PMID:10082377
| Citation |
Kelleher, NL, Taylor, SV, Grannis, D, Kinsland, C, Chiu, HJ, Begley, TP and McLafferty, FW (1998) Efficient sequence analysis of the six gene products (7-74 kDa) from the Escherichia coli thiamin biosynthetic operon by tandem high-resolution mass spectrometry. Protein Sci. 7:1796-801 |
|---|---|
| Abstract |
The 10(5) resolving power and MS/MS capabilities of Fourier-transform mass spectrometry provide electrospray ionization mass spectra containing >100 molecular and fragment ion mass values of high accuracy. Applying these spectra to the detection and localization of errors and modifications in the DNA-derived sequences of proteins is illustrated with the thiCEFSGH thiamin biosynthesis operon from Escherichia coli. Direct fragmentation of the multiply-charged intact protein ions produces large fragment ions covering the entire sequence; further dissociation of these fragment ions provides information on their sequences. For ThiE (23 kDa), the entire sequence was verified in a single spectrum with an accurate (0.3 Da) molecular weight (Mr) value, with confirmation from MS/MS fragment masses. Those for ThiH (46 kDa) showed that the Mr value (1 Da error) represented the protein without the start Met residue. For ThiF (27 kDa), MS/MS localized a sequence discrepancy to a 34 residue peptide. The first 107 residues of ThiC (74 kDa) were shown to be correct, with C-terminal heterogeneity indicated. For ThiG (predicted Mr = 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 codon for the first protein. MS/MS ions are consistent with 68 fragments predicted by the corrected ThiS/ThiG DNA sequences. |
| Links |
PubMed PMC2144080 Online version:10.1002/pro.5560070815 |
| Keywords |
Amino Acid Sequence; Bacterial Proteins/chemistry; Base Sequence; Escherichia coli/genetics; Mass Spectrometry; Molecular Sequence Data; Operon; Phosphotransferases (Phosphate Group Acceptor)/chemistry; Thiamine/biosynthesis; Thiamine/chemistry |
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