TEMPO-based FRIPS (Free Radical Initiated Peptide Sequencing) is a method using TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) as a radical initiator to sequence peptide by collision-activated dissociation.Peptides of interest were conjugated with o-TEMPO-Bz-C(O)-NHS at the position of the N-terminus for TEMPO-based FRIPS application.Here, two step collisional activations are needed to be implemented for FRIPS analysis by Iontrap instrument, that is, MS3 is required, since FRIPS can yield peptide backbone dissociations with two collisional activations;one for the radical generation (Bz-C(O)-peptides) and the other for peptide backbone dissociations.So, in order to reduce a collisional activation step, we used instruments such as Q-TOF, Orbitrap which can apply high-collisional energy.When high-collisional activation was applied to o-TEMPO-Bz-C(O)-peptide, not only homolytic cleavage in the bond between benzyl carbon and oxygen of the TEMPO group but also peptide backbone fragmentation was occurred.Consequently, peptide sequencing was possible in a single-step FRIPS was possible in the positive-ion mode by applying high-energy collisional activation.