Workflow of iTRAQ/TMT and MultiNotch MS Analysis

Quantitative proteomics is a pivotal field in contemporary biological research, extensively employed in disease mechanism studies, drug target identification, and biomarker discovery. iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) and TMT (Tandem Mass Tag) are widely used techniques that enable relative or absolute quantification of multiple samples through chemical labeling.

 

Sample Preparation

Sample preparation is the critical foundation of the entire workflow. The quality of the samples significantly influences the accuracy and reliability of subsequent analyses. Typically, samples undergo protein extraction, protein concentration determination, reduction, and alkylation. These procedures prepare the proteins for analysis, ensuring reduced complexity and enhanced analytical accuracy.

 

iTRAQ/TMT Labeling

Following protein extraction, the samples are evenly divided into multiple fractions. Each fraction is then enzymatically digested, most commonly using trypsin. Subsequently, each fraction is labeled with iTRAQ or TMT reagents. These reagents contain distinct mass tags, facilitating the simultaneous analysis of samples from different sources in a single mass spectrometry run.

 

High-Performance Liquid Chromatography (HPLC) Separation

The labeled peptide mixtures are separated by High-Performance Liquid Chromatography (HPLC). The objective of HPLC is to fractionate the complex peptide mixture into simpler components, thereby enhancing the sensitivity and accuracy of subsequent mass spectrometry analysis. Peptides are separated based on their physicochemical properties, such as hydrophilicity and hydrophobicity, using gradient elution.

 

Mass Spectrometry Analysis

The peptides separated by HPLC are introduced into the mass spectrometer for analysis. This step is the core of the entire workflow. The mass spectrometer measures the mass-to-charge ratio (m/z) of the peptides, and the resulting data is used to identify peptide sequences through database searches. The primary goal at this stage is to determine the relative abundance and identity of the parent proteins based on the mass spectrometry data of the peptides.

 

Data Analysis

After the mass spectrometry data is collected, it undergoes extensive bioinformatics analysis to extract biologically meaningful information. Data analysis typically involves several steps: processing the raw data, identifying proteins, quantifying protein abundance, and performing statistical analyses. By employing specialized software, researchers can discern differences in protein expression across different samples, providing critical insights for further biological interpretation.

 

Results Interpretation and Validation

Once data analysis is complete, the results need to be carefully interpreted. This process usually includes examining the changes in protein expression levels, conducting functional enrichment analyses, and annotating biological pathways. To ensure the robustness of the findings, experimental validation—such as Western blotting or quantitative PCR—is often conducted as needed.

 

The iTRAQ/TMT-based quantitative proteomics workflow is a powerful and precise tool for the simultaneous quantification of multiple protein samples. This process, which includes key steps such as sample preparation, peptide labeling, HPLC separation, mass spectrometry analysis, and data interpretation, is designed to maximize accuracy and reliability in protein quantification.