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Chemical Proteomics Strategies for Analyzing Protein Lipidation Reveal the Bacterial O -Mycoloylome.

Nicholas BanaheneTrenton M Peters-ClarkeKyle J BiegasEvgenia ShishkovaElizabeth M HartAmelia C McKitterickNikolas H KambitsisUlysses G JohnsonThomas G BernhardtJoshua J CoonBenjamin M Swarts
Published in: Journal of the American Chemical Society (2024)
Protein lipidation dynamically controls protein localization and function within cellular membranes. A unique form of protein O -fatty acylation in Corynebacterium , termed protein O -mycoloylation, involves the attachment of mycolic acids─unusually large and hydrophobic fatty acids─to serine residues of proteins in these organisms' outer mycomembrane. However, as with other forms of protein lipidation, the scope and functional consequences of protein O -mycoloylation are challenging to investigate due to the inherent difficulties of enriching and analyzing lipidated peptides. To facilitate the analysis of protein lipidation and enable the comprehensive profiling and site mapping of protein O -mycoloylation, we developed a chemical proteomics strategy integrating metabolic labeling, click chemistry, cleavable linkers, and a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method employing LC separation and complementary fragmentation methods tailored to the analysis of lipophilic, MS-labile O -acylated peptides. Using these tools in the model organism Corynebacterium glutamicum , we identified approximately 30 candidate O -mycoloylated proteins, including porins, mycoloyltransferases, secreted hydrolases, and other proteins with cell envelope-related functions─consistent with a role for O -mycoloylation in targeting proteins to the mycomembrane. Site mapping revealed that many of the proteins contained multiple spatially proximal modification sites, which occurred predominantly at serine residues surrounded by conformationally flexible peptide motifs. Overall, this study (i) discloses the putative protein O -mycoloylome for the first time, (ii) yields new insights into the undercharacterized proteome of the mycomembrane, which is a hallmark of important pathogens (e.g., Corynebacterium diphtheriae , Mycobacterium tuberculosis ), and (iii) provides generally applicable chemical strategies for the proteomic analysis of protein lipidation.
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