Tandem mass spectrometry is an important tool for structure elucidation of natural and synthetic organic products. Fragmentation of odd electron ions (OE+) generated by electron ionization (EI) was extensively studied in the last few decades, however there are only a few systematic reviews available concerning the fragmentation of even-electron ions (EE+/EE−) produced by the currently most common ionization techniques, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). This review summarizes the most important features of tandem mass spectra generated by collision-induced dissociation fragmentation and presents didactic examples for the unexperienced users.

Mass‐spectrometry coupled to liquid chromatography is an indispensable tool in the field of proteomics. In the last decades, more and more complex and diverse biochemical and biomedical questions have arisen. Problems to be solved involve protein identification, quantitative analysis, screening of low abundance modifications, handling matrix effect, and concentrations differing by orders of magnitude. This led the development of more tailored protocols and problem centered proteomics workflows, including advanced choice of experimental parameters. In the most widespread bottom‐up approach, the choice of collision energy in tandem mass spectrometric experiments has outstanding role. This review presents the collision energy optimization strategies in the field of proteomics which can help fully exploit the potential of MS based proteomics techniques. A systematic collection of use case studies is then presented to serve as a starting point for related further scientific work. Finally, this article discusses the issue of comparing results from different studies or obtained on different instruments, and it gives some hints on methodology transfer between laboratories based on measurement of reference species.

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.

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. Electronic supplementary material The online version of this article (10.1007/s13361-019-02271-x) contains supplementary material, which is available to authorized users.

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.

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The use of tandem mass spectrometry (MS/MS) is a fundamental prerequisite of reliable protein identification and quantification in mass-spectrometry-based proteomics. In bottom-up and middle-down proteomics, proteins are identified by the characteristic fragments of their constituting peptides. Post-translational modifications (PTMs) often further complicate proteome analyses. Citrullination is an increasingly studied PTM converting arginines to citrullines (Cit, X) and is implicated in several autoimmune and neurological diseases as well as different types of cancer. Confirmation of citrullination is known to be very challenging since it results in the same molecular mass change as Asn/Gln deamidation. In this study, we explore which MS/MS characteristics can be used for the reliable identification of citrullination. We synthesized several peptides incorporating Cit residues that model enzymatic cleavages of different proteins with verified or putative citrullination. Collision-induced dissociation was used to investigate the energy dependence of Byonic and Mascot scores and confirmed sequence coverage (CSC) along with the neutral loss of HNCO characteristic to citrulline side chains. We found that although the recommended values (19–45 V) for ramped collision energy settings cover the optimal Mascot, Byonic, or %CSC scores effectively, the diagnostic HNCO loss from precursors and fragments may reach their maximum intensities at lower and higher collision energies, respectively. Therefore, we suggest broadening the ramp range to ∼5–60 V to obtain more favorable identification rates for citrullinated peptides. We also found that Byonic was more successful in correctly identifying citrullinated peptides with deamidated residues than Mascot.

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.