Collision-Induced Dissociation of Citrullinated Peptide Anions

Peptideidentification by positive electrospray ionization (ES+)tandem mass spectrometry (MS/MS) is a well-established strategy inproteomics. Several research groups reported the usefulness of negativeelectrospray ionization (ES-) for gaining complementary structuralinformation on peptides and their post-translational modifications(PTM) compared to ES+. Fragmentation of citrullinated peptides hasnot been previously explored in ES-. In this study, 9 peptidescontaining citrulline residues were investigated in ES- bystepwise collision energy-dependent measurements on a QTOF instrumentand a Q-Orbitrap instrument. Our results of high resolution and massaccuracy show the favored citrulline-selective loss of HNCO from thesepeptide precursors and their fragments similarly to that inES+along with y-NH3/z, c, c-NH3/b sequenceions. Loss of HNCO from citrullinated peptides in ES- and aproposed mechanism for the reaction have been described here for thefirst time. HNCO loss intensities from precursors were generally evenhigher than that in ES+. Interestingly, the most intense fragmentscorresponded to neutral losses from sequence ions while intact sequenceions were usually minor components of the spectra. High-intensityions related to cleavages N-terminal to Asp and Gluresidues that have been previously reported were also observed. Onthe other hand, a relatively high number of peaks were observed, possiblydue to internal fragmentation and/or scrambling events. While (ES-)MS/MS spectra always require manual inspection and the annotationmay be ambiguous, the favorable loss of HNCO and the preferable cleavage N-terminal to Asp residues can be used to differentiatebetween citrullinated/deamidated sequences.