Collision-induced dissociation spectra obtained by Fourier transform ion cyclotron resonance mass spectrometry using a 13C, 15N-doubly depleted protein

Fourier transform ion cyclotron resonance mass spectra of C-13,N-15-doubly depleted cystatin A(2-98) M65L, produced by Escherichia coli grown on 99.9% [C-12]glucose and 99.99% [N-14]ammonium sulfate, showed salient monoisotopic peaks composed of C-12 and N-14. Collision-induced dissociation spectra were obtained by increasing the capillary-skimmer potential for the electrospray ionization and by extending the trapping time in a radio frequency-only hexapole ion guide. Fragment ions in the spectra could be readily assigned to the amino acid sequence, owing to their markedly improved resolution and sensitivity as compared to those with the natural isotopic composition. Detailed analyses of the fragmentation patterns, facilitated by the use of C-13,N-15-doubly depleted proteins, enabled the assignment of similar to 180 fragment ions to the sequence, while natural isotopic cystatin A allowed; the assignment of similar to 110 fragment ions. Interestingly, no fragmentation was detected between residues 50-61 and 62-67, which are stretches known to be involved in the antiparallel beta-sheet at the center of the protein.