Detection of Nα-terminally formylated native proteins by a pan-N-formyl methionine-specific antibody

Abstract

N-formyl methionine (fMet)-containing proteins are pro-duced in bacteria, eukaryotic organelles mitochondria and plastids, and even in cytosol. However, N alpha-terminally for-mylated proteins have been poorly characterized because of the lack of appropriate tools to detect fMet independently of downstream proximal sequences. Using a fMet-Gly-Ser-Gly-Cys peptide as an antigen, we generated a pan-fMet-specific rabbit polyclonal antibody called anti-fMet. The raised anti-fMet recognized universally and sequence context- independently Nt-formylated proteins in bacterial, yeast, and human cells as determined by a peptide spot array, dot blotting, and immunoblotting. We anticipate that the anti-fMet anti-body will be broadly used to enable an understanding of the poorly explored functions and mechanisms of Nt-formylated proteins in various organisms.