Phosphopeptide Enrichment Service
- Cancer Signaling Research: Map phosphorylation-driven activation of oncogenic kinases and downstream signaling nodes.
- Drug Mechanism of Action: Track changes in phosphoproteome in response to targeted inhibitors or drug candidates.
- Biomarker Discovery: Identify disease-specific phosphosites as potential diagnostic or prognostic indicators.
- Cell Signaling Pathway Analysis: Quantify temporal phosphorylation changes under different stimuli or stress conditions.
- Developmental Biology: Understand how dynamic phosphorylation regulates cellular differentiation and fate.
- Storage and Shipping: Samples should be stored at -80℃ and shipped with dry ice.
MtoZ Biolabs provides advanced and high-throughput phosphoproteomics solutions that enable deep profiling of phosphorylation events across a wide range of biological samples. To support reliable and reproducible MS-based phosphorylation analysis, MtoZ Biolabs offers a dedicated Phosphopeptide Enrichment Service designed to selectively enrich phosphorylated peptides from diverse biological matrices, including tissues, cells, plasma, serum, and more. This enrichment service serves as a robust foundation for downstream LC-MS/MS analysis, enabling confident identification and quantification of phosphosites with high specificity and depth.
Due to the substoichiometric nature of phosphorylation and the presence of highly abundant non-phosphorylated peptides, phosphopeptides often constitute only a minor fraction of the total peptide pool. Without selective enrichment, these low-abundance modified peptides are likely to be masked during MS analysis, leading to poor detection sensitivity and incomplete phosphosite coverage. Enrichment techniques not only increase the relative abundance of phosphopeptides but also improve signal-to-noise ratios, enhance mass spectrometric detection, and ultimately enable high-confidence identification of site-specific phosphorylation. Therefore, phosphopeptide enrichment is an essential preparatory step for any comprehensive phosphoproteomic investigation, particularly in studies aiming to elucidate dynamic signaling events or uncover novel phosphorylation-mediated regulatory mechanisms.
Service at MtoZ Biolabs
Given the typically low abundance and high dynamic range of phosphopeptides in complex biological samples, efficient enrichment is a prerequisite for high-quality phosphoproteomic analysis. MtoZ Biolabs' comprehensive Phosphopeptide Enrichment Service utilizes multiple strategies tailored to diverse experimental goals. Our protocols are optimized to selectively enrich phosphorylated peptides prior to LC-MS/MS analysis, thereby enhancing detection sensitivity and phosphoproteome coverage. In addition to phosphorylation, MtoZ Biolabs also offers enrichment and analysis services for other post-translational modifications, such as acetylation, ubiquitination, and glycosylation. Our platform supports both exploratory and targeted studies, accommodating diverse research needs from large-scale phosphosite mapping to specific kinase substrate investigations.
Phosphopeptide Enrichment Methods Provided by MtoZ Biolabs:
💠Strong Cation Exchange Chromatography (SCX)
SCX separates peptides based on their charge states under acidic conditions. Since phosphorylation reduces the overall positive charge of peptides, phosphopeptides typically elute earlier than their non-phosphorylated counterparts. SCX is often used as a pre-fractionation step to reduce sample complexity and improve enrichment efficiency when combined with downstream TiO₂ or IMAC protocols.
💠Metal Oxide Affinity Chromatography (MOAC)
MOAC, most commonly using titanium dioxide (TiO₂), exploits the affinity between phosphate groups and metal oxides. Phosphopeptides bind selectively to the metal oxide matrix through Lewis acid-base interactions, allowing their separation from non-phosphorylated peptides. TiO₂ enrichment is particularly effective for mono-phosphorylated peptides and is widely used in large-scale phosphoproteomic studies.
💠Immobilized Metal Affinity Chromatography (IMAC)
IMAC utilizes immobilized trivalent metal ions (e.g., Fe³⁺, Ga³⁺) to capture phosphorylated peptides via coordination with their phosphate groups. IMAC offers strong binding affinity and is suitable for enriching both mono- and multi-phosphorylated peptides. It is frequently applied to samples from cells, tissues, or biofluids where deeper phosphoproteome coverage is desired.
💠Immunoprecipitation (IP)
IP-based enrichment uses antibodies that specifically recognize phospho-serine, phospho-threonine, or phospho-tyrosine residues. This method allows for the selective isolation of phosphopeptides bearing specific motifs or PTMs and is especially valuable for studying low-abundance phospho-tyrosine events in signaling cascades. IP can also complement global enrichment methods to enhance target-specific insights.
Figure 1. The Commonly used Strategies for Phosphopeptide Enrichment
Analysis Workflow
1. Sample Preparation: Biological samples such as cells, tissues, or biofluids are lysed under conditions that preserve phosphorylation status, typically with phosphatase inhibitors included.
2. Protein Digestion: Extracted proteins are enzymatically digested (commonly with trypsin or Lys-C) to generate peptides suitable for enrichment.
3. Phosphopeptide Enrichment: Based on project needs, we apply IMAC, TiO₂, SCX, or phospho-specific immunoprecipitation methods to selectively enrich phosphopeptides.
4. LC-MS/MS Analysis: Enriched phosphopeptides are subjected to high-resolution liquid chromatography–tandem mass spectrometry for sensitive and accurate detection.
5. Data Processing: Raw MS data are processed to identify phosphopeptides, assign modification sites, and quantify phosphorylation intensities.
6. Bioinformatics Analysis: Functional annotation, pathway enrichment, motif analysis, and phosphosite mapping are performed to extract biological insight.
Figure 2. Workflow for Phosphopeptide Analysis
Service Advantages
☑️Experienced Team: Skilled scientists with extensive phosphoproteomics expertise ensure accurate and reproducible results.
☑️Customizable Enrichment Methods: Multiple enrichment strategies (e.g., IMAC, MOAC, SCX, IP) tailored to specific sample types and research goals.
☑️Advanced Analytical Platform: Cutting-edge LC-MS/MS systems deliver high sensitivity and site-specific resolution.
☑️End-to-End Workflow: From sample preparation to bioinformatics interpretation, we offer seamless, full-process support.
☑️High Compatibility: Suitable for diverse biological samples including tissues, cells, and biofluids.
Applications
Our Phosphopeptide Enrichment Service is applicable across diverse research areas:
Sample Submission Suggestions
*Note: If you have specific requirements or need guidance on sample preparation, please do not hesitate to contact us.
Deliverables
1. Comprehensive Experimental Details
2. Materials, Instruments, and Methods
3. Total Ion Chromatogram & Quality Control Assessment (project-dependent)
4. Data Analysis, Preprocessing, and Estimation (project-dependent)
5. Bioinformatics Analysis
6. Raw Data Files
Related Services
Peptide Enrichment Service | MS-compatible
Phosphoproteomics Analysis Service
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