High-resolution multistage MS, MS2, and MS3 matrix-assisted laser desorption/ionization FT-ICR mass spectra of...

By combined and repeated use of sustained off-resonance irradiation (SORI) for ion dissociation, stored waveform inverse Fourier transform (SWIFT) waveforms for ion isolation, and ion axialization and remeasurements techniques, we obtain for the first time MS, MS2, and MS3 FT-ICR mass spectra from peptide ions (enzymatic digest products of horse cytochrome c) produced from a single laser shot. The successive fragmentation of gas-phase ions detected from the same initial batch of ions increases the sensitivity of analysis of trace amounts of biological samples in structural mass spectrometry, and fragment identification is facilitated by resolution of carbon-13 isotopic distributions. The method is illustrated by analyses of subfemtomole amounts of crudely purified samples of tryptic digest solutions of horse cytochrome c and bovine cytochrome c. The high-resolution primary ion mass spectrum, along with the collision-induced dissociation (CID) and MSn capabilities of FT-ICR, help to determine the primary amino acid sequence of the fragment ions beyond what is obtained from enzymatic digestion alone, without prior chromatographic separation and purification.