Guanidination of lysine residue improves the sensitivity and facilitates the interpretation of free radical initiated peptide sequencing (FRIPS) mass spectrometry results

Lysine-containing peptides, which are often found in tryptic peptide mixtures, frequently undergo double conjugations with o-TEMPO-Bz-C(O)- groups in TEMPO-assisted free radical-initiated peptide sequencing (FRIPS) mass spectrometry applications because of the facile conjugation reaction at the ɛ-primary amino group of the lysine side chain. Doubly conjugated peptides required an additional collisional activation step because they include two free radical initiators, which is not favorable for the applications of the FRIPS approach in practical peptide MS analysis. To avoid this issue, this study introduces a new strategy of guanidinating lysine-containing peptides prior to the o-TEMPO-Bz-C(O)- conjugation. The guanidination step was found to be readily incorporated into our TEMPO-assisted FRIPS procedure with high yield. More importantly, the guanidinated peptides were demonstrated to undergo radical-driven peptide backbone fragmentations, similar to peptides that lack a lysine residue. In summary, combined with the guanidination step, the TEMPO-assisted FRIPS MS offers another practical tool that can be used to analyze and characterize peptides.