Principle of Targeted Proteomics

Targeted proteomics differs from traditional shotgun proteomics, which typically involves analyzing all detectable proteins in a sample through mass spectrometry. In contrast, it focuses on pre-selected target proteins that are often relevant to specific biological issues or diseases.

 

1. Main Methods

The core technologies typically include liquid chromatography-tandem mass spectrometry (LC-MS/MS) and selective reaction monitoring (SRM) or parallel reaction monitoring (PRM). Through these techniques, researchers can quantitatively analyze target proteins in complex biological samples and obtain their characteristic information.

 

Principles

1. Sample Preparation

Sample preparation is the first step, involving cell lysis, protein extraction, and purification. Effective sample preparation not only enhances the recovery of target proteins but also minimizes the influence of interfering substances in the sample. Common sample preparation methods include:

 

(1) Cell Lysis

Using appropriate lysis buffers to release proteins from cells.

 

(2) Protein Precipitation

Precipitating proteins using organic solvents (such as ethanol or acetone) to remove small molecules and salts.

 

(3) Enzymatic Digestion

Cleaving large protein molecules into peptide fragments to facilitate subsequent analysis.

 

2. Target Protein Selection

(1) Biological Relevance

Target proteins should be closely related to the research question or disease, potentially serving as biomarkers or therapeutic targets.

 

(2) Abundance

Target proteins should have a sufficiently high abundance in the sample to ensure they can be detected during analysis.

 

(3) Stability

Target proteins should remain stable under varying experimental conditions to facilitate reproducible experiments.

 

3. Separation and Detection

(1) Liquid Chromatography (LC)

Utilizing liquid chromatography to separate peptide fragments. Typically, reversed-phase liquid chromatography (RPLC) is employed to separate peptides based on polarity.

 

(2) Mass Spectrometry (MS)

The separated peptides are detected via mass spectrometry, recording their mass-to-charge ratio (m/z) and signal intensity. The sensitivity and resolution of mass spectrometry determine the accuracy of targeted analyses.

 

In targeted proteomics, selective reaction monitoring (SRM) or parallel reaction monitoring (PRM) techniques are commonly employed, allowing researchers to specifically monitor the signals of targeted peptide fragments, significantly enhancing the sensitivity and selectivity of analyses.

 

Applications

1. Discovery of Disease Biomarkers

By analyzing proteins associated with diseases, researchers can identify potential biomarkers.

 

2. Drug Development

In new drug development, researchers can monitor the effects of drugs on target proteins.

 

3. Systems Biology

Targeted proteomics helps researchers understand the complexity of biological systems, revealing interactions between proteins.

 

As an efficient protein analysis method, targeted proteomics provides powerful tools for biological and medical research. By precisely quantifying and analyzing specific proteins, researchers can gain deeper insights into biological processes and disease mechanisms, advancing related fields.