Ion trap tandem mass spectrometry of intact GTP-binding protein γ-subunits

Tandem mass spectrometry (MS), including both MS2 and MS3 product ion scans, has been applied for the structural characterization of intact γ-subunits of heterotrimeric GTP-binding proteins (G-Proteins). Intact γ-subunits were ionized by electrospray ionization and fragmented by collision-induced dissociation in a quadrupole ion trap instrument. Extensive fragmentation was observed for the multiply charged precursor ions of these 7–8 kDa proteins. Spectral interpretation was facilitated by loss of the labile isoprenyl modification and fragmentation at C-terminal proline residues. General rules for interpretation of product ion spectra were developed for determination of sequence and posttranslational modification. Second generation product ion scans were shown to be valuable for assisting in spectral interpretation. Analysis of charge state effects on product ion spectra indicated nearly identical fragmentation patterns with differing charges on resulting product ions and provided confirmation of product ion structure assignments. This approach can be used to identify specific γ-subunits and to determine the structures of posttranslational modifications without the need for proteolytic peptide mapping.