Workflow of DIA-PRM Proteomics

Data-Independent Acquisition - Parallel Reaction Monitoring (DIA-PRM) is a proteomics analysis method that combines the strengths of Data-Independent Acquisition (DIA) and Parallel Reaction Monitoring (PRM) mass spectrometry techniques. DIA enables the acquisition of spectral data from all analytes in a sample within a single run, while PRM allows for high-precision, quantitative analysis of specific target peptides during subsequent analyses. The integration of these two approaches allows the DIA-PRM method to achieve extensive proteome coverage along with highly sensitive and specific quantitative analysis.

 

Workflow of DIA-PRM Proteomics

1. Sample Preparation

The first step in DIA-PRM proteomics analysis is the preparation of the sample, and the quality of this preparation is critical for ensuring the accuracy and reliability of subsequent mass spectrometry analysis. Typically, samples undergo protein extraction followed by enzymatic digestion, such as trypsin digestion, which breaks down proteins into peptides. After desalting and purification, these peptides are then introduced into the mass spectrometer for analysis.

 

2. DIA Data Acquisition

Following the completion of sample preparation, the next crucial step is DIA data acquisition. The DIA method captures data from all peptides in the sample using a segmented acquisition approach. During mass spectrometry, the instrument scans across a broad range of m/z (mass-to-charge ratio) intervals, capturing signals from all potential peptides. The unbiased nature of DIA acquisition allows for the simultaneous detection of all peptides across multiple samples, greatly enhancing proteome coverage.

 

3. PRM Targeted Peptide Analysis

Once DIA data acquisition is completed, PRM analysis is conducted on specific target peptides, which are typically preselected based on DIA data. In PRM mode, the mass spectrometer selectively captures and analyzes these preselected target peptides. By utilizing high resolution and accurate mass measurement, PRM enables highly sensitive and specific quantification of these target peptides. The strength of PRM lies in its ability to reliably quantify low-abundance peptides even within complex samples.

 

4. Data Processing and Analysis

The final step in the DIA-PRM proteomics workflow is data processing and analysis. This begins with database searches using the DIA-acquired data to identify all peptides and proteins within the sample. Subsequently, PRM data is employed to validate and quantify the presence and relative abundance of the target peptides. This process typically involves complex data processing and algorithm applications, including intensity-based quantification, data normalization, and false discovery rate control.

 

5. Interpretation and Application of Results

Once data processing is completed, the final results must be interpreted in a biological context. This interpretation often involves functional analysis of target proteins, correlation with known signaling pathways, and exploration of potential underlying biological mechanisms. The strength of the DIA-PRM method lies in its ability to provide high-quality quantitative data, enabling researchers to gain a more accurate understanding of the role of proteins in various biological processes.

 

DIA-PRM proteomics effectively combines the advantages of DIA and PRM, optimizing the workflow for high-throughput, high-precision proteome analysis. This method is of significant importance in biomedical research, particularly for the discovery of disease biomarkers and validation of drug targets.