Charge state dependent ion trap collision-induced dissociation of reduced bovine and porcine trypsin cations

Abstract

The quadrupole ion trap collision-induced dissociation (CID) behavior of reduced bovine and porcine trypsin cations, formed via nanospray ionization, is reported for the odd-numbered precursor ion charge states over the range of +9 to +21. Dissociation of precursor ions in both bovine and porcine trypsin yield predominantly b- and y-type ion backbone cleavages with strong evidence for preferential cleavages C-terminal to aspartic acid at intermediate to low charge states, C-terminal to lysine residues at intermediate charge states, and N-terminal to proline residues across all charge states examined. Both homologs showed similarly rich fragmentation within the first dozen residues at the N-terminus and both homologs proved to be resistant to fragmentation within the roughly 40 residues from the C-terminal end of the protein. Spectra derived from single precursor ion charge states showed fragmentation at 15% or less of the 222 amide linkages, while cleavage at roughly 30% of the bonds was observed when all charge states examined were considered. Significant differences in the identities of the observed fragmentation channels for the different charge state precursor ions accounts for the larger sequence coverage associated with the integration of fragmentation information across all charge states. In terms of individual fragmentation channels, many similarities between the two protein homologs were noted. Many of the major differences can be accounted for on the basis of the sequence differences associated with the homologs. However, some notable differences were noted for cleavage sites where residue identities were the same. Observations of this type highlight the fact that numerous factors, in addition to primary structure, affect protein ion dissociation under ion trap collisional activation conditions.