Mechanism of DIA-PRM Proteomics

The development of proteomics technology has provided powerful tools for understanding protein functions, interactions, and regulatory mechanisms within cells. Among the various proteomics techniques, Data-Independent Acquisition (DIA) and Parallel Reaction Monitoring (PRM) have become increasingly favored by researchers. By combining the strengths of both methods, DIA-PRM has emerged as a powerful technique for high-throughput and highly sensitive protein quantification.

 

DIA is a novel mass spectrometry acquisition method that captures all ions without bias throughout the analysis, unlike traditional Data-Dependent Acquisition (DDA). This comprehensive approach enhances protein detection while reducing biases associated with selective acquisition. PRM, a targeted quantitative technique, provides highly specific and sensitive quantification by selecting specific precursor ions and monitoring all their fragment ions.

 

DIA-PRM merges the broad data acquisition capability of DIA with the high specificity and sensitivity of PRM, allowing for the comprehensive screening of numerous proteins in complex samples alongside precise quantification of specific targets.

 

Mechanisms of DIA-PRM

1. Sample Preparation and Digestion

DIA-PRM analysis typically begins with the extraction of proteins from biological samples, followed by enzymatic digestion into peptide mixtures, which form the basis for mass spectrometry analysis.

 

2. Mass Spectrometry Acquisition

In DIA mode, the mass spectrometer systematically captures all precursor ions, ensuring comprehensive detection of all peptides in the sample. The precursor ions are divided into multiple mass windows, each window is fragmented simultaneously, and the resulting fragment ions are detected.

 

3. Data Analysis

The large volume of data generated by DIA requires specialized software for analysis. These tools utilize database searches and spectral library matching to identify and quantify proteins, a process that is complex and computationally intensive due to the substantial data volume.

 

4. PRM Validation

After the initial DIA analysis, target proteins can be selected for PRM validation. PRM provides high specificity and sensitivity by monitoring all fragment ions of selected precursor ions, ensuring reliable and accurate quantification of target proteins.

 

5. Data Integration and Interpretation

Finally, data from both DIA and PRM are integrated and interpreted. By leveraging the comprehensive nature of DIA and the precision of PRM, researchers can conduct in-depth analyses of protein expression profiles in complex biological samples, revealing changes in protein expression and their roles in various biological processes.