Toward efficient analysis of >70 kDa proteins with 100% sequence coverage

Andrew J. Forbes, Matthew T. Mazur, Hiten M. Patel, Christopher T. Walsh, Neil L. Kelleher*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

49 Scopus citations


For complete characterization of larger proteins, primary structural analysis by mass spectrometry must be made more efficient. A straightforward approach is illustrated here using two proteins of 159 and 199 kDa with five and nine Lys residues, respectively. These proteins were degraded by Lys-C to mixtures of peptides ranging in size from 5 to 48 kDa, whose multiply charged ions (from electrospray ionization) are far more amenable than the intact proteins to direct interrogation in a Fourier-transform mass spectrometer. For the 199 kDa PchF of ∼60% purity, an unfractionated Lys-C digest gave 106 isotopic distributions from 71 components (most of which were below 6 kDa); 15% sequence coverage was obtained. For the >90% pure PchE (159 kDa), complete sequence coverage was obtained from six Lys-C peptides of 5, 8, 26, 32, 40 and 48 kDa, with all but the largest of these measured at isotopic resolution on a 4.7 Tesla instrument. Practical strategies for implementing this characterization strategy on a proteomic scale are considered.

Original languageEnglish (US)
Pages (from-to)927-933
Number of pages7
Issue number8
StatePublished - Aug 2001


  • Electrospray
  • Fourier transform mass spectrometry
  • Limited proteolysis
  • Peptide mapping

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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