New free radical-initiated peptide sequencing (FRIPS) mass spectrometry reagent with high conjugation efficiency enabling single-step peptide sequencing

A newly designed TEMPO-FRIPS reagent, 4-(2,2,6,6-tetramethylpiperidine-1-oxyl) methyl benzyl succinic acid N-hydroxysuccinimide ester or p-TEMPO–Bn–Sc–NHS, was synthesized to achieve single-step free radical-initiated peptide sequencing mass spectrometry (FRIPS MS) for a number of model peptides, including phosphopeptides. The p-TEMPO–Bn–Sc–NHS reagent was conjugated to target peptides, and the resulting peptides were subjected to collisional activation. The peptide backbone dissociation behaviors of the MS/MS and MS(3) experiments were monitored in positive ion mode. Fragment ions were observed even at the single-step thermal activation of the p-TEMPO–Bn–Sc–peptides, showing mainly a-/x- and c-/z-type fragments and neutral loss ions. This confirms that radical-driven peptide backbone dissociations occurred with the p-TEMPO–Bn–Sc–peptides. Compared to the previous version of the TEMPO reagent, i.e., o-TEMPO–Bz–C(O)–NHS, the newly designed p-TEMPO–Bn–Sc–NHS has better conjugation efficiency for the target peptides owing to its improved structural flexibility and solubility in the experimental reagents. An energetic interpretation using the survival fraction as a function of applied normalized collision energy (NCE) ascertained the difference in the thermal activation between p-TEMPO–Bn–Sc– and o-TEMPO–Bz–C(O)– radical initiators. This study clearly demonstrates that the application of the p-TEMPO–Bn–Sc– radical initiator can improve the duty cycle, and this FRIPS MS approach has the potential to be implemented in proteomics studies, including phosphoproteomics.