Phosphorylation Site Absolute Quantification Service
- Kinase-Substrate Interaction Studies: Determine kinase activity and substrate specificity by quantifying site-specific phosphorylation events.
- Signal Transduction Research: Measure phosphorylation site occupancy to reveal regulatory mechanisms in cellular signaling pathways.
- Drug Discovery and Kinase Inhibitor Evaluation: Assess drug effects on phosphorylation levels to validate kinase inhibitors and targeted therapies.
- Biomarker Discovery and Validation: Identify phosphorylation-based biomarkers for disease progression and therapeutic response.
- Pathway and Network Analysis: Quantify phosphorylation stoichiometry to understand pathway regulation and cellular function.
Phosphorylation site absolute quantification is a cutting-edge mass spectrometry-based approach that precisely measures the molar amount of phosphorylated peptides, rather than just relative changes. Absolute quantification enables researchers to determine the exact phosphorylation occupancy at specific sites. This level of precision is crucial for understanding kinase-substrate interactions, phosphorylation stoichiometry, and pathway regulation. To provide a comprehensive understanding of protein phosphorylation, MtoZ Biolabs offers a Phosphorylation Site Absolute Quantification Service that covers all phosphorylation sites, including serine (Ser), threonine (Thr), and tyrosine (Tyr) residues. Our approach ensures a full-spectrum analysis of phosphorylation events, allowing researchers to uncover their functional impact on protein activity, stability, and interactions.
Analysis Workflow
1. Protein Extraction and Digestion: Proteins are extracted while preserving phosphorylation states, then enzymatically digested into peptides for downstream analysis.
2. Stable Isotope-Labeled Internal Standards Addition: Synthetic isotope-labeled phosphopeptides (represented by the square box in Fig. 1) are added as internal standards for precise, site-specific quantification of endogenous phosphopeptides (represented by the circle in Fig. 1).
3. Phosphopeptide Enrichment: Phosphorylated peptides are selectively enriched using IMAC, TiO₂, or antibody-based methods to enhance detection sensitivity.
4. LC-MS/MS Analysis: Phosphopeptides are separated by nanoLC and analyzed using PRM or MRM, providing sensitive and specific phosphorylation site absolute quantification.
5. Calibration Curve and Quantification: A standard curve from known phosphopeptide concentrations enables accurate phosphorylation site occupancy measurement.
Figure 1. Workflow for Phosphorylation Site Absolute Quantification Service
Service Advantages
✔️High specificity and sensitivity for detecting low-abundance phosphorylated peptides in complex samples.
✔️Optimized phosphopeptide enrichment ensures comprehensive coverage of Ser, Thr, and Tyr phosphorylation sites.
✔️Absolute quantification eliminates variability, providing reproducible and accurate phosphorylation measurements.
✔️Advanced data analysis maps phosphorylation sites to kinases, pathways, and functional networks.
✔️Tailored workflows support kinase activity studies, drug evaluation, and biomarker discovery.
Applications
Our Phosphorylation Site Absolute Quantification Service provides precise, site-specific phosphorylation measurements, supporting diverse research applications across biological and clinical studies.
Case Study
Absolute Quantification of Rab10-Thr73 Phosphorylation in Parkinson’s Disease
LRRK2 mutations drive Parkinson’s disease (PD) by elevating Rab10-Thr73 phosphorylation, necessitating precise quantification for biomarker validation and drug evaluation. Traditional methods lack the accuracy to measure phosphorylation stoichiometry, prompting the use of Phosphorylation Site Absolute Quantification Service. Researchers employed stable isotope-labeled (SIL) phosphopeptides and targeted LC-MS/MS to measure pRab10 levels in neutrophils from PD patients carrying LRRK2 mutations, before and after inhibitor treatment. Results showed 1.9-fold higher pRab10 levels in G2019S and 3.7-fold in VPS35 D620N mutation carriers, with inhibitor treatment reducing pRab10, confirming its role as a PD biomarker. This study highlights the Phosphorylation Site Absolute Quantification Service as a precise, reproducible solution for biomarker research, drug response evaluation, and patient stratification in disease studies.
Karayel, Ö. et al. Mol Cell Proteomics. 2020.
FAQ
Q: What is the difference between absolute and relative quantification in phosphorylation analysis?
Relative quantification reports phosphorylation fold changes across different conditions but does not provide exact molar amounts of phosphorylated peptides. In contrast, absolute quantification uses stable isotope-labeled internal standards or reference peptides to measure precise site occupancy. This yields exact concentrations of phosphorylated peptides rather than ratio-based estimates, allowing deeper mechanistic insights, improved reproducibility, and reliable cross-experimental comparisons in phosphorylation studies.
Deliverables
1. Comprehensive Experimental Details
2. Materials, Instruments, and Methods
3. Total Ion Chromatogram & Quality Control Assessment
4. Data Analysis, Preprocessing, and Estimation
5. Bioinformatics Analysis
6. Raw Data Files
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
Related Services
Quantitative Phosphoproteomics Service
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