Methyl-Proteomics Service

When preparing samples, it's crucial to ensure purity and optimal concentration. Biological matrices, such as tissue or cell samples, should ideally be free from contaminants like nucleic acids, polysaccharides, and lipids, which can interfere with peptide separation and MS/MS detection. Samples should also avoid substances like SDS, thiourea, or strong ion salts unless you specify their presence, as these can impact ionization efficiency and downstream analysis. If you're submitting protein samples, check the buffer to ensure compatibility with the HPLC-MS/MS setup.

Creative Proteomics employs methylation-specific antibodies alongside techniques such as hydrophilic interaction chromatography (HILIC) and strong cation exchange (SCX) to selectively enrich methylated peptides. This multi-method approach minimizes non-specific binding, ensuring that enriched peptides truly reflect methylation events. Additionally, our antibodies are rigorously validated to detect methylated lysine or arginine residues with high specificity, thus enhancing the reliability of methylation site identification.

Our Q-Exactive and Orbitrap Fusion™ Tribrid™ systems provide ultra-high sensitivity, often detecting methylated peptides at the femtomole level. To enhance detection sensitivity, especially for low-abundance methylation sites, we recommend sample concentration adjustments based on initial test results. The incorporation of labeling techniques like SILAC or TMT/iTRAQ can also improve quantitation sensitivity, providing more robust data on differential methylation across samples.

Yes, non-histone protein methylation analysis can be challenging due to the vast diversity of these proteins and the lower abundance of methylation events compared to histones. Our service employs a refined enrichment process and high-resolution mass spectrometry to detect these low-abundance modifications. Additionally, using advanced data processing tools, we optimize the detection of subtle methylation events on transcription factors and other regulatory proteins, which may play crucial roles in cellular processes.

Absolutely. Our HPLC-MS/MS pipeline and advanced MS methods allow for the identification of mono-, di-, and trimethylation on both arginine and lysine residues within the same sample. By using immunoaffinity enrichment combined with high-resolution mass spectrometry, we can map various methylation states accurately, even within complex biological samples.

For reliable quantification, we recommend including several controls: 1) non-methylated or baseline samples to identify methylation-specific signals, 2) methylated peptide standards to calibrate the MS response and quantify relative abundance, and 3) technical replicates to ensure data reproducibility. These controls are essential for interpreting differential methylation data accurately and minimizing experimental variability.

Our bioinformatics team uses advanced software tools to identify and quantify methylation sites, map peptides to specific residues, and analyze patterns across experimental conditions. For complex datasets, we employ normalization and correction methods to handle variability and statistical analysis techniques to identify significant methylation changes. We also offer optional consulting to help interpret results, understand potential biological implications, and suggest follow-up experiments.

Methylation can be affected by environmental factors, so we recommend freezing samples immediately after collection and storing them at -80°C to maintain stability. Avoid freeze-thaw cycles, as these can degrade methylated peptides. During transport, ensure samples remain on dry ice. For long-term storage or sensitive samples, lyophilization may be considered to protect methylation integrity.

Low-abundance methylation sites can be challenging to detect due to low stoichiometry and background noise. Our enrichment techniques, including the use of high-specificity antibodies, increase the chances of detecting these rare modifications. For highly sensitive detection, we also recommend using SILAC or TMT labeling to boost the signal for low-abundance methylation events across different conditions.

Yes, our scientists can provide support in designing experiments tailored to specific research questions. This includes advising on sample preparation, selecting appropriate enrichment and labeling methods, and choosing optimal MS settings based on the type of methylation or target protein of interest. By understanding your research goals, we can help you obtain precise, relevant data to answer specific methylation-related hypotheses.