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.

Full mass spectra of high resolving power are obtained from 0.2 nL sample volumes of large (> 10 kDa) nucleotides and proteins using a new electrospray ionization (ESI) system combined with Fourier transform mass spectrometry. The ESI needles are fabricated by laser-heated pulling of fused-silica tubing (5-20 microns i.d.), followed by chemical etching and surface metalization. Total analyte loaded at the instrument of 8.6 fmol and 216 amol produces signal-to-noise ratios of 400:1 and 60:1, respectively, and resolving power of > 10(5) for full mass spectra, while the total amount of material consumed is approximately 150 and 10 amol, respectively.

Complete sequence information for an "unknown" 50-mer DNA and extensive sequence verification for another 50-mer and 42-, 51-, 55-, 60-, 72-, 100-, and 108-mer DNAs is obtained by electrospray ionization/Fourier transform mass spectrometry that supplies 10-100× higher accuracy and resolving-power data using nozzle-skimmer (NS), collisionally activated, and infrared multiphoton dissociation (IRMPD). In addition to the previously recognized 3′-and 5′-terminal (w and a) ions, internal ions (i) and MS/MS/MS of fragment ions provide unique structural information across the DNA. NS dissociation can also yield other new backbone cleavages (forming b, c, d, and r ions) that provide extensive 5′-end information. These spectra indicate that loss of the base T rarely triggers formation of w, a, or i fragment ions, a correlation of further sequencing utility. Point mutation screening is demonstrated using a modified 50-mer unknown; a 9.04 (theory 9.01) decrease in the molecular weight (M r ) value indicates A f T, while three IRMPD fragment ions pinpoint this mutation at base 27. Introduction (measurement time <1 min) of 8 × 10 -16 mol of the 50-mer gave an M r value with only a 0.2 error.

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Several experimental factors have been investigated that influence the efficiency of desorption and subsequent chemical ionization of nonvolatile, thermally labile molecules during laser-induced acoustic desorption/Fourier transform ion cyclotron resonance mass spectrometry (LIAD/FT-ICR) experiments. The experiments were performed by using two specially designed LIAD probes of different outer diameters (1/2 and 7/8 in.) and designs. Several improvements to the design of the "first generation" (1/2 in.) LIAD probe are presented. The larger diameter (7/8 in.) probe provides a larger surface area for desorption than the smaller diameter probe. Further, it was designed to desorb molecules on-axis with the magnetic field of the instrument. This is in contrast to the smaller probe for which desorption occurs 1.3 mm off-axis. This improved alignment, which provides better overlap between the desorbed molecules and trapped reagent ions, results in a substantial increase in the sensitivity of LIAD analyses. The thickness of the sample layer deposited on the irradiated metal foil and the number of laser shots fired on the backside of the foil were found to have a significant effect on the overall signal and the relative abundances of the ions formed in the experiment. Evaporation of a tetrapeptide, Val-Ala-Ala-Phe (VAAF), from Ag, Al, Au, Cu, Fe, and Ti foils, followed by protonation by protonated pyridine, revealed that the titanium foil provides the greatest signal. The importance of the laser power density was examined by desorbing a low MW polymer, polyisobutenyl succinic anhydride, at power densities ranging from 5.40 x 10(8) to 9.00 x 10(8) W/cm(2) at the backside of the foil. Higher laser power densities resulted in greater signals and an improved distribution for the higher molecular weight oligomers.