Citrullinated Proteins Analysis Service
Citrullination is a post-translational modification (PTM) catalyzed by the peptidylarginine deiminase (PAD) enzyme family. This modification converts arginine residues into citrulline in a calcium-dependent manner, resulting in a loss of positive charge and potential alteration of protein structure, function, and interaction capacity. Citrullination is irreversible and has been widely implicated in various physiological and pathological processes, including immune regulation, gene expression, apoptosis, and the development of autoimmune disorders. The study of citrullinated proteins has garnered significant interest in biomedical research, particularly in fields such as rheumatoid arthritis (RA), multiple sclerosis, cancer immunology, and neurodegeneration. Detection and characterization of citrullinated proteins can provide valuable insights into disease mechanisms, serve as biomarkers for early diagnosis, and even guide therapeutic development.
Figure 1. PAD-Mediated Citrullination
However, accurate identification and quantification of citrullinated sites remain technically challenging due to their subtle mass change (+0.984 Da), low stoichiometry, and the potential for isobaric interference. Hence, high-sensitivity and high-resolution mass spectrometry, coupled with optimized sample preparation and enrichment strategies, is critical for reliable citrullinated proteins analysis.
Service at MtoZ Biolabs
MtoZ Biolabs offers a comprehensive Citrullinated Proteins Analysis Service designed to support researchers in uncovering the biological roles and diagnostic value of protein citrullination. The Citrullinated Proteins Analysis Service integrates cutting-edge mass spectrometry technologies, specialized enrichment strategies, and robust workflows to ensure high sensitivity, accuracy, and reproducibility in the identification of citrullinated peptides and proteins. Our Citrullinated Proteins Analysis Service platform incorporates a suite of advanced analytical techniques, including:
💠Citrullinated Protein-Specific Enrichment Strategies
Citrullinated peptides often exist at low abundance and can be masked by non-modified peptides. To overcome this challenge, MtoZ Biolabs adopts two well-established strategies—Citrulline Reactive Beads (CRB) and Biotin-Based Enrichment (BPG)—each offering distinct advantages. CRB enables direct capture of citrullinated peptides via chemical immobilization, though potential background interference may occur. In contrast, BPG tagging followed by streptavidin-based purification provides cleaner enrichment, albeit through a more complex workflow. Both methods enhance the specificity and depth of citrullinated proteins analysis when combined with high-resolution LC-MS/MS.
Figure 2. Enrichment of Citrullinated Proteins Using Biotin-PG
💠Dual Fragmentation Strategy for Site-Specific Identification
To precisely identify citrullinated sites, MtoZ Biolabs combines collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation modes during LC-MS/MS analysis. While CID provides comprehensive fragmentation coverage, HCD offers superior resolution and facilitates the detection of low-abundance diagnostic ions associated with citrullinated residues. The complementary nature of these two fragmentation techniques enhances confidence in site localization.
💠SILAC-Based Quantification for Citrullinated Proteins
For projects involving relative quantification of citrullinated proteins under different biological conditions, we implement Stable Isotope Labeling by Amino acids in Cell culture (SILAC). This metabolic labeling strategy enables accurate comparison of citrullination levels across samples, offering insights into dynamic PTM regulation, disease progression, or therapeutic response.
💠DIA for Comprehensive Quantitative Proteomics
We also offer Data-Independent Acquisition (DIA)-based proteomic profiling. DIA provides deep proteome coverage and reproducible quantification across large sample cohorts, making it suitable for biomarker discovery, longitudinal studies, and unbiased citrullination landscape analysis. The DIA workflow is particularly advantageous in complex clinical or tissue-derived samples.
Analysis Workflow
1. Sample Preparation: Extraction, quantification, reduction, alkylation, and enzymatic digestion of protein samples.
2. Citrullinated Peptide Enrichment: Selective enrichment using CRB or BPG strategies to isolate citrullinated peptides.
3. Mass Spectrometry Analysis: Enriched peptides are separated by nano-LC and analyzed using high-resolution tandem mass spectrometry. This step enables precise detection of citrullinated peptides and comprehensive profiling of their abundance across samples.
4. Data Processing and Interpretation: Raw MS data are processed using professional software to identify citrullinated sites and quantify their expression levels. Results are annotated with functional insights such as GO terms, KEGG pathways, and protein interaction networks. A detailed report including spectra, modification maps, and optional biological interpretation is provided to support downstream research.
Service Advantages
✔️High-Resolution Mass Spectrometry: Advanced LC-MS/MS platforms ensure accurate detection and confident identification of citrullinated sites.
✔️Customized Workflow Design: Tailored protocols based on sample properties, experimental objectives, and desired depth of analysis.
✔️Expert Bioinformatics Support: Comprehensive data analysis including site localization, functional annotation, and pathway enrichment.
✔️Comprehensive Reporting: Delivers raw data, modification maps, quantitative comparisons, and publication-ready visual outputs.
Applications
1. Autoimmune Disease Research
Identify citrullinated autoantigens involved in diseases like rheumatoid arthritis, multiple sclerosis, and lupus; support biomarker discovery and pathogenesis studies.
2. Cancer Biology
Explore aberrant citrullination in tumors; assess PAD enzyme expression and its impact on tumor immunity and progression.
3. Neurodegenerative Disorders
Investigate citrullinated proteins in Alzheimer's, Parkinson's, and other neuroinflammatory conditions; understand protein misfolding and neuronal damage.
4. Infectious and Inflammatory Responses
Characterize histone citrullination and NETosis-related mechanisms during infection and immune activation.
5. Biomarker Discovery in Clinical Samples
Identify citrullinated peptides in plasma, synovial fluid, or tissues as potential diagnostic or prognostic biomarkers.
FAQ
Q1: Can I submit crude biological samples for citrullination analysis?
Yes. We accept a wide range of sample types including cell lysates, plasma, and tissues. Pre-treatment such as protein extraction and buffer exchange may be required, which we can handle upon request.
Q2: Can I request both qualitative and quantitative analysis?
Absolutely. We support both identification of citrullinated sites and relative quantification using label-free or isobaric labeling methods.
Q3: What is the detection limit for citrullinated peptides?
Using our optimized protocols and high-resolution instruments, we can detect citrullinated peptides in the low femtomole range under optimal sample conditions.
Whether you are mapping disease-associated citrullinated proteins or validating drug targets, MtoZ Biolabs delivers the technical precision and analytical depth your study demands. Our scalable platform supports discovery through to validation, ensuring reproducible and reliable results. Contact us today to discuss your project or request a quote. Let MtoZ Biolabs be your trusted partner in PTM proteomics.
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