A quantitative and site-specific atlas of the citrullinome reveals widespread existence of citrullination and insights into PADI4 substrates

Title: A quantitative and site-specific atlas of the citrullinome reveals widespread existence of citrullination and insights into PADI4 substrates

Journal: Nature Structural & Molecular Biology

Published: 2024

Background

Citrullination, a post-translational modification catalyzed by peptidylarginine deiminases (PADs), plays essential roles in gene regulation, immune responses, and disease development, particularly in autoimmune conditions like rheumatoid arthritis. Despite its importance, a comprehensive and quantitative mapping of the citrullinome in human cells has been lacking, limiting our understanding of its biological functions and disease relevance. This study addresses this gap by systematically profiling citrullinated proteins and their regulatory dynamics, providing a crucial resource for citrullination-related biomedical research.

Materials & Methods

Cell lysis and protein digestion

The cells were pelleted by centrifugation and the Lockes buffer was removed. Cell pellets were then lysed in 10 pellet volumes of Lysis Buffer (6M guanidine-HCl, 50 mM TRIS, pH 8.5). Rapid cell lysis was achieved by alternating vigorous vortexing and vigorous shaking of the samples, 5 seconds per cycle and for 30 seconds in total, after which the lysates were snap frozen in liquid nitrogen. The lysates were stored at −80℃ until further sample processing. Lysates were thawed at room temperature (RT) and homogenized using a microtip sonicator, via two pulses of 10 seconds at 30 W. The homogenized lysates were reduced and alkylated, by the addition of Tris(2 carboxyethyl)phosphine (TCEP) and chloroacetamide (CAA), both to a final concentration of 5mM, and incubation for one hour at RT. Proteins were digested using Lysyl Endopeptidase (Lys-C, 1:100 w/w) (cat. 129-02541, Wako Chemicals) for 3 hours at RT. Following a three-fold dilution with 50mM Tris, a second round of Lys-C (1:200 w/w) digestion was performed overnight at RT. Following digestion, samples were acidified via addition of trifluoroacetic acid (TFA) to a final concentration of 0.5% (v/v).

Purification of peptides

Peptides were purified using reversed-phase C18 cartridges (SepPak Classic, 350 mg, Waters). Cartridges were activated with 5 mL acetonitrile (ACN) and equilibrated three times with 5 mL of 0.1% TFA, after which samples were loaded. Sample loading was accelerated using a vacuum manifold, maintaining two-thirds atmospheric pressure. Following loading, cartridges were washed three times with 5 mL of 0.1% TFA, after which peptides were eluted using 4 mL of 30% ACN in 0.1% TFA. The eluted peptides were frozen overnight at −80 °C in 15 mL tubes with small holes punctured into the caps, after which the frozen peptides were lyophilized for 96 h. Lyophilized peptides were dissolved in 25 mM ammonium bicarbonate pH 8.5, and the peptide concentration was estimated through absorbance at 280 nm, using a NanoDrop instrument.

Results

Citrullination Service Figure 1: The number of cumulative peptides detected and across the conditions; The number of cumulative proteins detected and across the conditions; Abundance of PADI2 and PADI4 across conditions, based on label-free quantification (LFQ) intensity.

Citrullination Service Figure 2: Representative MS/MS spectrum, showing localized citrulline.

Citrullination Service Figure 3: Number of citrullination sites and occupancy of citrullination detected in the five conditions by MS/MS and matching; Gene Ontology term enrichment analysis for biological process of citrullination target proteins in HL60 cells and the NLC+Cal30' condition.

Conclusions

This study presents the first large-scale, quantitative map of the human citrullinome, identifying over 14,000 citrullinated sites across 4,000 proteins in HL60 leukemia cells. Using a robust mass spectrometry approach combined with PADI4 inhibition, the researchers revealed dynamic and site-specific regulation of citrullination, including its enrichment in chromatin-associated proteins and known autoantigens. These findings not only highlight the biological complexity and regulatory potential of citrullination but also provide a valuable resource for understanding its role in diseases such as rheumatoid arthritis and cancer.

Reference

Rebak, A. S., Hendriks, I. A., Elsborg, J. D., Buch-Larsen, S. C., Nielsen, C. H., Terslev, L., Kirsch, R., Damgaard, D., Doncheva, N. T., Lennartsson, C., Rykær, M., Jensen, L. J., Christophorou, M. A., & Nielsen, M. L. (2024). A quantitative and site-specific atlas of the citrullinome reveals widespread existence of citrullination and insights into PADI4 substrates. Nature structural & molecular biology, 31(6), 977–995. https://doi.org/10.1038/s41594-024-01214-9

* For Research Use Only. Not for use in diagnostic procedures.

Our customer service representatives are available 24 hours a day, 7 days a week. Inquiry

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

Online Inquiry

Please submit a detailed description of your project. We will provide you with a customized project plan to meet your research requests. You can also send emails directly to for inquiries.

Great Minds Choose Creative Proteomics