Tandem mass spectrometry-based approaches are effective for the detection and identification of peptides in peptidome studies. This study investigates the use of an N-terminal derivatization strategy that improves the accuracy of de-novo sequence determination. Derivatization to create an N-terminal imine reduces sequence scrambling, decreases the complexity of fragmentation spectra, and increases the yield of N-terminal, sequence-specific ions. In the present study, density functional theory (DFT) was used to determine the protonation sites, and structures of key intermediates and transition states to provide a better understanding of the mechanism(s) of, and energetics for, the dissociation reactions of the derivatized peptides. The working hypothesis is that the basicity of the imine may enhance the tendency for the N-terminal dissociation products (bn- and an-type ions) to retain the ionizing proton. The general fragmentation of AGG, GAG, and GGA in native and derivatized form was investigated using tandem mass spectrometry with collision-induced-dissociation (MSn CID). The experimental data confirms that there is a greater diversity of bn and an fragment ions generated from the derivatized peptides. In addition, the relative intensities of the fragmentation ions are greater for the modified peptides compared to the unmodified analogues. In MSn experiments, dissociation occurs through the bn/an series to reveal the complete peptide sequence. The computational investigation surveyed the protonation sites of the unmodified and N-terminally derivatized peptides. Candidate precursor ion structures included those for which the N-terminal imine is protonated, as well as “charge-solvated” structures in which the amide carbonyl O atoms are protonated, and isomers that are protonated at amide N atoms. For the derivatized peptides, those structure in which the imine is protonated are lowest in energy. Calculations show that the proton at the N-terminal imine is mobilized, and the b/y fragmentation pathway activated in a similar way for both the modified and unmodified peptides.

Computational investigation of the intrinsic conformation and fragmentation of peptides modified to include N-terminal imine groups

ACS Spring 2022

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