Detection of PTMs in Arginine-and Lysine-Rich Peptides via Sequential IP and Proteinase K Digestion of Tagged Protein

This is the authors' abstract. We don't add key points for this paper.

Post-translational modifications (PTMs) are critical regulators of protein function, especially within intrinsically disordered and arginine/lysine-rich regions such as nuclear localization signals and RNA-binding motifs. However, the dense clustering of arginine (R) and lysine (K) residues often complicates protease-based peptide recovery for downstream mass spectrometry (MS). Here, we describe a method optimized for detecting PTMs in such regions, particularly focusing on the peptide sequence RKKRRQRR, which resists cleavage by Proteinase K. The method combines FLAG-based immunoprecipitation, in-bead Proteinase K digestion, and a secondary antibody pull-down against the target peptide. The protocol includes specific strategies to eliminate co-eluted antibody contaminants and prepares samples for high-resolution LC-MS/MS analysis. This workflow facilitates reliable identification of PTMs in peptides refractory to traditional enzymatic digestion.