Workflow of Multiple Reaction Monitoring

Multiple Reaction Monitoring (MRM) is an important method in mass spectrometry, widely applied in biomedical research and drug development. MRM enables high sensitivity and specificity analysis in complex biological samples, making it a core tool in quantitative proteomics and metabolomics.

 

MRM Workflow

1. Sample Preparation

Sample preparation is a crucial first step in multiple reaction monitoring analysis. Effective sample preparation can significantly enhance the sensitivity and accuracy of subsequent analyses. This step usually includes several key aspects:

 

(1) Sample Collection and Processing

Choose suitable biological samples (e.g., serum, urine, cell extracts) and process them promptly after collection to minimize degradation or changes in analytes. Samples should be stored at low temperatures to maintain stability.

 

(2) Protein Extraction and Purification

Use appropriate solvents and extraction methods to isolate target proteins from the samples. Common extraction methods include precipitation, membrane filtration, and affinity chromatography. After extraction, further purification can be achieved through ultrafiltration and column chromatography.

 

(3) Digestion and Labeling

Enzymatically digest the extracted proteins (e.g., using trypsin) to convert them into peptide segments. This process is often coupled with labeling reagents to enhance detection sensitivity, especially in quantitative analysis.

 

2. Chromatographic Separation

Chromatographic separation is a key step in the MRM workflow, effectively isolating target compounds from other components in the sample, thereby reducing background interference. Common chromatographic techniques include:

 

(1) High-Performance Liquid Chromatography (HPLC)

HPLC is a widely used separation technique that allows the separation of compounds with different polarities and molecular weights by selecting appropriate columns and mobile phase conditions.

 

(2) Gas Chromatography (GC)

For volatile small molecules, gas chromatography is ideal. This method vaporizes the samples through heating and combines them with inert gases for separation through a chromatographic column.

 

During the chromatographic process, the separated peptides are introduced into the mass spectrometric detection stage.

 

3. Mass Spectrometric Detection

(1) Ionization

In the mass spectrometer, the peptides in the sample are converted into charged particles through the ionization process. Common ionization techniques include Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption Ionization (MALDI).

 

(2) Targeted Reaction Monitoring

In mass spectrometry, MRM monitors target analytes by selecting specific precursor and product ion pairs. After setting the precursor ion, the mass spectrometer fragments it to generate product ions, allowing for highly specific and sensitive quantification by monitoring certain product ion signals.

 

(3) Data Acquisition

The mass spectrometer collects the intensity signals of ions in real-time and records the corresponding mass-to-charge ratio (m/z) data. The accuracy and sensitivity of this process directly influence the reliability of the analytical results.

 

4. Data Analysis

(1) Signal Intensity Calculation

Calculate the concentration of target analytes based on the detected product ion signal intensities. This is typically achieved through the establishment of a standard curve.

 

(2) Quantitative and Qualitative Analysis

Compare the signal intensities in the samples with standard samples for quantitative analysis, and correct the results using other data (e.g., background interference, internal standard calibration).

 

(3) Data Interpretation and Reporting

Interpret the analytical results in terms of biological significance, usually requiring a combination of experimental design and biological context to form a final research report.

 

The workflow of multiple reaction monitoring is an essential tool for the quantitative analysis of target analytes in biological samples. Through rigorous sample preparation, effective chromatographic separation, precise mass spectrometric detection, and scientific data analysis, MRM provides reliable technical support for biomedical research.