的蛋白质污染。高灵敏度的MS分析然后能够检测到各种类型的翻译后修饰。蛋白激酶C(PKC)由11种密切相关的异构体组成，参与多种信号转导机制。在PKC亚型中，PKC-δ是独一无二的，因为它的过度表达会导致细胞生长抑制。我们发现，缺乏PKC-δ的小鼠在没有刺激的情况下，表现出外周淋巴器官增大，B淋巴细胞数量增加，以及淋巴组织中存在大量生发中心。我们试图开发一种新的方法，名为“胚胎和基因工程蛋白质组学”，以揭示PKC-δ缺失小鼠的变化。在这项研究中，我们建立了高效和大规模的蛋白质磷酸化和泛素化方法。此外，我们还将SILAC方法应用于磷蛋白质组的定量分析。较少

The accumulation of genome sequence data has facilitated the establishment of new approaches to systematic characterization of gene expression profiles at the mRNA level (transcriptome). Such strategies do not, however, necessarily provide direct information about the profile of protein expression (proteome), given that the abundance of a given mRNA does not necessarily correlate with that of the encoded protein. Furthermore, numerous characteristics of proteins, including their subcellular localization, interactions with other molecules, stability, and posttranslational modification, are amenable to study only at the protein level. Recent advances in methods for protein identification based on MS and searches of protein or DNA sequence databases have allowed high-throughput analysis of the proteome. Posttranslational modification, including phosphorylation, regulates the functions of proteins by affecting their interactions with other molecules, their enzymatic activity, or their subc … More ellular localization and is pivotal to the control of many cellular processes. Such modification is difficult to identify by standard proteomics approaches, however, because the modified proteins usually constitute a small proportion of all protein molecules. It is therefore necessary first to concentrate such modified proteins and to prevent contamination by highly abundant proteins. Highly sensitive MS analysis is then able to detect various types of posttranslational modification. Protein kinase C (PKC), which comprises 11 closely related isoforms, has been implicated in a wide variety of signaling mechanisms. Among PKC isotypes, PKC-δ is unique in that its overexpression results in inhibition of cell growth. We showed that mice that lack PKC-δ exhibit enlargement of peripheral lymphoid organs, expansion of the B lymphocyte population, as well as the presence of numerous germinal centers in lymphoid tissues in the absence of stimulation. We tried to develop a new approach designated "Proteomics with Embryonic and Genetic Engineering (PGEM)" to uncover the changes in PKC-δ-null mice. In this study, we established efficient and large-scale methods for phosphorylation and ubiquitylation of proteins. Furthermore, we also appled the SILAC method to quantitative analysis of the phosphoproteome. Less