Workflow of DIA in Protein Quantification

Data-Independent Acquisition (DIA) technology is a widely used quantitative analysis method in proteomics research. Unlike the traditional Data-Dependent Acquisition (DDA), DIA technology allows for the simultaneous monitoring of all detectable peptides, thus enhancing data comprehensiveness and quantification accuracy.

 

Sample Preparation

Sample preparation is a crucial step in proteomics analysis. The biological samples are first subjected to protein extraction and purification to eliminate potential contaminants that might interfere with the analysis. Common procedures include cell lysis, protein centrifugation, and further separation and purification using SDS-PAGE or chromatographic techniques. The precision of this process directly affects the accuracy of the subsequent analyses.

 

Protein Digestion

After sample preparation, protein digestion is carried out. Typically, trypsin is used to cleave the proteins into smaller peptides. The conditions for digestion, such as temperature, time, and pH, must be strictly controlled to ensure the proteins are fully digested.

 

Peptide Separation

The digested peptides are then separated to reduce sample complexity and minimize interference in mass spectrometry detection. Reversed-phase high-performance liquid chromatography (RP-HPLC) is the most commonly used method, separating peptides based on differences in hydrophobicity and thereby enhancing the resolution in mass spectrometry analysis.

 

Mass Spectrometry Data Acquisition (DIA)

During the mass spectrometry phase, DIA technology stands out due to its data acquisition approach. In DIA mode, the mass spectrometer divides all peptides in the sample into multiple mass range windows. It then fragments the ions in each window sequentially and collects their tandem mass spectra. This process does not require the preselection of specific target peptides, thereby avoiding data bias and ensuring comprehensive and accurate results.

 

Data Analysis

Once mass spectrometry data acquisition is complete, specialized software is used to process the DIA data. Researchers typically begin by constructing or selecting a reference spectral library, matching the experimental peptide tandem mass spectra with the theoretical spectra in the library. Then, quantitative analysis algorithms are employed to calculate the abundance of each peptide precisely, leading to quantitative information for the proteins in the sample.

 

Result Validation and Interpretation

After obtaining the protein quantification results, it is essential to validate and interpret the results. This generally involves using standards or independent experiments to verify the quantification results of key proteins. Additionally, researchers need to interpret the data in the context of biological background to reveal potential biological significance.