Quantitative Analysis of Serum Proteins Based on LC-MS/MS

As modern biology and medical research advance, the quantitative analysis of serum proteins has become an essential tool for evaluating physiological and pathological states. LC-MS/MS (liquid chromatography-tandem mass spectrometry) is a highly sensitive, selective, and accurate quantitative method widely used in serum protein analysis. It enables the identification and quantification of low-abundance proteins in serum, providing strong support for biomarker discovery and disease mechanism research.

 

Technical Principle of LC-MS/MS

LC-MS/MS is a technique that combines liquid chromatography (LC) with tandem mass spectrometry (MS/MS). LC separates complex serum protein samples, and MS/MS performs qualitative and quantitative analysis of the protein fragments. The working principle of LC-MS/MS involves two main components: first, LC separates proteins based on their polarity differences; second, the mass spectrometer ionizes the separated proteins through electrospray ionization (ESI) and detects them based on their mass-to-charge ratio (m/z). Finally, with secondary mass spectrometry (MS/MS), precise identification and quantification of specific proteins or peptide fragments can be achieved.

 

Workflow of Serum Protein Quantification

1. Sample Preparation

Total proteins are extracted from the serum, followed by denaturation, reduction, and alkylation of the proteins. Proteins are then digested with trypsin to generate a peptide mixture.

 

2. Liquid Chromatography Separation

The digested peptide samples are injected into a high-performance liquid chromatography (HPLC) system, where peptides are separated based on their hydrophilicity and hydrophobicity.

 

3. Mass Spectrometry Detection

Using electrospray ionization (ESI), the peptides are ionized and enter the tandem mass spectrometer for m/z detection, generating a first-level mass spectrum (MS1). Next, selected peptides of interest undergo second-level mass spectrometry (MS2) for more precise structural information.

 

4. Data Analysis and Quantification

The mass spectrometry data are processed using specialized bioinformatics software, matching the results to protein databases, allowing for quantitative analysis of serum proteins. Common quantification methods include labeled quantification (such as iTRAQ, TMT) and label-free quantification.

 

Advantages of LC-MS/MS in Serum Protein Quantification

1. High Sensitivity and Resolution

LC-MS/MS can detect low-abundance proteins in serum at extremely low concentrations, which is crucial for early disease diagnosis.

 

2. High Throughput

With automated operation on the mass spectrometry platform, a large number of samples can be analyzed simultaneously, improving experimental efficiency.

 

3. Accurate Quantification

LC-MS/MS achieves high precision in protein quantification, regardless of whether labeled or label-free methods are used, ensuring high reproducibility of the results.

 

Applications

LC-MS/MS has a wide range of applications in serum protein quantification, particularly in biomarker discovery, drug target research, and disease mechanism exploration:

 

1. Biomarker Discovery

By quantitatively analyzing protein differences in serum, potential biomarkers related to specific diseases can be identified.

 

2. Drug Target Research

LC-MS/MS precisely measures protein changes under drug treatment, helping to reveal drug targets and mechanisms.

 

3. Disease Mechanism Exploration

Quantitative comparisons of serum proteins between healthy and diseased states provide insights into the molecular pathogenesis of diseases.