Shotgun and targeted proteomics of Mycolicibacterium smegmatis highlight the role of arginine phosphorylation in the functional adaptation to its environment

Although the phosphorylation of serine (S), threonine (T), and tyrosine (Y) is well-established, arginine phosphorylation (pR) has recently garnered significant attention due to its crucial role in bacteria pathogenicity and stress response. Mycolicibacterium smegmatis, a nonpathogenic surrogate of Mycobacterium tuberculosis, serves as a model for studying mycobacterial pathogenesis. A recent proteomics study identified six pR proteins in M. smegmatis. To gain a more comprehensive understanding, we performed pR profiling using mass spectrometry in combination with two distinct phosphopeptide enrichment strategies: titanium-immobilized metal ion affinity chromatography (Ti⁴⁺-IMAC) and Fe-NTA cartridge purification. This approach led to the identification of 1192 shared pR peptides with 1553 pR sites in M. smegmatis following both competitive and non-competitive scoring assessments for pR and pS/T/Y. Further stringent filtering through manual verification resulted in 58 high-confident pR sites across 57 proteins. These confirmed pR-proteins are functionally related, particularly in DNA binding and ATP binding. Alterations in the modification of three pR sites during the logarithmic and stationary phases at the phosphorylation level, but not at the total cell protein level, further suggest the role of pR in the bacterium's functional adaptation to its environment.

Significance

Our findings reveal that pR proteins are prevalent and play roles in DNA-binding and ATP-binding activities, providing insights into the broader biological functions of pR peptides in other genetically diverse species. The reliable identification of bacterial pR events in M. smegmatis not only propels the study of pR within the realm of proteomics but also paves the way for exploring its detailed function in bacteria.