A pan-N-formylmethionine-specific antibody as a tool for analyzing Nα-terminal formylation

Formylmethionine (fMet) plays crucial roles across bacterial and eukaryotic systems, contributing to protein translation, degradation, complex formation, stress adaptation, disease progression, and immune response. However, detecting fMet-bearing (fMet-) peptides and proteins has remained challenging due to the lack of effective anti-pan-fMet antibodies. We developed a polyclonal pan-fMet-specific antibody using a single antigen peptide, fMet-Gly-Ser-Gly-Cys pentapeptide, and a mixed antigen peptide, fMet-Xaa-Cys (Xaa, any of the 20 amino acids) tripeptides, as the immunogen. The resulting antibodies were rigorously evaluated through immunoblotting of bacterial and eukaryotic cell lysates, as well as enzyme-linked immunosorbent assays using synthetic fMet-peptides and their unformylated counterparts. The pan-fMet-specific antibodies provide a robust, cost-effective tool for detecting fMet-bearing proteins across species. Furthermore, this approach lays the groundwork for developing antibodies against other N-terminal modifications—such as acylation, alkylation, oxidation, and arginylation—enhancing our understanding on protein modifications across diverse biological contexts.