Top Down versus Bottom Up Protein Characterization by Tandem High-Resolution Mass Spectrometry

Abstract

Characterization of larger proteins by mass spectrometry (MS) is especially promising because the information complements that of classical techniques and can be obtained on as little as 10 - 17 mol of protein. Using MS to localize errors in the DNA-derived sequence or modifications (posttranslational, derivatized active sites, etc.) usually involves extensive proteolysis to yield peptides of <3 kDa, with separation and MS/MS to compare their sequences to those expected (the “bottom up” approach). In contrast, an alternative “top down” approach limits the dissociation (proteolysis or MS/MS) to yield larger products from which a small set of complementary peptides can be found whose masses sum to those of the molecule. Thus a disagreement with the predicted molecular mass can be localized to a fragment(s) without examining all others, with further dissociation of the fragments in the same way providing further localization. Using carbonic anhydrase (29 kDa) as an example, Fourier transform mass spectrometry is unusually effective for the bottom up approach, in that a single spectrum of an extensive chymotryptic digest identifies 64 expected peptides, but these only cover 95% of the sequence; 20 fragment masses are unassigned so that any set whose masses sum to that of the molecule would be misleading. Extensive Lys-C dissociation yields 17 peptides, 23 unassigned masses, and 96% coverage. In the contrasting “top down” approach, less extensive initial dissociation by Lys-C, MS/MS, or CNBr in each case provides 100% coverage, so that modified protein fragment(s) could easily be recognized among the complementary sets. MS/MS of such a fragment or more extensive proteolysis provide further localization of the modification. The combined methods cleaved 137 of the 258 amide bonds between residues.