Overview of Label-Free Proteomics Experimental Workflow

Label-free proteomics is a method for analyzing protein expression that does not require prior labeling of samples. The advantage of this method is its simplicity, cost-effectiveness, and applicability to a wide range of sample types. Here are the typical steps in a label-free proteomics experiment.

 

Sample Preparation

1. Protein Extraction

Proteins are extracted from cells, tissues, or biological fluids. This step usually includes cell lysis, protein precipitation, and purification.

 

2. Protein Quantification

The protein concentration is determined by using BCA, Bradford, or other protein quantification methods.

 

Protein Digestion

1. Enzymatic Digestion

The protein sample is treated with an enzyme (usually trypsin) to hydrolyze the proteins into peptides.

 

Liquid Chromatography Separation

1. Peptide Separation

Peptides are separated by their hydrophobic properties by using reversed-phase high-performance liquid chromatography (HPLC) or other chromatographic techniques.

 

Mass Spectrometry Analysis

1. MS Analysis

Peptides are analyzed by mass spectrometry to determine their mass/charge ratio.

 

2. Tandem Mass Spectrometry (MS/MS)

Selected peptides are further fragmented to obtain information for protein identification and quantification.

 

Data Processing and Analysis

1. Data Processing

Mass spectrometry data is processed by using specialized software such as Proteome Discoverer, MaxQuant, or others.

 

2. Peptide Identification

Peptide mass spectra are matched to known protein sequences by database search algorithms.

 

3. Protein Quantification

Relative quantification of proteins is performed based on peptide signal intensity.

 

Bioinformatics Analysis

1. Data Interpretation

Protein expression patterns are analyzed to identify significantly up- or down-regulated proteins.

 

2. Biological Function Annotation

Using bioinformatics tools such as DAVID, STRING, etc., the discovered proteins are subjected to biological pathway and function analysis.

 

Validation Experiments

1. Validation

Key findings are validated by techniques such as Western blot, enzyme-linked immunosorbent assay (ELISA), etc.