Top down versus bottom up protein characterization by tandem high-resolution mass spectrometry

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 lo calize errors in the DNA-derived sequence or modifications (posttranslation al, derivatized active sites, etc.) usually involves extensive proteolysis to yield peptides of <3 kDa, with separation and MS/MS to compare their seq uences to those expected (the "bottom up" approach). In contrast, an altern ative "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 wi th the predicted molecular mass can be localized to a fragment(s) without e xamining all others, with further dissociation of the fragments in the same way providing further localization. Using carbonic anhydrase (29 kDa) as a n example, Fourier transform mass spectrometry is unusually effective far t he bottom up approach, in that a single spectrum of an extensive chymotrypt ic digest identifies 64 expected peptides, but these only cover 95% of the sequence; 20 fragment masses are unassigned so that any set whose masses su m to that of the molecule would be misleading. Extensive Lys-C dissociation yields 17 peptides, 23 unassigned masses, and 96% coverage. In the contras ting "top down" approach, less extensive initial dissociation by Lys-C, MS/ MS, or CNBr in each case provides 100% coverage, so that modified protein F ragment(s) could easily be recognized among the complementary sets. MS/MS o f such a fragment or more extensive proteolysis provide further localizatio n of the modification. The combined methods cleaved 137 of the 258 amide bo nds between residues.