Efficient sequence analysis of the six gene products (7-74 kDa) from the Escherichia coli thiamin biosynthetic operon by tandem high-resolution mass spectrometry

The 10(5) resolving power and MS/MS capabilities of Fourier-transform mass spectrometry provide electrospray ionization mass spectra containing >100 m olecular and fragment ion moss values of high accuracy, Applying these spec tra to the detection and localization of errors and modifications in the DN A-derived sequences of proteins is illustrated with the thiCEFSGH thiamin b iosynthesis operon from Escherichia coli. Direct fragmentation of the multi ply-charged intact protein inns produces large fragment ions covering the e ntire sequence; further dissociation of these fragment ions provides inform ation on their sequences. For ThiE (23 kDa), the entire sequence was verifi ed in a single spectrum with an accurate (0.3 Da) molecular weight (M-r) va lue, with confirmation from MS/MS fragment masses. Those for ThiH (46 kDa) showed that the M-r value (1 Da error) represented the protein without the start Met residue, For ThiF (27 kDa), MS/MS localized a sequence discrepanc y to a 34 residue peptide, The first 107 residues of ThiC (74 kDa) were sho wn to be correct, with C-terminal heterogeneity indicated. For ThiG (predic ted M-r = 34 kDa), ESI/FTMS showed two components of 7,310.74 (ThiS) and 26 ,896.5 Da (ThiG); MS/MS uncovered three reading frame errors and a stop cod on for the first protein. MS/MS ions are consistent with 68 fragments predi cted by the corrected ThiS/ThiG DNA sequences.