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Andrew J. Forbes, Matthew T. Mazur and Neil L.
Kelleher*
Department of Chemistry, 600 S. Mathews Avenue, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA E-mail:
kelleher@scs.uiuc.edu
Hiten M. Patel and Christopher T. Walsh
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,
Boston, MA 02115, USA |
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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. |
