Mass spectrometric characterization of the covalent modification of the nitrogenase Fe-protein in Azoarcus sp. BH72.
Nitrogenase Fe-protein modification was analyzed in the endophytic beta-proteobacterium Azoarcus sp. BH72. Application of modern MS techniques localized the modification in the peptide sequence and revealed it to be an ADP-ribosylation on Arg102 of one subunit of nitrogenase Fe-protein. A double digest with trypsin and endoproteinase Asp-N was necessary to obtain an analyzable peptide because the modification blocked the trypsin cleavage site at this residue. Furthermore, a peptide extraction protocol without trifluoroacetic acid was crucial to acquire the modified peptide, indicating an acid lability of the ADP-ribosylation. This finding was supported by the presence of a truncated version of the original peptide with Arg102 exchanged by ornithine. Site-directed mutagenesis verified that the ADP-ribosylation occurred on Arg102. With our approach, we were able to localize a labile modification within a large peptide of 31 amino acid residues. The present study provides a method suitable for the identification of so far unknown protein modifications on nitrogenases or other proteins. It represents a new tool for the MS analysis of protein mono-ADP-ribosylations.
Oetjen
Janina
J
General Microbiology, Faculty of Biology and Chemistry, University Bremen, Germany.
Rexroth
Sascha
S
Reinhold-Hurek
Barbara
B
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D020627:Azoarcus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007501:Iron / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational
D001426:Bacterial Proteins; D010455:Peptides; D001120:Arginine; D007501:Iron; D010088:Oxidoreductases; C029130:nitrogenase reductase