Absolute Quantification in Targeted Proteomics

Targeted proteomics is one of the critical technological approaches in modern biological research, utilizing mass spectrometry (MS) to quantify specific proteins or protein groups. Unlike discovery-based studies in traditional proteomics, targeted proteomics focuses on the precise measurement of known proteins, enabling deeper insights into their biological functions and clinical applications. Within targeted proteomics, absolute quantification (AQ) is a key approach, allowing the precise determination of the absolute concentration of target proteins in biological samples.

 

Principles of Absolute Quantification in Targeted Proteomics

Absolute quantification in targeted proteomics relies on techniques such as Selected Reaction Monitoring (SRM) or Multiple Reaction Monitoring (MRM), by selecting specific peptides as target molecules for quantification in a mass spectrometer. The method often involves using standards, which can be synthetic isotope-labeled peptides or recombinant proteins. By comparing known concentrations of standards with target proteins in the sample, the absolute concentration of the target protein in the sample can be determined.

 

The key to absolute quantification lies in optimizing mass spectrometer parameters, including peptide selection, fragment ion choice, and calibration of response curves. Ion-selective detection in the mass spectrometer ensures the precise quantification of target proteins, significantly enhancing data reliability and reproducibility. Moreover, absolute quantification methods can overcome matrix effects between samples, ensuring accurate quantification of target proteins in complex biological matrices.

 

Major Absolute Quantification Methods and Their Comparison

Absolute quantification methods mainly include isotope dilution-based methods and label-based quantification methods. Below is a detailed description of the two methods:

 

1. Isotope Dilution Method

The isotope dilution method is among the most widely used techniques in absolute quantification. By adding a known concentration of isotope-labeled peptides to the sample, during mass spectrometry analysis, both the labeled peptides and the corresponding native peptides are detected simultaneously. By calculating the response ratio between the two, the concentration of the target peptides in the sample can be inferred. This method is characterized by high accuracy and sensitivity, making it particularly suitable for quantifying low-abundance proteins.

 

2. Label-Based Quantification Methods

Label-based quantification methods include chemical labeling (e.g., TMT, iTRAQ) and isotope labeling (e.g., SILAC). These methods label target peptides with isotopic tags through chemical reactions, and the relative abundance of target proteins between different samples is determined by mass spectrometry analysis, with correction to achieve absolute quantification results. Compared to isotope dilution, label-based quantification is suitable for parallel analysis of multiple samples, greatly improving analysis efficiency.

 

Applications of Absolute Quantification in Biomedical Research

1. Validation of Biomarkers

In clinical research, the validation of protein biomarkers requires highly accurate quantitative data. Absolute quantification in targeted proteomics allows precise measurement of candidate biomarkers, providing scientific evidence for their application in disease diagnosis and prognosis.

 

2. Drug Mechanism of Action Studies

Absolute quantification enables researchers to understand the effects of drugs on specific proteins in the body. By monitoring target protein concentrations at different time points and doses, it is possible to uncover the mechanism of action of drugs, providing crucial data for new drug development.

 

3. Systems Biology Studies

In systems biology, understanding the quantitative relationships within protein networks is fundamental to constructing biological network models. Absolute quantification provides high-precision protein concentration data, aiding in the quantitative characterization of protein interactions and dynamic changes.

 

The absolute quantification method in targeted proteomics offers a powerful tool for protein research. Its high sensitivity and precision make it indispensable in biomedical research. Although the application of absolute quantification techniques still faces challenges such as the high cost of mass spectrometers and time-consuming method development, with technological advances and wider adoption of mass spectrometers, absolute quantification is poised to play an increasingly crucial role in proteomics research.