Dataset Information

Characterization of Citrullination Sites in Neutrophils and Mast Cells Activated by Ionomycin via Integration of Mass Spectrometry and Machine Learning

ABSTRACT: Citrullination is an important post-translational modification implicated in many diseases including rheumatoid arthritis (RA), Alzheimer's disease and cancer. Neutrophil and mast cells have different protein-arginine deiminases expression profile and ionomycin induced activation make them the ideal cellular models to study proteins susceptible to citrullination. We performed high resolution mass spectrometry and stringent data filtration to identify citrullination sites in neutrophil and mast cells treated with and without ionomycin. We identified a total of 831 validated citrullination sites on 393 proteins. Several of these citrullinated proteins are important component of pathways involved in innate immune responses. Using this benchmark primary sequence dataset, we developed machine learning models to predict citrullination in neutrophil and mast cells proteins. Our neutrophil protein citrullination prediction model achieved greater than 76% accuracy and 0.39 Matthews correlation coefficient (MCC) on an independent validation set. In summary, this study provides the largest number of validated citrullination sites in neutrophil and mast cell proteins. The use of our novel motif analysis approach to predict citrullination sites will facilitate the discovery of novel protein substrates of protein-arginine deiminases (PADs), which may be key to understanding immunopathologies of various diseases.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Leukocyte, Cell Culture

SUBMITTER: Raghothama Chaerkady

LAB HEAD: Sonja Hess

PROVIDER: PXD023131 | Pride | 2022-01-12

REPOSITORIES: Pride

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Characterization of Citrullination Sites in Neutrophils and Mast Cells Activated by Ionomycin via Integration of Mass Spectrometry and Machine Learning.

Chaerkady Raghothama R Zhou Yebin Y Delmar Jared A JA Weng Shao Huan Samuel SHS Wang Junmin J Awasthi Shivangi S Sims Dorothy D Bowen Michael A MA Yu Wen W Cazares Lisa H LH Sims Gary P GP Hess Sonja S

Journal of proteome research 20210519 6

Citrullination is an important post-translational modification implicated in many diseases including rheumatoid arthritis (RA), Alzheimer's disease, and cancer. Neutrophil and mast cells have different expression profiles for protein-arginine deiminases (PADs), and ionomycin-induced activation makes them an ideal cellular model to study proteins susceptible to citrullination. We performed high-resolution mass spectrometry and stringent data filtration to identify citrullination sites in neutroph ...[more]

PMID: 34008986

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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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