Tagging lipoproteins in mycobacteria via metabolic incorporation of modified fatty acids

Lipoproteins are lipid-modified proteins and critical components of the mycobacterial cell envelope with diverse functions related to virulence and survival. In Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, lipoproteins account for ∼3 % of the Mtb proteome, yet their precise functions remain poorly characterized. Prior experimental efforts have addressed the validation and preliminary functional annotation of specific lipoproteins. Whereas these studies elucidated aspects of lipoprotein localization and involvement in host-pathogen interactions, the methodologies employed do not allow further analysis such as targeted enrichment or on a proteome-wide level. To overcome these limitations, we developed a method in which modified fatty acids are metabolically incorporated into lipoproteins. These fatty acids are functionalized with click-compatible and photo-activatable chemical handles, thereby enabling downstream detection and identification of lipoproteins. The method described here provides a facile and versatile approach for further investigating mycobacterial lipoprotein synthesis, identity, and function.