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

Tandem mass spectrometry (MS/MS) is often employed for the sequencing of protein ions and the annotation of protein post-translational modification. Collision-induced dissociation (CID) and electron transfer dissociation (ETD) are the most frequently utilized MS/MS techniques, but both have significant limitations. CID is often funneled into a few kinet-ically labile bonds limiting the effectiveness of sequencing and ETD, is only applicable to multiply charged ions and re-quires specialized instrumentation. Free radical-initiated peptide sequencing (FRIPS) is an alternative fragmentation technique that generates sequence informative ions via collisionally-initiated radical chemistry. Its main advantages over the previously mentioned fragmentation techniques include charge state independence, improved efficiency of sequence ion generation, and applicability to most MS instrumentation. Collision activation (CA) homolytically cleaves the radical precursor, initiates radical formation, extensive hydrogen atom transfer, and peptide backbone dissociation. Although the FRIPS technique shows great promise when applied to cations, radical initiation competes directly with typical CID path-ways. When multiply charged ions of conjugated ACTH 1-14, b-amyloid 10-20, substance p, and melittin are collisionally activated, 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 instigated by the ortho-orientation of the radical precursor undergoing fac-ile conversion into five- or six-membered intermediates and that the para-precursor 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 the complete elimination of these undesired collisional processes and the significant increase in radi-cal precursor ion abundance. Moreover, we see a significant increase in the sequence coverage generated when the p-TEMPO-Bz is utilized. From these results, p-TEMPO-Bz significantly improves the performance of positive-ion mode FRIPS and may be a suitable alternative to the currently utilized ortho-TEMPO-Bz-based FRIPS.