Absolute and Relative Quantitative Proteomics Analysis

Absolute and relative quantitative technologies, as two major quantitative methods in proteomics, have revealed changes in protein abundance and functional characteristics within organisms. Proteins are one of the most important molecules in biological organisms, playing a crucial role in cell functions and biological processes. In order to gain an insight into protein composition, expression levels, and functional regulation, scientists have conducted research in proteomics.

 

The Principle and Method of Absolute Quantification

Absolute quantification refers to the determination of protein expression levels by measuring the absolute abundance of specific peptides or proteins in protein samples. The core principle of absolute quantification is to use an internal standard substance (such as an isotope marker or a specific peptide) to compare with the proteins in the sample to be tested, thereby determining the absolute abundance of the tested protein.

 

Common methods of absolute quantification include isotope labeling methods and quantitative SWATH-MS (Sequential Window Acquisition of All Theoretical Mass Spectra-Mass Spectrometry) techniques. The isotope labeling method divides the sample into different treatment groups, uses different isotope-labeled internal standard substances, and then measures the ratio of isotope markers to the protein to be tested through mass spectrometry analysis, thereby determining the absolute abundance of the protein. The quantitative SWATH-MS technique continuously scans a series of preset mass-to-charge ratio windows through the mass spectrometer, obtains all possible peptide spectral maps, and conducts quantitative analysis by comparing the spectral map library.

 

The Principle and Method of Relative Quantification

Relative quantification refers to the comparison of protein expression levels under different conditions by measuring the relative abundance changes of proteins in protein samples. Relative quantification methods are mainly based on mass spectrometry technology, such as the Label-Free method and TMT (Tandem Mass Tagging) method in quantitative proteomics.

 

The Label-Free method quantifies the relative abundance of proteins by measuring the peak area or peak height of proteins in the sample. This method does not require additional markers, and the relative expression level of proteins can be inferred by comparing the peak area or peak height of specific peptides in different samples.

 

The TMT method uses chemical markers to separately label the proteins in different samples, then mixes the labeled samples, and measures the abundance ratio of the labeled peptides through mass spectrometry analysis, thereby comparing the relative abundance of proteins in different samples.

 

Applications of Absolute and Relative Quantification

1. Biomedical Research

Absolute and relative quantification technologies have a wide range of applications in biomedical research. They can help reveal changes in protein expression during the development of diseases, identify disease markers, and gain a deeper understanding of disease mechanisms.

 

2. Drug Development

Absolute and relative quantification technologies play an important role in drug development. They can assess the impact of drugs on the proteome, reveal the targets and mechanisms of drug action, and provide a basis for drug development.

 

3. Personalized Medicine

Absolute and relative quantification technologies can help to identify differences in proteins between individuals, providing basic data for personalized medicine. By comparing the differences in protein expression in individual samples, precision diagnosis and personalized treatment can be achieved.

 

As important means of quantitative analysis in proteomics, absolute and relative quantification technologies have expanded our quantitative understanding of the proteome. By accurately measuring changes in protein abundance, we can reveal the expression regulation and functional characteristics of proteins within biological organisms. Absolute and relative quantification technologies have important application prospects in biomedical research, drug development, and personalized medicine.