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

For complete characterization of larger proteins, primary structural analys is by mass spectrometry must be made more efficient. A straightforward appr oach is illustrated here using two proteins of 159 and 199 kDa with five an d 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 mult iply-charged ions (from electrospray ionization) are far more amenable than the intact proteins to direct interrogation in a Fourier-transform mass sp ectrometer. For the 199 kDa PchF of similar to 60% purity, an unfractionate d 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 strat egies for implementing this characterization strategy on a proteomic scale are considered.