Mass spectrometric determination of a novel modification of the N-terminus of histidine-tagged proteins expressed in bacteria.
Two proteins, FKBP, and Spo0F, were expressed in bacteria as histidine-tagged fusion proteins and isolated under native conditions. MALDI-TOF-MS analysis revealed that each protein preparation contained two components, neither of which corresponded to the molecular weights predicted from DNA sequences. The difference in molecular weight between the two FKBP components and two Spo0F components was approximately 178 +/- 14 Da. Site-specific proteolytic cleavage resulted in the release of 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 weights. The histidine-tagged peptide released from both fusion proteins displayed two distinct peaks by MALDI-FT-MS corresponding to monoisotopic molecular weights of 2269. 027 Da and 2447.087 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 ESI-MS-MS and found to be a truncated histidine tag resulting from an initiator methionine deletion. ESI-MS-MS analysis of the peptide at 2447.087 Da indicated 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 modifications. A synthetic peptide with the identical sequence of the isolated his-tag M-G-H-H-H-H-H-H-H-H-H-H remained unmodified during the protein purification process, suggesting that modification of the initiator methionine was carried out in vivo, rather than the result of a chemical reaction from the isolation procedure.
Yan
Z
Z
R. W. Johnson Pharmaceutical Research Institute, Spring House, Pennsylvania 19477-0776, USA.
Caldwell
G W
GW
McDonell
P A
PA
Jones
W J
WJ
August
A
A
Masucci
J A
JA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002478:Cells, Cultured; D004926:Escherichia coli; D015951:Factor Xa; D006639:Histidine / Q000737:chemistry*; D020104:Immunophilins / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D017421:Sequence Analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D022021:Tacrolimus Binding Proteins
D001426:Bacterial Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C080951:Spo0F protein, Bacillus subtilis; D006639:Histidine; D015951:Factor Xa; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins