COLLISION-INDUCED DISSOCIATION SPECTRA OBTAINED BY FOURIER-TRANSFORM ION-CYCLOTRON RESONANCE MASS-SPECTROMETRY USING A C-13, N-15-DOUBLY DEPLETED PROTEIN

Fourier transform ion cyclotron resonance mass spectra of C-13,N-15-do ubly depleted cystatin A(2-98) M65L, produced by Escherichia coli grow n on 99.9% [C-12]glucose and 99.99% [N-14]ammonium sulfate, showed sal ient monoisotopic peaks composed of C-12 and N-14. Collision-induced d issociation spectra were obtained by increasing the capillary-skimmer potential for the electrospray ionization and by extending the trappin g time in a radio frequency-only hexapole ion guide. Fragment ions in the spectra could be readily assigned to the amino acid sequence, owin g 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 de pleted proteins, enabled the assignment of similar to 180 fragment ion s to the sequence, while natural isotopic cystatin A allowed; the assi gnment of similar to 110 fragment ions. Interestingly, no fragmentatio n was detected between residues 50-61 and 62-67, which are stretches k nown to be involved in the antiparallel beta-sheet at the center of th e protein.