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.