Near-Zero Missed Cleavages with a High-Fidelity Recombinant Arg-C Zero for Mass Spectrometry-Based Proteomics

Abstract

Proteolytic digestion remains a critical step in bottom-up proteomics workflows, with enzyme specificity and efficiency directly impacting peptide identification and protein sequence coverage. Here, we present the comprehensive characterization of Arg-C Zero, a recombinant arginyl endopeptidase derived from Porphyromonas gingivalis that exhibits exceptional fidelity in cleaving specifically at the C-terminus of arginine residues. Unlike conventional serine proteases such as Trypsin, Arg-C Zero utilizes a histidine-cysteine catalytic dyad mechanism, achieving near-zero missed cleavage rates (>99% efficiency) under standard proteomics conditions. Through systematic evaluation using HeLa protein extracts, we demonstrate that Arg-C Zero maintains consistent performance across varying digestion times. The enzyme shows robust activity across a broad pH range and tolerates up to 4M urea, making it ideally suitable for a diverse range of proteomics sample preparation workflows. While Trypsin/LysC combinations remain superior for comprehensive proteome coverage, Arg-C Zero offers unique advantages for applications requiring high specificity and reproducible arginine-specific cleavage patterns, particularly for analysis of post-translational modifications (PTMs). Here, we demonstrate how Arg-C Zero aids comprehensive mapping of histone PTMs, and when used in low-pH workflows help preserve labile ADP-ribosylation sites, expanding the analytical capabilities of mass spectrometry for characterizing these challenging modifications. The enzymes resistance to proline-adjacent cleavage sites and compatibility with standard mass spectrometry buffers position it as a valuable addition to the proteomics enzyme toolkit.