Aberrant post-translational modifications (PTMs) on arginine residues, namely methylation and citrullination, are closely linked to oncogenic processes. Detection of these PTMs is challenging with current technologies and can be hampered by low abundance and mass differences that are difficult to resolve by mass spectrometry. To overcome these hurdles, we developed methods for PTM detection using Quantum-Si’s PlatinumTM single-molecule protein sequencing platform.
To demonstrate the detection and differentiation of arginine PTMs, we applied our platform to distinguish between not only asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) but also between citrulline and native arginine residues. Sequencing traces revealed that arginine and ADMA bind the arginine recognizer with similar pulse duration (PD), whereas SDMA exhibited no binding. These results provide a clear kinetic difference between these isomeric arginine PTMs. Interestingly, arginine dimethylation also influenced the recognition of the upstream residue. Moreover, citrulline and arginine side chains also exhibited distinguishable kinetic signatures.
Citrullination eliminated N-terminal arginine recognition and resulted in a large increase in the median PD of preceding amino acid residues. Taken together, single-molecule protein sequencing offers an alternative approach to detection of arginine PTMs that is not based on m/z, but rather on the kinetic signature of binding between recognizers and N-terminal amino acids (NAAs). The ability to directly detect arginine PTMs offers potential for biomedical research. We envisage applications using Quantum-Si’s platform to key areas of cancer research including biomarker development, PTM crosstalk, and drug discovery.