Add Subproject Protein phosphorylation is one of the major posttranslational modifications that regulates protein expression and activity in bacteria. Although serine, threonine and tyrosine phosphorylation have been well-established, arginine phosphorylation has gained attention in recent decades due to its global importance in processes such as protein degradation, competence, stringent response, and other stress response. Mycolicibacterium smegmatis, a nonpathogenic surrogate of Mycobacterium tuberculosis, is a predominate model for studying the basic biology of mycobacterial pathogenesis. A recent study confirmed the presence of arginine phosphorylation in M. smegmatis and identified six arginine phosphorylated proteins based on shotgun proteomics analysis. Here, to provide a more comprehensive understanding, we performed a global, in-depth analysis of the M. smegmatis arginine phosphoproteome using high-accuracy mass spectrometry in combination with an optimized titanium-immobilized metal ion affinity chromatography (Ti4+-IMAC) phosphopeptide enrichment strategy.