Mechanism of MRM/PRM in Protein Quantification

In biomedical research, the quantitative analysis of proteins is crucial for understanding biological processes and disease mechanisms. Multiple Reaction Monitoring (MRM) and Parallel Reaction Monitoring (PRM) are two high-sensitivity mass spectrometry techniques widely used in proteomics research. These techniques enable the quantification of specific proteins and their peptide segments, assisting researchers in identifying and quantifying target proteins in complex biological samples.

 

Basic Principles of MRM and PRM

Both MRM and PRM are based on mass spectrometry and rely on the selective monitoring of specific ions.

 

1. Mechanism of MRM

In MRM, the sample ions are first generated through an ion source. These ions are then directed to a mass analyzer. During this process, MRM selectively monitors two specific mass-to-charge ratios (m/z): one for the parent ion (the protein or peptide of interest) and the other for the product ion (specific ions produced from the fragmentation of the parent ion). By accurately selecting these masses, MRM allows for the specific detection of target molecules.

 

(1) Selectivity and Sensitivity

MRM can selectively monitor target peptides in complex matrices while optimizing conditions to enhance sensitivity, making it particularly effective for detecting low-abundance proteins.

 

(2) Quantitative Analysis

MRM can provide accurate quantification by comparing with known concentrations of standards. This method's advantage lies in its ability to rapidly and efficiently process large numbers of samples.

 

2. Mechanism of PRM

PRM operates similarly to MRM but differs in data acquisition and analysis. In PRM, the mass spectrometer can simultaneously monitor multiple product ions, allowing PRM to obtain more quantitative information in a single experiment.

 

(1) High-Throughput Quantification

PRM is capable of analyzing multiple target peptides simultaneously in a single experiment, making it especially useful in studies requiring the quantification of multiple biomarkers.

 

(2) High Resolution

PRM typically uses high-resolution mass spectrometers, which provide greater mass resolution, reducing cross-reactivity and interference and enhancing the reliability of the analysis.

 

3. Comparison of MRM and PRM

MRM and PRM have distinct advantages regarding sensitivity, specificity, and throughput. MRM excels in low-abundance samples, while PRM is better suited for high-throughput and multi-peptide analyses. In practical applications, the choice of technology depends on the specific goals and sample types of the experiment.

 

4. Applications of MRM/PRM

MRM and PRM are extensively applied in biomarker discovery, early disease diagnosis, and drug metabolism studies. For example, in cancer research, quantifying tumor markers can provide strong evidence for disease progression. Additionally, in drug development, MRM/PRM techniques assist in assessing the bioavailability and safety of drugs.

 

5. Mechanism Analysis

(1) Data Processing

In mass spectrometry data analysis, MRM and PRM rely on complex data processing algorithms. By comprehensively analyzing parameters such as mass, abundance, and retention time, the accuracy and reproducibility of quantitative results can be improved. Software tools like MaxQuant and Proteome Discoverer are widely used to process and analyze mass spectrometry data, extracting quantitative information on target peptides.

 

(2) Standardization and Calibration

To ensure the reliability of quantitative results, MRM and PRM techniques often require standardization and calibration. Common methods include the internal standard method and external standard method. The internal standard method corrects for detection differences by adding known concentrations of isotopically labeled peptides, while the external standard method establishes standard curves for quantification.

 

MRM and PRM, as essential components of modern mass spectrometry techniques, provide powerful tools for protein quantification analysis. By selectively monitoring specific peptides and integrating advanced data processing and standardization methods, these techniques offer high sensitivity and specificity in quantitative protein analysis.