Determining Linear Free Energy Relationships in Peptide Fragmentation Using Derivatization and Targeted Mass Spectrometry.

The sequence complexity of a proteome is utilized with rational chemical derivatization to establish the linear free energy relationship (LFER) in order to investigate the collision-induced dissociation (CID) of peptides. The derivatization produces groups of peptides that have varying designer residues of aliphatic amino acids at the N-terminus but stay invariable for the rest of sequences, which are naturally occurring and uncontrolled. Collisional fragmentation of the derivatized peptides is advantageously monitored by liquid-chromatography multiple-reaction-monitoring mass spectrometry. Systematically tuning the gas-phase basicity of the N-termini of peptides establishes LFERs that report the structural similarities and differences in CID of all the backbone amides of doubly protonated tryptic peptides. For the cleavage of an internal or C-terminal amide, the peptide N-terminus mainly affects the mobility of the N-terminal proton instead of directly participating in the amide cleavage. In contrast, the terminal residue plays more pronounced roles in the cleavage of the first and second amide bonds. LFERs for the competition between the symmetric and asymmetric cleavage of the second amide bond support the protonated oxazolone structure for N-terminal fragments. This competition is affected locally by the chemistry of the first three residues and remotely by charge repulsion between the two protonation sites.