Itaconation Analysis – LC-MS/MS Mapping of Protein Itaconation Sites

Protein itaconation is a metabolite-driven post-translational modification formed by covalent attachment of itaconate to reactive cysteine residues, linking immune metabolism to redox regulation and signal transduction. Creative Proteomics provides integrated metabolic labeling–based enrichment and high-resolution LC-MS/MS for sensitive, site-specific identification and quantitative profiling of protein itaconation.

Comprehensive detection: Sensitive identification and quantitative profiling of itaconated proteins and site-specific cysteine modifications.
Advanced technology: Metabolic labeling combined with high-resolution LC-MS/MS to ensure accuracy, specificity, and proteome-wide coverage.
Functional insights: Integrated bioinformatics analysis for motif discovery, pathway enrichment, and protein interaction network interpretation.
Research impact: Elucidate regulatory roles of protein itaconation in immune and metabolic signaling, providing mechanistic insights and potential therapeutic targets in inflammatory and cardiovascular diseases.

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What Is Itaconation?

Itaconation is a recently identified metabolite-driven post-translational modification in which the immunometabolite itaconate covalently modifies proteins, predominantly at cysteine residues. This modification arises from the accumulation of itaconate produced by IRG1 (also known as ACOD1) during inflammatory activation of immune cells, especially macrophages. Owing to its α,β-unsaturated carboxylic acid structure, itaconate acts as an electrophile and reacts with nucleophilic cysteine thiols via Michael addition, leading to stable cysteine itaconation. Functionally, itaconation alters protein activity, redox sensitivity, and protein–protein interactions, thereby reshaping cellular metabolic and inflammatory signaling. Proteins involved in glycolysis, oxidative stress responses, and innate immune pathways have been identified as key targets. Through modulation of enzymes and signaling regulators such as KEAP1 and inflammatory mediators, itaconation serves as an important molecular link between metabolic reprogramming and anti-inflammatory immune regulation^[1].

Itaconation Service at Creative Proteomics

Itaconation is an emerging metabolite-driven post-translational modification that plays a critical role in immunometabolic regulation and inflammatory signaling. However, due to its low abundance, cysteine reactivity, and dynamic nature, itaconation is difficult to detect using conventional proteomic workflows, making specialized identification strategies essential. We provide a dedicated itaconation identification service designed to enable sensitive and reliable profiling of this modification. Our service is based on metabolic labeling strategies combined with high-resolution LC–MS/MS analysis, allowing selective incorporation and detection of itaconate-derived modifications on target proteins. By integrating optimized sample preparation, enrichment-compatible workflows, and advanced mass spectrometry, we support identification of itaconylated proteins and confident site-level characterization. This service facilitates systematic investigation of itaconation dynamics, target proteins, and functional relevance in immune and metabolic studies.

Itaconation Research Platforms

Thermo Fisher Easy-nLC 1000 and Thermo Fisher LTQ Obitrap ETD
(Figure from Thermo Scientific)

Workflow of Itaconation Analysis

Advantages Our Itaconation Service

Comprehensive and Accurate Detection: We provide sensitive and high-precision identification of protein itaconation sites, ensuring reliable results for downstream analysis.
Advanced identification technology: Adopting advanced metabolic labeling and LC-MS/MS, we achieve in-depth profiling of protein itaconation modifications.
Customizable Experimental Design: We can provide customized experimental procedures for different experiments, which are flexible and applicable.
Reliable and Reproducible Results: Rigorous quality control ensures consistency across experiments, supporting confident scientific conclusions.
Functional Insight and Pathway Analysis: We offer comprehensive bioinformatics analysis to link itaconation events with cellular pathways and protein functions.

Applications of Itaconation

Anti-inflammatory and Immunomodulatory

Itaconate as a key immunometabolite with potent anti-inflammatory properties, capable of suppressing pro-inflammatory cytokine production and alleviating chronic inflammatory responses through metabolic reprogramming^[1].

Regulation of Immune Signaling

Itaconate exerts its biological effects partly through covalent modification of cysteine residues on target proteins (protein itaconation), such as KEAP1, leading to activation of the Nrf2 antioxidant and anti-inflammatory signaling pathway and modulation of cellular stress responses^[1][2][3].

Control of Immune Cell Function

Itaconate regulates the activation and functional state of immune cells, particularly macrophages, by integrating metabolic control with redox and inflammatory signaling, thereby shaping overall immune responses^[1].

Autoimmune and Inflammatory Diseases

Due to its combined anti-inflammatory, antioxidant, and immune-regulatory effects, itaconate and its derivatives are proposed as promising therapeutic candidates for autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus^[1][4].

Demo Result of Itaconation Service

PCA plot

Heat map

GO analysis

Heatmap

The plot is used to show the enrichment of itaconated proteins in different pathways.

KEGG analysis

The protein-protein interaction network demonstrated the interactions between all itaconated differential proteins.

PPI

FAQs of itaconation Service

Which sample types are accepted by the service？

We only accept samples of cell. We recommend that customers provide fresh or frozen samples, which should avoid repeated freezing and thawing, and we will be responsible for subsequent protein extraction and pretreatment.

Whether the sample needs pretreatment？

If you need this service, please contact us before cell culture. We will provide the probes for co-incubation with cells and the labeling methods.

What information is included in the final report?

The final report includes identified itaconated proteins and sites, quantitative results, quality control metrics, and bioinformatics analyses such as functional annotation and pathway enrichment.

Learn about other Q&A.

Case Study

Article

Publications

Here are some publications in Proteomics research from our clients:

Parkinson’s disease-associated LRRK2-G2019S mutant acts through regulation of SERCA activity to control ER stress in astrocytes. 2019. https://doi.org/10.1016/j.yjmcc.2007.03.738
The cytoplasmic microtubule array in Neurospora crassa depends on microtubule-organizing centers at spindle pole bodies and microtubule+ end-depending pseudo-MTOCs at septa. 2022. https://doi.org/10.1016/j.fgb.2022.103729
BRCA1 antibodies matter. 2021. https://doi.org/10.7150/ijbs.63115
Metasecretome and biochemical analysis of consortium PM-06 during the degradation of nixtamalized maize pericarp. 2023. https://doi.org/10.1016/j.bcab.2023.102634
Trypanosoma cruzi DNA Polymerase β Is Phosphorylated In Vivo and In Vitro by Protein Kinase C (PKC) and Casein Kinase 2 (CK2). 2022. https://doi.org/10.3390/cells11223693

More Publications

Itaconation Service Sample Requirements

Sample	Sample Quantity
Cell	suspension cell	> 3x10⁸
Cell	adherent cell	> 3x10⁸

References

Xie, Y., Cheng, Q., Xu, M. L., Xue, J., Wu, H., & Du, Y. (2025). Itaconate: A Potential Therapeutic Strategy for Autoimmune Disease. Scandinavian journal of immunology, 101(5), e70026. https://doi.org/10.1111/sji.70026
Zeng, L., Wang, Y., Huang, Y., Yang, W., Zhou, P., Wan, Y., Tao, K., & Li, R. (2025). IRG1/itaconate enhances efferocytosis by activating Nrf2-TIM4 signaling pathway to alleviate con A induced autoimmune liver injury. Cell communication and signaling : CCS, 23(1), 63. https://doi.org/10.1186/s12964-025-02075-5
Song, J., Zhang, Y., Frieler, R. A., Andren, A., Wood, S., Tyrrell, D. J., Sajjakulnukit, P., Deng, J. C., Lyssiotis, C. A., Mortensen, R. M., Salmon, M., & Goldstein, D. R. (2023). Itaconate suppresses atherosclerosis by activating a Nrf2-dependent antiinflammatory response in macrophages in mice. The Journal of clinical investigation, 134(3), e173034. https://doi.org/10.1172/JCI173034
Aso, K., Kono, M., Kanda, M., Kudo, Y., Sakiyama, K., Hisada, R., Karino, K., Ueda, Y., Nakazawa, D., Fujieda, Y., Kato, M., Amengual, O., & Atsumi, T. (2023). Itaconate ameliorates autoimmunity by modulating T cell imbalance via metabolic and epigenetic reprogramming. Nature communications, 14(1), 984. https://doi.org/10.1038/s41467-023-36594-x

Proteomics Sample Submission Guidelines

Ensure your samples are prepared and submitted correctly by downloading our comprehensive Proteomics Sample Submission Guidelines. This document provides detailed instructions and essential information to facilitate a smooth submission process. Click the link below to access the PDF and ensure your submission meets all necessary criteria.

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From Our Clients

"I recently used their proteomics service for a project analyzing protein interactions in yeast models. The team was very responsive and helped clarify the methodology they employed, which made me feel confident in the results. The data quality was solid, with clear identification of several key proteins involved in our study. Their thorough analysis enabled me to pinpoint specific interactions that I hadn't considered before, which significantly improved the direction of my research. I appreciate their professionalism and support throughout the process."

Sarah Thompson, University of California, Berkeley

"Our lab collaborated with them on a project studying cancer biomarkers. The proteomics analysis provided was detailed and focused, specifically highlighting the differential expression of proteins between healthy and tumor samples. Their clear explanations of the data helped my team understand the biological implications. I also appreciated their willingness to revise the reports based on our feedback, ensuring that we had everything we needed for our publication. This collaborative spirit was invaluable."

Emily Rodriguez, Stanford University

"Our lab worked with them on a project studying the effects of diet on gut microbiota using proteomics. They used a label-free quantification method to analyze proteins in fecal samples before and after dietary intervention. The results showed significant changes in protein expression linked to microbial activity. This was pivotal for our hypothesis about diet-microbiota interactions. The clarity of their data presentation made it easy for our team to integrate these findings into our ongoing research."

Dr. Lisa Wong, University of Toronto

"My experience with Creative Proteomics during the mass spectrometry analysis was excellent. We sent in human saliva and mouse brain tissue samples, which they expertly analyzed using both LC-MS and GC-MS techniques. The results were invaluable, revealing key metabolites in the saliva and identifying biomarkers linked to brain function in the brain tissue."

Dr. Emily Carter, Senior Research Scientist

"The overall service from Creative Proteomics was outstanding. They made the entire process seamless and efficient, allowing us to focus on our research. We worked with leaf and root samples from various Arabidopsis genotypes for targeted metabolomics analysis. Their thorough profiling of primary and secondary metabolites gave us important insights into how the plants respond metabolically to environmental stress."

Dr. Laura Henderson, Plant Physiologist

"We had a pleasant collaboration with Creative Proteomics on mass spectrometry analysis of lipids. They conducted a detailed analysis of lipid species, providing us with important insights into lipid metabolism and its relationship with metabolic syndrome disease states."

Dr. Sarah Mitchell, Research Scientist

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