Development of an enhanced anti-pan-N-formylmethionine-specific antibody

Both bacterial and eukaryotic ribosomes can initiate protein synthesis with formylmethionine (fMet), but detecting fMet-bearing peptides and fMet-bearing proteins has been challenging due to the lack of effective anti-pan-fMet antibodies. Previously, we developed a polyclonal anti-fMet antibody using a fMet-Gly-Ser-Gly-Cys pentapeptide that detects those fMet-bearing peptides and fMet-bearing proteins regardless of their sequence context. In this study, we significantly improved the antibodys specificity and affinity by using a mixture of fMet-Xaa-Cys (fMXC) tripeptides (Xaa, any of the 20 amino acids) as the immunogen. This newly optimized anti-fMet antibody is a powerful, cost-effective tool for detecting fMet-bearing proteins across species. Furthermore, this approach provides a foundation for developing anti-pan-specific antibodies targeting other N-terminal modifications through acylation, alkylation, oxidation, or arginylation, etc. METHOD SUMMARYfMet-Gly-Ser-Gly-Cys (fMGSGC), fMet-dPEG4-Cys (fMdPEG4C), and fMet-Xaa-Cys (fMXC; Xaa, any of the 20 amino acids) were used as antigens to generate anti-pan-fMet-specific antibodies (anti-fMet antibodies). The quality of the raised antibodies was evaluated by immunoblotting using lysates from Escherichia coli (E. coli) DH5 cells and human kidney HK2 cells, as well as by enzyme-linked immunosorbent assay (ELISA) with purified fMet-bearing (fMet-) proteins and their unformylated counterparts.