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Peptide identification by positive electrospray ionization (ES+) tandem mass spectrometry (MS/MS) is a well-established strategy in proteomics. Several research groups reported the usefulness of negative electrospray ionization (ES-) for gaining complementary structural information on peptides and their post-translational modifications (PTM) compared to ES+. Fragmentation of citrullinated peptides has not been previously explored in ES-. In this study, 9 peptides containing citrulline residues were investigated in ES- by stepwise collision energy-dependent measurements on a QTOF instrument and a Q-Orbitrap instrument. Our results of high resolution and mass accuracy show the favored citrulline-selective loss of HNCO from these peptide precursors and their fragments─similarly to that in ES+─along with y-NH 3 /z, c, c-NH 3 /b sequence ions. Loss of HNCO from citrullinated peptides in ES- and a proposed mechanism for the reaction have been described here for the first time. HNCO loss intensities from precursors were generally even higher than that in ES+. Interestingly, the most intense fragments corresponded to neutral losses from sequence ions while intact sequence ions were usually minor components of the spectra. High-intensity ions related to cleavages N -terminal to Asp and Glu residues that have been previously reported were also observed. On the other hand, a relatively high number of peaks were observed, possibly due to internal fragmentation and/or scrambling events. While (ES-) MS/MS spectra always require manual inspection and the annotation may be ambiguous, the favorable loss of HNCO and the preferable cleavage N -terminal to Asp residues can be used to differentiate between citrullinated/deamidated sequences.

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.

Tandem mass spectrometry is an indispensable tool in proteomics used for protein sequencing and quantitation. On the basis of the sequential fragments usually generated from peptide ions via collision-induced dissociation, electron-transfer dissociation, or a combination of the two, probabilistic database search engines could be used for the identification of the peptides. The correct localization of posttranslational modifications (PTMs) poses a more challenging problem than the general identification of proteins. Histones are involved in the regulation of DNA transcription via the wealth of PTMs on their N-terminal tail. In this study, we analyzed the histone H4 peptide SGRGK incorporating four different posttranslational modifications: citrullination, acetylation, phosphorylation, and arginine methylation at various positions. The pentapeptides model the enzymatic cleavage of the N-terminal tail of human histone H4 protein by LysC protease. Fragmentation of the peptides was investigated using higher-energy collisional dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer higher-energy collisional dissociation (EThcD) on an ultrahigh resolution and mass accuracy instrument. We found that while all three techniques have their unique characteristics, advantages, and pitfalls, EThcD generated the most fragment ion-rich spectra. Despite potential ambiguities regarding exact fragment identities, full sequence coverage and PTM mapping may also be achievable. We also found novel neutral losses from the charge-reduced precursors characteristic to citrullination in ETD and EThcD which may be used in proteomic applications. N-Terminal acetylation and arginine methylation could also be confirmed by their characteristic neutral losses from the charge-reduced precursors.

Tandem mass spectrometry of peptides is of utmost importance in proteomics. Collision-induced dissociation usually generates y type fragment ion series from tryptic peptides, carrying information on their primary structure. Amino acid side chains or differences in their basicity could alter fragmentation processes considerably. The well-known proline effect is a cleavage preference at the N-terminus of proline residues in peptides, usually yielding a very abundant y ion while suppressing others. Previously, we reported a similar phenomenon occurring at the C-terminus of citrulline residues and coined the term Cit effect. To confirm the presence of Cit effect in large proteomic datasets, we analyzed 293 peptides containing Cit residues based on the human proteome database mining work of Lee et al. (2018). The occurrence of Cit effect was found to be 44%. Comparing bond scissions at the amide linkage between Cit-Zzz (citrulline followed by a specified residue) to Aaa 1 -Aaa 2 (Aaa can be any residue except Cit), 5 Cit-Zzz cleavages were significantly (CL = 95.0%) more frequent in > 85% of the cases in terms of relative sequential base beak occurrence. We used Pro effect to compare with Cit effect and obtained very similar results. On the other hand, our study showed that Cit effect is slightly inferior in the overall incidence to Pro effect (50% vs. 33%, CL = 95%) among deiminated peptides when Pro residues were also present in the sequence. Our results suggest that Cit effect is a characteristic feature and a possible biasing factor of deiminated peptides which can confirm the position of citrullination sites.

Rationale: Protein citrullination (deimination) is a post-translational modification of proteins converting arginine(s) into citrulline(s). "Overcitrullination" could be associated with severe pathological conditions. Mass spectrometric analysis of modified proteins is hindered by several problems. A comprehensive study of the fragmentation of deiminated peptides is not yet available. In this paper we have made an attempt to describe the characteristics of these processes, based on the studies of epitope model oligopeptides derived from clinically relevant proteins., Methods: Solutions of purified model peptides containing either one or two citrulline residues as well as their native variants were injected directly into the electrospray source of a high accuracy and resolution quadrupole-time-of-flight instrument and were analysed by tandem mass spectrometry using low-energy collision-induced dissociation., Results: Loss of isocyanic acid from citrulline residues is a preferred fragmentation route for deiminated peptides, which yields ornithine residues in the sequence. However, simultaneous detection of both the isocyanic acid loss and sequence fragments is often compromised. A preferential cleavage site was observed between citrulline and any other following amino acids yielding intensive complementary b- and y-type ions. Also, citrulline positioned at the C-termini displays a preferential cleavage N-terminal to this residue yielding characteristic y 1 ions. These phenomena are described here for the first time and are referred to as the "citrulline effect"., Conclusions: We found that the citrulline effect is very pronounced and could be used as a complementary tool for the confirmation of modification sites in addition to losses of isocyanic acids from the protonated molecules or from fragment ions. Low collision energy applied to peptide ions having partially mobile protons reveals the site of modification by generating specific and intensive fragments of the sequence. On the other hand, fragmenting precursor ions with mobile protons usually allows full sequence coverage, although citrulline-specific fragments may exhibit lower intensities compared to other fragments., (Copyright © 2018 John Wiley & Sons, Ltd.)