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Protein citrullination and homocitrullination profiling in mice using LC-MS/MS

ABSTRACT: Citrullination and homocitrullination are two post-translational modifications (PTMs) that may cause protein structural and functional changes. Although they have been linked to various biological processes and disease development, the understanding is still poor because of limitations to enrich, detect and localize them. Herein, we report a biotin thiol tag that enables derivatization, enrichment and confident identification of these two PTMs coupled with mass spectrometry. We apply this method to profile these modifications in different tissues of mice. In total, we identify 1,198 citrullination sites and 108 homocitrullination sites from 619 and 79 proteins, respectively, which is the largest dataset to date. We reveal previously unknown distribution and functions of these two PTMs. We also achieve multiplexing quantitative analysis by combing this method with isotopic labeling strategies. This study contributes to an improved understanding of protein citrullination and homocitrullination, and processes the potential to further decipher their physiological and pathological roles.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Spleen, Heart, Brain, Liver, Lung, Pancreas, Kidney

SUBMITTER: Zihui Li

LAB HEAD: Lingjun Li

PROVIDER: PXD023733 | Pride | 2022-12-05

REPOSITORIES: Pride

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Project description:Citrullination is a post-translational modification of arginine catalyzed by five peptidylarginine deiminases (PADs) in humans. The loss of a positive charge may cause structural or functional alterations and while the modification has been linked to several diseases including rheumatoid arthritis and cancer, its physiological or pathophysiological roles remain largely unclear. In part this is owing to limitations in available methodology able to robustly enrich, detect and localize the modification. As a result, only few citrullination sites have been identified on human proteins with high confidence. In this study, we mined data from mass spectrometry-based deep proteomic profiling of 30 human tissues to identify citrullination sites on endogenous proteins. Database searching of ~70 million tandem mass spectra yielded ~13,000 candidate spectra which were further triaged by spectrum quality metrics and the detection of the specific neutral loss of isocyanic acid from citrullinated peptides to reduce false positives. Because citrullination is easily confused with deamidation, we synthetised ~2,200 citrullinated and 1,300 deamindated peptides to build a library of reference spectra. This led to the validation of 375 citrullination sites on 209 human proteins. Further analysis showed that >80% of the identified modifications sites were new and for 56% of the proteins, citrullination was detected for the first time. Sequence motif analysis revealed a strong preference for Asp and Gly, residues around the citrullination site. Interestingly, while the modification was detected in 26 human tissues with the highest levels found in brain and lung, citrullination levels did not correlate well with protein expression of the PAD enzymes. Even though the current work represents the largest survey of protein citrullination to date, the modification was mostly detected on high abundance proteins arguing that the development of specific enrichment methods would be required in order to study the full extent of cellular protein citrullination.

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Protein citrullination and homocitrullination profiling in mice using LC-MS/MS

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