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

项目摘要 蛋白质精氨酸甲基化是一种翻译后修饰（PTM），其调节许多生物学活性， 这些过程包括转录、信号转导、代谢和增殖。精氨酸甲基化 存在三种形式，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很重要， 相对于其他PTM，仍然研究不足。目前，该领域的主要障碍是缺乏强大的 以及通过质谱鉴定蛋白质精氨酸甲基化位点的成本有效的工具。鉴定 蛋白质精氨酸甲基化位点的质谱分析 因为在细胞裂解物中甲基化肽与非甲基化肽的化学计量比低。 然而，目前用于甲基肽富集的现有技术缺乏特异性， 再现性差，成本高。新的甲基肽亲和试剂的发明将使 更便宜、更可重复、更全面地发现精氨酸甲基化位点。解决这个 问题，我们建议使用mRNA展示来设计针对这三种蛋白质的新型蛋白质亲和试剂。 蛋白质精氨酸甲基化的形式：MMA、ADMA和SDMA。mRNA展示是一种强有力的体外选择 该技术可以从高达1014的文库中选择具有高结合亲和力（nM Kd）的蛋白质序列 序列的我们建议使用这些试剂从细胞裂解物中富集甲基肽，然后 使用液相色谱-质谱法（LC-MS）鉴定精氨酸甲基化位点 蛋白质组学我们假设mRNA展示产生的蛋白质亲和试剂将上级 目前的甲基肽富集策略在成本、再现性和覆盖范围方面的优势。我们 因此，我们将追求以下两个目标：1）我们将开发针对单甲基- 目的2：我们将开发针对甲基蛋白质组学的亲和纯化试剂， 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。所有 所开发的试剂将针对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mRNA展示来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展示从未被用于 开发特异性识别甲基精氨酸修饰肽的试剂或亲和试剂， 蛋白质组学如果成功的话，改进的甲基肽亲和试剂将代表技术上的进步 在目前的艺术状态，从而使科学界能够追求的研究， 以前是不可能的，包括全面的鉴定和研究蛋白质精氨酸 甲基化位点调节生物学功能。

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.