Metabolic Labeling of Clostridioides difficile Proteins

The introduction of stable isotopes in vivo via metabolic labeling approaches (SILAC or ¹⁵N-labeling) allows, after combination of differentially treated labeled and unlabeled cells or protein extracts, for correction of protein quantification errors implemented during elaborated sample preparation workflows. The SILAC-based approach uses heavy arginine and lysine to incorporate the label into bacterial strains and cell lines, whereas ¹⁵N-metabolic labeling is achieved by cultivation in ¹⁵N-salt containing media. In case of Clostridioides difficile, the lack in arginine and lysine auxotrophy as well as the Stickland dominated metabolism makes metabolic labeling challenging. Here, a step-by-step guideline for the metabolic labeling of C. difficile is described, which combines cultivation in liquid ¹⁵N-substituted medium followed by cultivation steps on solid ¹⁵N-substituted medium. The described procedure results in a label incorporation rate higher than 97%. Cells prepared by the following method can be used as standard for relative quantification approaches of, e.g., the membrane or surface proteome of C. difficile.