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

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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

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 lysine (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)81-87
Number of pages7
JournalEuropean Journal of Mass Spectrometry
Volume7
Issue number2
DOIs
StatePublished - Jan 1 2001

Keywords

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

ASJC Scopus subject areas

  • Atomic and Molecular Physics, and Optics
  • Spectroscopy

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