Improved Performance of Positive-ion mode Free Radical-Initiated Peptide Sequencing with para-TEMPO-Bz

Free radical-initiated peptide sequencing (FRIPS) is a tandem mass spectrometry (MS/MS) technique that generates sequence informative ions via collisionally-initiated radical chemistry. Collision activation homolytically cleaves an installed radical precursor, initiates radical formation, extensive hydrogen atom transfer, and peptide backbone dissociation. While the FRIPS technique shows great promise, when applied to multiply charged derivatized peptide ions, a series of high abundance mass losses are observed which syphon ion abundance from radically generated sequence ions. This loss of ion abundance reduces the sequence coverage generated by FRIPS fragmentation. In this work, we hypothesized that these mass losses were assisted by the ortho-orientation of the radical precursor undergoing facile conversion into five- or six-membered intermediates or products and that when combined with the lower bond dissociation energy of the para-precursor, conjugated peptides would not undergo this chemistry. To test this assertion, we synthesized para-TEMPO-Bz, conjugated it to these peptides, and collisionally activated each. And indeed, we see dramatic attenuation of these undesired collisional processes and the significant increase in radical precursor ion abundance. The increase in ion abundance leads to a significant increase in the sequence coverage generated. These results demonstrate that p-TEMPO-Bz significantly improves the performance of positive-ion mode FRIPS and may be a compelling alternative to the currently utilized ortho-TEMPO-Bz-based FRIPS.