Detection of Proteins Using Parallel Reaction Monitoring

Parallel Reaction Monitoring (PRM) is a mass spectrometry (MS) technique used for the quantitative analysis of specific proteins or peptides in complex biological samples. As an alternative to Selected Reaction Monitoring (SRM), PRM employs high-resolution, high-mass accuracy mass spectrometers, often based on Orbitrap or Q-TOF systems. PRM is characterized by its ability to detect multiple target peptides simultaneously within a single run and accurately analyze fragment ions using high-resolution detectors. Compared to traditional SRM methods, PRM enhances the sensitivity and specificity of detection, making it particularly suitable for quantifying low-abundance proteins.

 

Principles of PRM

PRM involves selecting target peptides through the quadrupole (Q1) of a mass spectrometer and then analyzing these peptides' fragment ions with a high-resolution MS. The process includes:

 

1. Peptide Selection

Representative peptides from the target proteins are chosen for PRM assays. These peptides must be stable, have suitable ionization efficiency, and be unique to the protein of interest.

 

2. Selection of Precursor Ions and Fragmentation

The quadrupole (Q1) selects precursor ions of the target peptides, which are then fragmented in the collision cell.

 

3. High-Resolution MS Detection

The fragment ions are detected and quantified using a high-resolution mass spectrometer (e.g., Orbitrap or Q-TOF). Unlike SRM, PRM records all fragment ion information under high resolution, eliminating the need for pre-optimization of each fragment ion before analysis.

 

4. Data Analysis and Quantification

Quantitative analysis is performed by evaluating the abundance of detected fragment ions, which allows researchers to infer the concentration of the target peptide and, subsequently, the expression level of the target protein.

 

Applications of PRM in Protein Detection

PRM has been widely applied in various biomedical research areas, particularly in the following:

 

1. Quantitative Studies of Biomarkers

PRM is effective in quantifying disease-related protein biomarkers, especially low-abundance biomarkers. Its high sensitivity makes it a crucial tool for screening and validating clinical biomarkers.

 

2. Protein-Protein Interaction Studies

In studies of protein interaction networks, PRM is used to quantify the composition of specific protein complexes, thus revealing functional connections between proteins.

 

3. Signal Pathway Research

PRM enables the quantitative analysis of key signaling pathway proteins, helping researchers understand the mechanisms of signal transduction and their alterations under different conditions, such as in cancer and other diseases.

 

Advantages of PRM

1. High Resolution and High Selectivity

PRM relies on high-resolution MS detection, allowing effective discrimination between closely related or isobaric peptides in complex samples, enhancing detection accuracy.

 

2. No Need for Fragment Ion Preselection

Unlike SRM, PRM does not require pre-selection of fragment ions during data acquisition, simplifying the method development process and reducing errors due to improper preselection.

 

3. High Sensitivity and Wide Dynamic Range

PRM can detect low-abundance proteins while providing quantitative analysis across a wide dynamic range, making it advantageous for analyzing complex samples like serum or tissue extracts.

 

Future Directions

1. Multiplexed Quantitative Analysis

Combining PRM with Data-Independent Acquisition (DIA) techniques could further enhance the capability for multi-target protein quantification, supporting larger-scale proteomic studies.

 

2. Integration with Emerging Technologies

PRM combined with single-cell MS or spatial proteomics may enable high-precision protein quantification at the single-cell level, offering deeper insights into complex biological processes.

 

3. Clinical Translation

PRM has great potential in early disease diagnosis and therapeutic monitoring. By detecting quantitative changes in disease-associated proteins, PRM could become a valuable tool for personalized medicine.