Background: Arginine citrullination is emerging as a widespread post-translational modification with diverse biological functions. To date, the citrullinated proteins have been reported only in a limited number of studies and while the modification itself has been linked to several diseases, including rheumatoid arthritis and cancer, its physiological or pathophysiological roles remain largely unclear. The bottleneck is limitations in available methodology to robustly enrich, detect, and localize the citrullinated residues. To overcome these limitations, we generated a mouse hyper-citrullinated spectral library and set up coordinates to confidently identify and validate citrullinated sites.
Methodologies: Generation of large-scale multiorgan mouse hyper-citrullinated spectral library that can be used to enable the robust detection of citrullinated peptides using DIA-MS. The step-by-step library generation workflow is based on creation of the hyper-citrullinated samples, collection of the high-quality fragment ion spectra in data dependent acquisition mode, spectral matching of modified peptides to their non-citrullinated form, along with the delta retention time shift (∆RT), as a signature for citrullination. The validation steps of citrullinated peptides include detection of neutral loss of isocyanic acid in peptides in CID spectra and Skyline validation.
Findings: Using this workflow, we detect ten-fold increase in citrullinated proteome coverage across six mouse organs compared to the current state-of-the art techniques. Our data reveals that the subcellular distribution of citrullinated proteins is tissue-type dependent and that citrullinated targets are involved in fundamental physiological processes, including metabolic process.
Conclusions: Presented a novel approach for the generation of hyper-citrullinated library and detection of citrullinated proteins with the citrullinated site provides a rich resource of candidates for hypothesis generation and will open new avenues for large-scale investigations of citrullinated proteins in clinical research.