New Methods of Phosphoproteomics

描述(申请人提供)：蛋白质的磷酸化/去磷酸化的可逆过程是翻译后的蛋白质修饰，对细胞间信号转导至关重要。放松对信号转导级联的调控会扰乱这一平衡良好的系统，并与癌症、II型糖尿病、囊性纤维化、阿尔茨海默病、中风、心脏病等疾病有关。尽管人类基因组图提供了对我们基因结构和序列的宝贵见解，但它对这些关键的翻译后蛋白质修饰提供的洞察力有限。不幸的是，与基因组技术相比，与阐明信号转导相关的蛋白质组技术一直缺乏发展。目前用于浓缩磷酸化底物的最常见技术是基于磷酸基团与固定在IMACTM离子交换树脂上的金属离子结合部位的配位。虽然这种方法已经成功地应用于几个系统，但它并不是没有实验限制和蛋白质选择性方面的缺陷。我们提出的计划旨在开发一种改进的方法，用于高效和特异地固定化磷酸化的蛋白质和多肽。我们的方法部分基于磷酸化基团与固体载体壁上的共价结合，而不需要金属离子连接，从而消除了IMAC系统中经常遇到的并发症，在IMAC系统中，磷酸盐以外的基团与金属离子中心络合，从而降低了选择性。我们计划的另一个独特方面是设计用于固定化磷酸化蛋白质的有机功能介孔结构二氧化硅(OMS)。这种载体具有刚性的开放骨架结构、非常高的比表面积和非常窄的孔径分布。与基于树脂的支持系统相比，后一种特性提供了重要的优点，例如，消除了特定溶剂的膨胀和进入基质的需要，并通过更高的功能位点的表面密度更有效地固定化蛋白质。在下一个筹资周期中，我们计划实现以下具体目标： 1.评价介孔固定化金属层析(MIMC)作为一种改进的基于树脂的IMAC方法，通过离子亲和结合机制来富集磷酸化底物。2.建立了一种新的固相富集方法(SPE)，作为对IMAC和MIMC方法的改进，建立了一种磷酸肽富集法。 3.进一步发展了以单珠状有机功能介孔结构二氧化硅(OMS)为固相载体的固相萃取富集磷蛋白/多肽的新方法。 4.评价和应用细胞调控低氧诱导因子(HIFs)的新技术。

DESCRIPTION (provided by applicant): The reversible process of phosphorylation/dephosphorylation of proteins is a post-translational protein modification that is crucial for intercellular signal transduction. Deregulation of the signal transduction cascade upsets this well balanced system and has been implicated in diseases such as cancer, type II diabetes, cystic fibrosis, Alzheimer's disease, stroke, heart disease and many more. Even though the human genome map presents invaluable insight into the structure and sequence of our genes, it offers limited insight into these critical post-translational protein modifications. Unfortunately, proteomic techniques relevant to the elucidation of signal transduction have been lacking in development in comparison to genomic technologies. The most common technique currently being practiced for the enrichment of phosphorylated substrates is based on the coordination of phosphate groups to metal ion binding sites immobilized on an IMAC (tm) ion exchange resin. Although this approach has been successfully applied to several systems, it is not without experimental limitations and drawbacks in protein selectivity. Our proposed program is aimed at developing an improved method for the efficient and specific immobilization of phosphorylated proteins and peptides. Our approach is based in part on the covalent attachment of phosphorylated groups to the walls of a solid support without the need for metal ion linkages, thus eliminating the complications often encountered in IMAC systems wherein groups other than phosphate complex with the metal ion centers and compromise selectivity. Another unique aspect of our program is focused on the design of organofunctional mesostructured silicas (OMS) for the immobilization of phosphorylated proteins. This support has rigid open framework structures, very high surface areas, and very narrow pore size distributions. These latter features offer important advantages in comparison to resin-based support systems, including, for example, the elimination of the need for specific solvents to swell and access the matrix and the more efficient immobilization of protein by virtue of a higher surface density of functional sites. During the coming funding cycle, we plan to address the following specific aims: 1. Evaluate Mesoporous Immobilized Metal Chromatography (MIMC) as an improvement over the resinbased IMAC approach for the enrichment of phosphorylated substrates through an ion affinity binding mechanism. 2. Develop a phosphopeptide enrichment method using a new solid phase enrichment procedure (SPE) as an improvement over both IMAC and MIMC methodologies. 3. Further advance our new SPE approach to phosphoprotein/peptide enrichment using organofunctional mesostructured silica (OMS) in single bead form as the solid phase host. 4. Evaluate and apply the new technology towards the elucidation the cellular regulation hypoxia inducible factors (HIFs).