- Service Details
- Case Study
What is Protein methylation
Protein methylation modification occurs more frequently on arginine (Arg) and lysine (Lys). At the Lys site, the ε-amino group of Lys can undergo mono-, di- or trimethylation catalyzed by lysine methyltransferase (KMTs) using S-Adenosylmethionine (SAM) as the methyl donor. The side chain of Arg residue can be mono-, symmetric or asymmetric di-methylation by protein Arg methyltransferases (PRMTs) in a SAM dependent manner.
Figure 1. Chemical structures of six distinct methylated modification on Arg or Lys residues.
The post-translational modification of histones, as well as DNA methylation, are pivotal epigenetic events that exert influence on gene expression. Protein methylation also modifies non-histone proteins, such as transcription factors. It involves in diverse biological processes including signal transduction, transcriptional regulation, DNA repairing, gene activation, gene repression, RNA processing and protein stability. Accordingly, the deregulation of protein methylation has been associated with a wide range of human disorders, such as metabolic, neurodegenerative, and muscular disorders. Therefore, the acquisition of a more comprehensive comprehension of methylation is imperative in order to unravel intricate biological processes and, ultimately, to advance the development and enhancement of disease treatments. However, the global profiling of methylated sites in cells is challenging and often complicated, due to: 1) low abundance and low stoichiometry; 2) lack of efficient biochemical strategies for the enrichment of methylated peptides; 3) several amino acid substitutions are isobaric to methylation.
Our Methyl-proteomics Service
Creative Proteomics has established advanced strategies for highly specific enrichment of methyl (Lysine)-peptides or methyl (Arginine)-peptides, along with a sensitive HPLC-MS/MS pipeline capable of analyzing various types of methylation in both eukaryotic and prokaryotic organisms. In addition, the methyl-proteomics service protocol has been optimized, facilitating a more efficient and precise site mapping for methyl-sites. Classically, the workflow of this service includes protein isolation from biological samples, proteolytic digestion into peptides, methyl-peptide enrichment utilizing immunoaffinity, hydrophilic interaction chromatography (HILIC), or Strong Cation exchange (SCX), identification of methylation sites, and comprehensive quantification of relative abundance changes for methylation sites. Additionally, the application of LC-MS/MS analysis can be complemented by incorporating iTRAQ/TMT labeling or SILAC labeling techniques to enhance the precision of relative quantitation between samples.
Figure 2. General Workflow for Methyl-proteomics Analysis.
- Professional detection and analysis capability: Experienced PTM research team, strict quality control system, together with ultra-high resolution detection system and professional data pre-processing and analysis capability, ensure reliable and accurate data.
- Reproducible: Obtain consistent and reproducible inter- and intra- assay results for data analysis.
- High specificity: Use methylation-specific antibodies for methyl-peptides enrichment.
- Multiplex, high-throughput: Deeper coverage of methylation site identification.
- High resolution and sensitivity: Q-Exactive, Q-Exactive HF, Orbitrap Fusion™ Tribrid™.
Tissue: animal tissue > 50 mg;
fresh plant > 100 mg;
Cell: suspension cell > 2 x 107;
adherent cell > 2 x 107;
microorganism > 50 mg or 2 x 107 cells;
Body fluid: Serum/plasma > 500 μL;
Protein: Total protein >1 mg and concentration >1 μg/μL.
Note: If you provide proteins please inform the buffer components whether it contains thiourea, SDS, or strong ion salts to ensure the test results. In addition, the sample should not contain components such as nucleic acids, lipids, and polysaccharides, which will affect the separation effect.
- Detailed report, including experiment procedures, parameters, etc.
- Raw data and data analysis results
How to place an order:
At Creative Proteomics, many excellent and experienced experts will optimize the experimental protocol according to your requirement to guarantee the high-quality results for protein acetylation. Creative Proteomics provides a broad range of technologies for methylation research that enable quantification of methylation protein modification. Please feel free to contact us by email to discuss your specific needs. Our customer service representatives are available 24 hours a day, from Monday to Sunday.
- Hyun K, Jeon J, Park K, et al. Writing, erasing and reading histone lysine methylations. Experimental & Molecular Medicine. 2017 Apr 28;49(4): e324.
- Blanc RS, Richard S. Arginine Methylation: The Coming of Age. Molecular Cell. 2017 Jan 5;65(1):8-24.
Deep Protein Methylation Profiling by Combined Chemical and Immunoaffinity Approaches Reveals Novel PRMT1 Targets.
Journal: Molecular & Cellular Proteomics
Main Technology: immunoaffinity purification (IAP), high pH strong cation exchange (SCX), and label-free quantitative proteomics.
- Enrichment of methyl peptides using two orthogonal techniques.
- Knockdown of PRMT1 leads to substantial changes in protein arginine "methylome".
- Discrimination of ADMA (asymmetric dimethyl arginine) and SDMA (symmetric dimethyl arginine) using characteristic neutral losses.
- Identification of PRMT1 targets and substrate scavenged by other PRMTs in the absence of PRMT1 activity.
Protein methylation has been implicated in many important biological contexts including signaling, metabolism, and transcriptional control. Despite the importance of this post-translational modification, the global analysis of protein methylation by mass spectrometry-based proteomics has not been extensively studied because of the lack of robust, well-characterized techniques for methyl peptide enrichment.
The identification results
Compared two methods for methyl peptide enrichment: IAP and SCX. In total, we identified 1720 methylation sites on 778 proteins. Notably, it revealed that these methods are largely orthogonal and quantitatively reproducible, suggesting that both methods are required for global analysis of protein methylation. PRMT1 (protein arginine methyltransferase 1) knockdown resulted in significant changes to 127 arginine methylation sites on 78 proteins. In contrast, only a single lysine methylation site was significantly changed. In PRMT1 knockdown cells, 114 monomethyl arginine sites that were either significantly down- or up-regulated on proteins enriched for mRNA metabolic processes.
Through integrative analysis of MMA and DMA, we identified a list of 18 PRMT1 substrates and 12 substrates scavenged by other PRMTs in the absence of PRMT1 activity. Taken together, our results describe a general method for deep profiling of protein methylation and identify novel potential MMA and ADMA methylation targets of PRMT1.
Figure 1. Workflow of quantitative proteomics of protein methylation modification.