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

Neil L. Kelleher, Sean V. Taylor, David Grannis, Cynthia Kinsland, Hsiu J U Chiu, Tadhg P. Begley, Fred W. McLafferty*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

90 Scopus citations

Abstract

The 105 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 M(r) 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 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 codon for the first protein. MS/MS ions are consistent with 68 fragments predicted by the corrected ThiS/ThiG DNA sequences.

Original languageEnglish (US)
Pages (from-to)1796-1801
Number of pages6
JournalProtein Science
Volume7
Issue number8
DOIs
StatePublished - 1998

Keywords

  • Electrospray
  • Escherichia coli
  • Fourier-transform mass spectrometry
  • Operon
  • Sequence
  • Thiamin biosynthesis
  • thiCEFSGH

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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