Generating Chimeric Peptides for Increasing Sample Throughput and Probing Fragmentation in Mass Spectrometry

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Shen, Yuanyuan
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Chemical derivatization of peptides with reagents of natural amino acids and utilities of the derivatization are presented. Derivatized peptides are chimeric, having a designer aliphatic residue at the N-terminal and a naturally occurring, uncontrolled sequence for the remaining residues. The peptide derivatization uses commercially available reagents, N-(fluorenylmethoxycarbonyl)-L-amino acid N-hydroxysuccinimde esters, thus allowing for convenient assembly of multiplexed mass spectrometric quantification.Utilizing chimeric peptides, a novel strategy of linear free energy relationship (LFER) is established for analyzing collision-induced dissociation of peptides. Systematic tuning the gas-phase basicity of the N-terminus of the chimeric peptides allows for probing the similarity and difference in transition states for collisional induced dissociation of backbone amides. In the symmetric bond cleavage study, the LFER analysis reveals two possibly different transition states for cleavage of the second amide bond from the N-terminus, in agreement with the bifurcating fragmentation of doubly-protonated tryptic peptides upon collision. The asymmetric cleavage of the amide bond for doubly-protonated Arg-peptide is also studied. Bifurcating behavior is not observed and the impact of peptide length is insignificant. The computational calculation and LFERs with the chimeric peptides strongly support the “protonation oxazolone” pathway for both symmetric and asymmetric bond dissociation.