TEMPO-Assisted Free-Radical-Initiated Peptide Sequencing Mass Spectrometry for Ubiquitin Ions: An Insight on the Gas-Phase Conformations

TEMPO ((2,2,6,6-tetramethylpiperidine-1-yl)oxyl)-assisted free-radical-initiated peptide sequencing mass spectrometry (FRIPS MS) is applied to the top-down tandem mass spectrometry of guanidinated ubiquitin (UB(Gu)) ions, i.e., p-TEMPO-Bn-Sc-guanidinated ubiquitin (UBT(Gu)), to shed a light on gas-phase ubiquitin conformations. Thermal activation of UBT(Gu) ions produced protein backbone fragments of radical character, i.e., a-/x- and c-/z-type fragments. It is in contrast to the collision-induced dissociation (CID) results for UB(Gu), which dominantly showed the specific charge-remote CID fragments of b-/y-type at the C-terminal side of glutamic acid (E) and aspartic acid (D). The transfer of a radical “through space” was mainly observed for the +5 and +6 UBT(Gu) ions. This provides the information about folding/unfolding and structural proximity between the positions of the incipient benzyl radical site and fragmented sites. The analysis of FRIPS MS results for the +5 charge state ubiquitin ions shows that the +5 charge state ubiquitin ions bear a conformational resemblance to the native ubiquitin (X-ray crystallography structure), particularly in the central sequence region, whereas some deviations were observed in the unstable second structure region (β₂) close to the N-terminus. The ion mobility spectrometry results also corroborate the FRIPS MS results in terms of their conformations (or structures). The experimental results obtained in this study clearly demonstrate a potential of the TEMPO-assisted FRIPS MS as one of the methods for the elucidation of the overall gas-phase protein structures.