Developing novel affinity reagents for methyl-arginine proteomics using mRNA display

项目总结 蛋白质精氨酸甲基化是一种翻译后修饰(Ptm)，它调节许多生物 包括转录、信号转导、新陈代谢和增殖等过程。精氨酸甲基化 以三种形式出现，单甲基精氨酸(MMA)、不对称二甲基精氨酸(ADMA)和对称 二甲基精氨酸(SDMA)尽管精氨酸甲基化在人类生物学和疾病中具有重要意义 相对于其他的PTM，研究还不够深入。目前，该领域的主要障碍是缺乏强有力的 以及通过质谱学识别蛋白质精氨酸甲基化位点的经济高效的工具。身份识别 利用蛋白质组学研究蛋白质精氨酸甲基化位点需要甲基肽的浓缩 因为在细胞裂解产物中甲基化的多肽与非甲基化的多肽的化学计量比很低。 然而，目前最先进的甲基肽浓缩技术缺乏特异性， 重复性差，成本高。新的甲基肽亲和试剂的发明将使 更便宜、更重复性和更全面的精氨酸甲基化位点的发现。要解决这个问题 针对这一问题，我们提出利用mRNA显示技术设计新型蛋白亲和试剂来对抗这三种病毒 蛋白质精氨酸甲基化的形式：MMA、ADMA和SDMA。MRNA展示是一种强大的体外选择 可以从多达1014个文库中选择具有高结合亲和力(NM KD)的蛋白质序列的技术 序列。我们建议使用这些试剂从细胞裂解物中富集甲基肽，然后 液质联用法鉴定精氨酸甲基化位点 蛋白质组学。我们假设，由mRNA展示产生的蛋白质亲和试剂将优于 目前的甲基肽浓缩策略在成本、重复性和覆盖面的广度方面。我们 为此，我们将追求以下两个目标：1)开发针对单甲基的亲和纯化试剂- 精氨酸(MMA)用于甲基蛋白质组学；2)目标2：我们将开发亲和纯化试剂 不对称二甲基精氨酸(ADMA)和对称二甲基精氨酸(SDMA)用于甲基蛋白质组学。全 开发的试剂将与最先进的甲基精氨酸蛋白质组学进行验证。如果成功，这些 研究将证明使用信使核糖核酸显示技术开发一套新的蛋白质亲和力的可行性。 甲基蛋白质组学试剂。我们的方法是高风险的，因为mRNA展示从未被用来 开发专门识别甲基精氨酸修饰的多肽或亲和试剂的试剂 蛋白质组学。如果成功，改进的甲基肽亲和试剂将代表着一项技术进步 从而使科学界能够进行具有以下特点的研究 以前是不可能的，包括全面鉴定和研究蛋白质精氨酸 调节生物功能的甲基化位点。

PROJECT SUMMARY Protein arginine methylation is a post-translational modification (PTM) that regulates numerous biological processes including transcription, signal transduction, metabolism, and proliferation. Arginine methylation occurs in three forms, monomethyl-arginine (MMA), asymmetric dimethyl-arginine (ADMA), and symmetric dimethyl-arginine (SDMA). Despite its importance in human biology and disease, arginine methylation remains understudied relative to other PTMs. Currently, the major obstacle in the field is the lack of robust and cost-effective tools to identify protein arginine methylation sites by mass spectrometry. Identification of protein arginine methylation sites using mass spectrometry proteomics requires methyl-peptide enrichment because of the low stoichiometry of methylated peptides to non-methylated peptides in cell lysates. However, current state-of-the-art technologies for methyl-peptide enrichment suffer from lack of specificity, poor reproducibility, and high cost. The invention of new methyl-peptide affinity reagents would enable cheaper, more reproducible, and more comprehensive discovery of arginine methylation sites. To solve this problem, we propose to use mRNA display to engineer novel protein affinity reagents against the three forms of protein arginine methylation: MMA, ADMA, and SDMA. mRNA display is a powerful in vitro selection technology that can select protein sequences with high binding affinity (nM Kd) from libraries of up to 1014 sequences. We propose to use these reagents to enrich methyl-peptides from cell lysates, followed by identification of arginine methylation sites using liquid chromatography-mass spectrometry (LC-MS) proteomics. We hypothesize that protein affinity reagents generated by mRNA display will be superior to current methyl-peptide enrichment strategies in terms of cost, reproducibility, and breadth of coverage. We will thus pursue the following two aims: 1) We will develop affinity purification reagents against monomethyl- arginine (MMA) for methyl-proteomics; 2) Aim 2: We will develop affinity purification reagents against asymmetric dimethyl arginine (ADMA) and symmetric dimethyl arginine (SDMA) for methyl-proteomics. All developed reagents will be validated against state-of-the-art methyl-arginine proteomics. If successful, these studies will demonstrate the feasibility of using mRNA display to develop a new suite of protein affinity reagents for methyl proteomics. Our approach is high-risk in that mRNA display has never been used to develop agents that specifically recognize methyl-arginine-modified peptides or affinity reagents for proteomics. If successful, improved methyl-peptide affinity reagents would represent a technical advance over the current state of the art, thereby enabling the scientific community to pursue studies that have previously been impossible, including comprehensive identification and investigation of protein arginine methylation sites that regulate biological function.