Phosphoproteomics
Phosphoproteomics
What Is Phosphorylation Proteomics?
MetwareBio’s Phosphorylation Proteomics Service combines optimized phosphopeptide enrichment techniques with advanced 4D label-free LC-MS/MS analysis on the Bruker timsTOF platform. This enables high-confidence phosphosite identification, precise localization, and quantitative profiling across thousands of phosphorylation events. Our platform supports a wide range of applications, including signal pathway mapping, kinase substrate analysis, drug mechanism studies, and phospho-network discovery, making it an ideal tool for advancing both basic and translational research.
Functional properties triggered by protein phosphorylation (Weber 2010)
Why Choose MetwareBio for Phosphorylation Analysis?
Phosphorylation Proteomics Workflow with Ti-IMAC and 4D LC-MS/MS
Enrichment
Detection
Phosphoproteome Analysis Deliverables
Experience in High-Coverage Phosphorylation Proteomics
Number of Phosphorylation Sites Identified Across Various Animal and Plant Samples
Applications of Phosphorylation Proteomics
Phosphorylation proteomics is a powerful tool for studying cell signaling, immune activation, and disease-related pathways. Abnormal phosphorylation patterns are widely associated with cancer, neurodegenerative disorders, cardiovascular disease, and autoimmune conditions. Phosphoproteome profiling enables the discovery of biomarkers, kinase targets, and drug response mechanisms, supporting translational research and precision medicine.
In model organisms such as mice, zebrafish, and Drosophila, phosphorylation regulates processes like development, cell proliferation, neural activity, and inflammation. Quantitative phosphoproteomics helps dissect in vivo signaling networks, track disease progression, and evaluate therapeutic intervention effects, advancing functional genomics and experimental medicine.
Phosphorylation plays a central role in plant signal transduction, influencing hormone signaling, growth regulation, and responses to abiotic and biotic stress. Phosphoproteomics reveals phosphorylation dynamics in drought tolerance, pathogen resistance, and photosynthetic regulation, supporting crop improvement and agricultural resilience research.
In microbes, phosphorylation controls metabolism, virulence, and antibiotic resistance. During infection, host and pathogen phosphoproteomes undergo rapid remodeling. Phosphorylation analysis in microbial systems or host–microbe models uncovers infection strategies, immune evasion mechanisms, and host defense pathways, supporting research in microbiology, immunology, and infectious disease.
Case Study of Phosphoproteomics
Phosphoproteomics Reveals Integrin-Driven Platelet Activation by Gut Microbial Metabolite
In a study published in Cell Metabolism (2024), researchers explored how the gut microbial co-metabolite 2-methylbutyrylcarnitine (2MBC) promotes platelet hyperactivity and thrombosis. Elevated in patients with COVID-19 and cardiovascular disease, 2MBC was found to bind and activate integrin α2β1, initiating downstream pro-thrombotic signaling. To dissect the molecular mechanisms, the team employed MetwareBio’s phosphoproteomics service to profile phosphorylation changes in 2MBC-treated platelets. The analysis identified differentially regulated phosphosites and highlighted enriched pathways such as integrin signaling, platelet activation, and cytosolic phospholipase A2 (cPLA2) activation, providing critical evidence that integrin α2β1 functions as the receptor mediating 2MBC’s effect.
This case highlights the value of phosphoproteomics in uncovering dynamic signaling events and functional receptor targets in complex disease processes. MetwareBio’s platform enables deep, site-specific phosphorylation profiling, supporting research in cardiovascular biology, immune signaling, and host–microbiome interactions.
2MBC directly binds to and potentiates integrin a2b1 activation (Huang et al., 2024)
Sample Requirements of Phosphoproteomics
| Sample Type | Samples | Recommended Sample Size | Minimum Sample Size |
| Human/Animal Tissue | Normal tissues (heart, liver, spleen, lungs, intestines, kidneys, etc.) | 50mg | 30mg |
| Fatty tissue | 1g | 500mg | |
| Brain tissue | 100mg | 50mg | |
| Bone | 1g | 500mg | |
| Hair | 1g | 500mg | |
| Plant Tissue | Young tissue (young leaf, seedling, petal, etc.) | 500mg | 200mg |
| Mature tissue (root, stem, fruit, pericarp, etc.) | 2g | 1.5g | |
| Liquid | Joint fluid, Lymph fluid | 1ml | 500μL |
| Cerebrospinal fluid | 1ml | 500μL | |
| Amniotic fluid | 5ml | 2ml | |
| Urine | 50mL | 20mL | |
| Fungi | 1g | 500mg | |
| Cells | Primary Cells | 2×10^7 | 1×10^7 |
| Transmissible cells | 1×10^7 | 5×10^6 | |
| Sperm, Platelets | 5×10^7 | 2×10^7 | |
| Protein | Protein | 1000μg | 500μg |
FAQ on Phosphorylation Proteomics
Phosphorylated peptides are often low in abundance and display poor ionization efficiency. Without specific enrichment, they are easily masked by non-modified peptides. Therefore, specialized phosphopeptide enrichment and optimized mass spectrometry workflows are essential for reliable detection and quantification of phosphorylation events.
Phosphopeptide enrichment is essential for successful phosphoproteomics due to the low abundance of phosphorylated peptides. Common enrichment methods include IMAC (Immobilized Metal Affinity Chromatography), TiO₂ chromatography, and Ti-IMAC (Titanium Ion Metal Affinity Chromatography). At MetwareBio, we use Ti-IMAC materials, which offer high specificity and recovery efficiency, allowing for sensitive and reproducible capture of both mono- and multi-phosphorylated peptides across diverse sample types.
Yes. By comparing phosphorylation levels across conditions or time points, and integrating with total protein abundance data, our analysis can help distinguish stimulus-responsive phosphorylation from baseline or structural phosphorylation, enabling deeper insight into signal transduction dynamics.
We offer paired quantitative proteome + phosphoproteome analysis, allowing normalization of phosphosite intensity to corresponding protein abundance. This helps distinguish true phosphorylation regulation from changes driven by protein expression differences.
We recommend starting with at least 200–300 µg of total protein per sample after lysis and quantification. For precious or limited samples, please consult us for low-input or optimized enrichment protocols.
Absolutely. We provide phosphorylation motif analysis, helping identify sequence preferences around phosphosites, which can suggest upstream kinase family activity, post-translational cross-talk, or regulatory hotspots.
Yes. Phosphorylation profiling is a powerful tool for mechanism-of-action studies, target deconvolution, and off-target effect assessment in drug-treated cells, tissues, or organoids—supporting pharmacodynamic evaluation in both early and late-stage research.
Our expert team can assist with custom data slicing, focusing on specific phosphosite groups, pathways, or gene sets. Whether you're interested in immune signaling, cell cycle regulation, or stress responses, we tailor analysis outputs to match your biological questions.
Reference
1. Weber, T. J. (2010). Protein kinases. In C. A. McQueen (Ed.), Comprehensive toxicology (2nd ed., pp. 473–493). Elsevier.https://doi.org/10.1016/B978-0-08-046884-6.00225-6
2. Huang, K., Li, Z., He, X., Dai, J., Huang, B., Shi, Y., Fan, D., Zhang, Z., Liu, Y., Li, N., Zhang, Z., Peng, J., Liu, C., Zeng, R., Cen, Z., Wang, T., Yang, W., Cen, M., Li, J., Yuan, S., … Chen, S. (2024). Gut microbial co-metabolite 2-methylbutyrylcarnitine exacerbates thrombosis via binding to and activating integrin α2β1. Cell metabolism, 36(3), 598–616.e9. https://doi.org/10.1016/j.cmet.2024.01.014
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