Quantification of the Effect of Citrulline and Homocitrulline Residues on the Collision-Induced Fragmentation of Peptides

Abstract

Posttranslational modifications of proteins like citrullination and carbamylation are associated with several diseases. Detailed analytical characterization of citrullinated and carbamylated proteins or peptides could be difficult due to the low concentration of the analytes in complex biological samples. High structural similarity and chemical behavior of citrullinated and carbamylated residues also pose a challenge. We previously reported the “citrulline effect” phenomenon that is manifested in the generation of intense y type ions originating from Cit-Zzz amide bond scissions in collision-induced dissociation tandem mass spectra of citrullinated tryptic peptides. In this study, we created a rigorous tryptic-like model system of both citrulline and homocitrulline-containing peptides that included appropriate and well-defined controls and fragment analogues to quantify the citrulline effect and investigate whether there is an effect for homocitrulline residues as well. Our results show that citrulline residues significantly increased fragmentation at their C-terminus relatively independent of the identity of the following amino acid. In comparison, homocitrulline residues displayed inconclusive results at the same energies. However, the strength of effects was dependent on collision energy and the position of citrulline and homocitrulline in the sequences. As newer software algorithms tend to observe structure–intensity relationships during annotation, this finding increases reliable identification of modified proteins/peptides.

Stepwise Collision Energy-Resolved Tandem Mass Spectrometric Experiments for the Improved Identification of Citrullinated Peptides

“…This phenomenon was preserved in EThcD experiments as well for even Cit-containing peptides bearing other PTMs at the same time . The citrulline effect was found not to be influenced considerably by the identity of the residue following or preceding Cit in our study involving more than 100 Cit-containing model peptides . Besides the neutral loss of isocyanic acid and the Cit effect, another advantageous characteristic would have been the use of the iminium ion of Cit [X] to further confirm the presence of the modification.…”

“…The curves of HNCO losses from precursors as a function of CE also tend to be broken down very quickly. Note the abundant y 6 ion in the spectra that is caused by the so-called “citrulline effect”, an enhanced cleavage C -terminal to citrulline residues. , The Cit effect was also observed for IXIF­DLGR, VXV­FQAT­XGK (2+) and to a lesser extent, VLXD­NIQG­ITK­PAIR peptides. The full series of y ions showing HNCO losses presented here is unfortunately not a characteristic feature for all peptide precursors.…”

“…An increased cleavage preference has been observed N-terminal to Pro residues in CID. 28 Our group also described an amino acid effect for citrulline residues in CID, 29 and to a lesser extent in HCD, 30 that results in a favorable cleavage C-terminal to these residues. This phenomenon was preserved in EThcD experiments as well for even Cit-containing peptides bearing other PTMs at the same time.…”

“…31 The citrulline effect was found not to be influenced considerably by the identity of the residue following or preceding Cit in our study involving more than 100 Cit-containing model peptides. 30 Besides the neutral loss of isocyanic acid and the Cit effect, another advantageous characteristic would have been the use of the iminium ion of Cit [X] to further confirm the presence of the modification. These iminium ions have been exploited during the deep proteomic profiling of 30 human tissues to validate citrullination sites; 32 however, it was found that the false negative rate could be high.…”