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

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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.