| Here, new methodologies are described which employ electron capture dissociation and computer automation in conjunction with the high resolution and mass accuracy of Fourier transform mass spectrometry (FTMS). First, a program for automated data reduction of high resolution mass spectra of proteins and peptides (called “THRASH”) has been developed. This program reduces a spectrum of a GluC digest of a 191 kDa protein to 824 monoisotopic masses corresponding to 528 different peptides in ∼2 h on a 270 MHz computer.; A computer program for automated de novo sequence prediction using data from both electron capture dissociation (ECD) and collisionally-activated dissociation (CAD) spectra is described. The sequence for ubiquitin (8.6 kDa, 76 amino acids) and melittin (2.8 kDa, 26 amino acids) are both correctly predicted using data corrected to within 1 and 15 ppm error, respectively. For cytochrome c (12.4 kDa, 104 amino acids), 92% of the sequence was predicted correctly; only the region containing the heme could not be sequenced.; ECD is effected for proteins >17 kDa by first exciting the proteins either by collisions with a background gas or with laser irradiation immediately before electron capture, termed “activated ion” (AI) ECD. Using “in-beam” collisions, 116/258 bonds were cleaved for carbonic anhydrase (29 kDa), 60/283 bonds were cleaved for thiazole kinase, 47/380 bonds were cleaved for thiaminase I (42 kDa). This method is also superior to “conventional” ECD even for proteins smaller than 17 kDa.; ECD was used to locate phosphate groups attached to proteins and peptides. For three peptides, the site of the phosphate was identified, while for β–casein (25 kDa), one site was identified and three others were localized to within five sites.; Electron capture dissociation was used to monitor the unfolding and refolding of cytochrome c using blackbody and laser infrared radiation to activate the ions. Unfolding was especially dramatic on the N-terminal side of threonine residues and within possible salt bridge structures. The 9+ of cytochrome c refolds over two minutes in the gas phase after infrared activation, while the 15+ refolds in less than five seconds. |
