How to use mass spectrometry to conduct label-free quantitative proteomics analysis?
In life sciences research, proteomics is an essential tool for deciphering the functions of biological systems, and quantitative analysis is crucial for uncovering changes in protein expression, pathway regulation, and disease mechanisms. Label-Free Quantitative Proteomics is increasingly favored by researchers due to its high adaptability to samples, simplicity of operation, and lower cost. With the aid of high-resolution mass spectrometry, researchers can accurately capture subtle changes in protein abundance without the need for chemical labels, and it is widely applied in areas such as biomarker screening, disease mechanism research, and drug target discovery.
1. What is Label-Free Quantitative Proteomics?
Label-Free Quantification (LFQ) is a method of protein quantification based on mass spectrometry intensity signals. Unlike isotopic labeling methods (e.g., SILAC, iTRAQ), LFQ does not require the introduction of chemical or metabolic labels at the sample preparation stage, but rather calculates relative protein abundance by comparing ion intensities or spectral counts of peptides across different samples.
Mass Spectrometry (MS): A core instrument for the identification and quantification of proteins, capable of high-precision analysis of peptides.
Label-Free Quantification (LFQ): Direct protein quantification based on mass spectrometry data without the need for chemical labels.
2. Core Principles of Label-Free Quantification
Label-Free Quantitative Proteomics mainly relies on two core concepts:
1. Peptide Ion Peak Intensity (MS1 Intensity)
By comparing the MS1 level signal intensities of the same peptide across different samples, protein abundance is indirectly reflected. This requires that mass spectrometry acquisition has high reproducibility and stability.
2. Spectral Counting
Counting the number of spectra (MS/MS spectra) in which each protein is identified in the sample, often used for rough quantification or low-resolution data.
In practical applications, MS1 Intensity is widely considered more precise, especially when combined with high-resolution mass spectrometers (e.g., Orbitrap) and advanced data processing algorithms.
3. Analysis of Label-Free Quantitative Experiment Workflow
Conducting a Label-Free Quantitative Proteomics project generally includes the following key steps:
1. Sample Preparation
(1) Protein Extraction: Ensuring consistent extraction conditions between different samples to avoid introducing systematic bias.
(2) In-gel/On-gel Digestion: Typically using trypsin for proteolysis of proteins into peptides.
(3) Desalting Purification: Using C18 solid-phase extraction columns to remove impurities and enhance mass spectrometry signal quality.
2. LC-MS/MS Analysis
(1) Liquid Chromatography (LC): Separating peptides to reduce complexity.
(2) High-Resolution Mass Spectrometry (e.g., Orbitrap Exploris 480): Achieving high-sensitivity detection and peptide identification.
3. Data Processing and Quantification
(1) Software Platforms: Such as MaxQuant, Proteome Discoverer, etc., for automatic extraction of peak intensities, retention time alignment, and protein quantification.
(2) Data Normalization: Normalizing signal intensities between samples to eliminate systematic errors.
(3) Differential Analysis: Using statistical methods (e.g., t-test, ANOVA) to screen for significantly differentially expressed proteins.
4. Advantages and Challenges of Label-Free Quantitative Proteomics
1. Advantages
(1) Wide Applicability: Does not rely on labels, suitable for any type of biological sample.
(2) No Limitation on Sample Number: Flexibly accommodates multi-omics samples and clinical cohorts.
(3) Lower Cost: Saves on labeling reagents and sample preparation time.
2. Challenges
(1) Quantification Precision Depends on Mass Spectrometry Performance: Instrument stability and sensitivity directly impact quantification quality.
(2) Large Technical Variations Between Samples: Requires strict standardization and process control.
(3) Prominent Missing Data Issue: Some low-abundance proteins may not be detected in certain samples.
5. Bethel Biotech's Label-Free Quantitative Proteomics Solutions
At Bethel Biotech, we understand the technical challenges and research needs of label-free quantification. Based on years of project experience and platform optimization, we offer the following advantageous services:
1. High-Resolution Mass Spectrometry Platform
Equipped with Thermo Scientific Orbitrap Exploris 480 and timsTOF Pro, achieving high sensitivity and wide dynamic range in peptide capture capabilities.
2. Optimized Sample Processing Solutions
Standardized protein extraction and digestion processes ensure data consistency and reproducibility, adaptable to various sample sources (tissues, cells, body fluids, etc.).
3. Professional Data Analysis Services
Using various data processing strategies such as MaxQuant + Perseus, DIA-NN, to flexibly customize differential protein screening, enrichment analysis, pathway annotation, and other modules based on research objectives.
4. Downstream Integration Services
Expandable to protein interaction networks, metabolomics combined analysis, biomarker discovery, and other research directions, providing clients with one-stop omics solutions.
Label-Free Quantitative Proteomics, as a high-throughput, flexible, and economical quantitative strategy, has been widely applied in basic research and translational medicine fields. With continuous improvements in mass spectrometry performance and data processing algorithms, the quantification precision and stability of LFQ will continue to enhance. At Bethel Biotech, we are committed to supporting researchers in efficiently conducting proteomics research, uncovering biological insights, and advancing scientific discovery through high-quality mass spectrometry platforms and customized research services. If you are interested in learning more about the technical details or cooperation opportunities in label-free quantitative proteomics, please contact us.
Bethel Biotech - Quality Service Provider for Bioproduct Characterization, Multi-Omics Mass Spectrometry Detection
Related Services:
How to order?
