Mass spectrometric determination of a novel modification of the N-terminusof histidine-tagged proteins expressed in bacteria

Two proteins, FKBP, and Spo0F, were expressed in bacteria as histidine-tagg ed fusion proteins and isolated under native conditions. MALDI-TOF-MS analy sis revealed that each protein preparation contained two components, neithe r of which corresponded to the molecular weights predicted from DNA sequenc es. The difference in molecular weight between the two FKBP components and two Spo0F components was approximately 178 +/- 14 Da. Site-specific proteol ytic cleavage resulted in the release off histidine-tagged peptide from the recombinant proteins. MALDI mass spectra of the cleaved proteins showed a single molecular ion peak for each species with the predicted molecular wei ghts. The histidine-tagged peptide released from both fusion proteins displ ayed two distinct peaks by MALDI-FT-MS corresponding to monoisotopic molecu lar weights of 2269.027 Da and 2447.1087 Da, respectively, which were both inconsistent with the predicted peptide sequence M-G-H-H-H-H-H-H-H-H-H-H-S- S-G-H-I-E-G-R of 2400.055 Da. The peptide at 2269.027 Da was sequenced by E SI-MS-MS and found to be a truncated histidine tag resulting from an initia tor methionine deletion. ESI-MS-MS analysis of the peptide at 2447.087 Da i ndicated a moiety of 178.0 Da attached to the second residue glycine of the histidine tag. This alteration of the N-terminus does not fit any known mo difications. A synthetic peptide with the identical sequence of the isolate d his-tag M-G-H-H-H-H-H-H-H-H-H-H remained unmodified during the protein pu rification process, suggesting that modification of the initiator methionin e was carried out in vivo, rather than the result of a chemical reaction fr om the isolation procedure. (C) 1999 Academic Press.