PRM-based Post-translational Modification Site Analysis Service

Post-translational modifications (PTMs) are critical biological processes that regulate protein structure and function. They are extensively involved in signaling pathways, metabolic regulation, and gene expression control. Common types of PTMs, such as phosphorylation, acetylation, methylation, and ubiquitination, typically occur at specific sites and profoundly influence cellular function. Therefore, accurate identification and quantification of these modification sites are essential for elucidating disease mechanisms, discovering biomarkers, and developing targeted therapeutics.

 



Yang YH, et al. Mol Med. 2023.

Figure 1. Functions and Effect of Post-Translational Modifications in Physiology and Pathology

 

Conventional data-dependent acquisition (DDA)-based proteomics plays a fundamental role in initial PTM screening. However, it has limitations in detecting low-abundance modified peptides, performing site-specific quantification, and ensuring reproducibility. In recent years, the development of targeted mass spectrometry has provided a more sensitive and precise approach for PTM research. Among these, Parallel Reaction Monitoring (PRM) stands out as an ideal tool for monitoring dynamic changes in known modification sites due to its high specificity and resolution.

 

Leveraging advanced high-resolution mass spectrometry platforms and extensive experience in PTM analysis, MtoZ Biolabs offers PRM-based Post-translational Modification Site Analysis Service to provide researchers with end-to-end quantitative solutions—from method development to data reporting.

 

Technical Principles

PRM is a high-resolution, targeted quantification technique based on mass spectrometry systems such as Orbitrap. It is designed to accurately identify and quantify known target peptides. In PTM analysis, PRM allows for simultaneous monitoring of all fragment ions from a modified peptide, enhancing qualitative accuracy and quantitative stability. Compared to MRM, PRM eliminates the need to predefine multiple transition pairs, simplifying method development and making it more suitable for high-precision analysis of complex biological samples.

 



Barthélemy, NR. et al. Front Aging Neurosci. 2019.

Figure 2. Principle of the Parallel Reaction Monitoring (PRM) Experiment

 

PRM is compatible with various types of PTMs, including phosphorylation, acetylation, methylation, and ubiquitination. When combined with stable isotope-labeled peptides, it supports both relative and absolute quantification. This approach is particularly effective for validating candidate modification sites and assessing their dynamic changes under different physiological or pathological conditions.

 

Analysis Workflow

MtoZ Biolabs adheres to internationally recognized protocols to deliver reliable and high-quality mass spectrometry services. The standard workflow for the PRM-based Post-translational Modification Site Analysis Service includes the following steps:

1. Sample Preparation and Quality Control

Receive samples from clients (tissue, cells, or purified proteins) and perform total protein quantification and quality assessment.

 

2. Digestion and Enrichment of Modified Peptides

Apply appropriate enzymatic digestion (e.g., trypsin) and use enrichment strategies based on the modification type (e.g., TiO₂, IMAC, or specific antibodies) to isolate target modified peptides.

 

3. Standard Peptide Design and Method Development

Synthesize stable isotope-labeled peptides based on target modification sites to establish and optimize the PRM method.

 

4. Mass Spectrometry Detection

Perform PRM acquisition using high-resolution instruments such as Orbitrap Fusion Lumos, capturing comprehensive fragment ion data.

 

5. Data Analysis and Reporting

Use software like Skyline to extract chromatographic data, integrate peak areas, and perform quantification, followed by generation of detailed reports and visual summaries.

 

Service Advantages

1. High Sensitivity and Specificity: PRM enables precise detection of low-abundance modified peptides, ideal for quantification in complex matrices.

2. Site-level Quantification: Offers accurate estimation of modification ratios at individual sites rather than total modification levels.

3. Excellent Reproducibility: High-resolution platforms ensure consistent results across batches.

4. Compatibility with Multiple PTM Types: Supports analysis of various modifications to meet diverse research needs.

5. Comprehensive Deliverables: Clients receive quantitative tables, annotated spectra, raw data files, and complete analytical reports.

 

Applications

This service is suitable for a wide range of research and application areas, including but not limited to:

1. Disease Mechanism Studies: Investigate changes in specific modification sites associated with cancer, neurodegenerative disorders, autoimmune diseases, etc.

2. Signaling Pathway Analysis: Explore how key protein modifications regulate cellular signaling cascades.

3. Drug Mechanism Validation: Monitor changes in modification levels in response to small molecules or antibody therapies.

4. Biomarker Discovery: Identify potential diagnostic or prognostic biomarkers by quantifying site-specific modifications.

5. Multi-omics Integration: Combine with proteomics, transcriptomics, and metabolomics for systems biology research.

 

Case Study

1. Analysis of Phosphorylation Site Occupancy of Peptides Based on a Chemical Derivatization Method

This study presents a two-step chemical derivatization method combined with LC-MS/MS for the quantitative analysis of phosphorylation site occupancy in peptides. By specifically labeling phosphorylated residues, the method reduces the charge disparity between phosphorylated and non-phosphorylated peptides, enabling their simultaneous detection. The approach was successfully applied to bovine casein and goldfish tissues, demonstrating its feasibility and reproducibility in complex biological samples, particularly for detecting low-occupancy and tissue-specific phosphorylation sites. PRM-based Post-translational Modification Site Analysis Service provides accurate quantification of post-translational modification sites, enabling comparative studies of modification states in complex samples, with support for low-abundance site detection and multi-sample modification level assessment.

 



Wang, X. et al. Anal Methods. 2025.

Figure 3. The MS/MS Spectra and XICs for a Phosphorylated and Non Phosphorylated Peptide

 

2. Tau Phosphorylation Rates Measured by Mass Spectrometry Differ in the Intracellular Brain vs. Extracellular Cerebrospinal Fluid Compartments and Are Differentially Affected by Alzheimer’s Disease

This study developed a highly sensitive PRM-based mass spectrometry method to quantify site-specific phosphorylation of tau protein in human brain and cerebrospinal fluid (CSF), comparing individuals with and without Alzheimer’s disease (AD). A total of 29 phosphorylation sites were identified in brain tau and 12 in CSF tau. Distinct phosphorylation patterns were observed between compartments, with some sites hyperphosphorylated in CSF even in non-AD individuals, and further increased in AD. The findings highlight differential tau phosphorylation as a compartment- and disease-specific process. PRM-based Post-translational Modification Site Analysis Service enables accurate quantification of modification rates at specific sites across biological compartments, supporting comparative analysis between physiological and pathological conditions such as neurodegenerative diseases.

 



Barthélemy, NR. et al. Front Aging Neurosci. 2019.

Figure 4. Detection of Phosphorylation Sites in the Mid-domain and C-terminus of Brain p-tau Protein

 

FAQ

Q1: Do I need to provide known modification site information?

A1: Yes. PRM is a targeted analysis technique and typically requires prior knowledge of the modification sites. If such information is not available, we recommend performing preliminary DDA screening.

 

Q2: What sample types are accepted?

A2: We accept fresh or frozen tissues, cultured cells, protein solutions, and immunoprecipitated products. Please consult our technical team for guidance on sample handling based on specific types.

 

Q3: Can multiple modification sites be analyzed?

A3: Yes. If a peptide contains multiple modification sites, PRM can differentiate and quantify them based on their fragment ion patterns.

 

Q4: Is the data publication-ready?

A4: Yes. Our workflow complies with international standards, and the data quality is suitable for publication. Many of our clients have used the results in high-impact journal submissions.

 

To customize your PRM-based Post-translational Modification Site Analysis Service, please contact MtoZ Biolabs. We will tailor the analytical solution to fit your research objectives and sample characteristics, helping accelerate your scientific discovery.