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

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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 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.

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