Mass Spectrometry: Method to Sequence Proteins

The mass spectrometry analysis has become the mainstream method for determining the amino acid sequence of proteins. With appropriate pre-treatment and mass spectrometry techniques, thousands to tens of thousands of proteins can be identified and quantified from complex samples. Here are the basic steps to analyze the protein sequence using mass spectrometry.

 

Protein Extraction and Purification

Proteins are extracted from cells, tissues, or other samples.

 

Protein Digestion

Proteins are broken down into peptide segments using proteases (such as Trypsin or Lys-C).

 

Liquid Chromatography Separation

Before mass spectrometry analysis, complex peptide mixtures are usually separated using liquid chromatography (such as reverse-phase high-performance liquid chromatography, RP-HPLC).

 

Mass Spectrometry Analysis

1. MS1 Stage

Primary mass spectrometry scan to determine the parent ion mass-to-charge ratio of peptide segments.

 

2. MS2 Stage (Also Known As Tandem Mass Spectrometry, MS/MS)

Select specific parent ions for fragmentation to generate daughter ions, and analyze the mass-to-charge ratio of the daughter ions. This step provides sequence information of peptide segments.

 

Data Analysis

1. Use mass spectrometry data analysis software (such as MaxQuant, PEAKS, Mascot, etc.) to process data.

2. The software will compare the experimental data with the protein database to determine the identity of the peptide segments and proteins.

3. Further analysis of protein quantification, modification, and interaction information can be performed.

 

Data Validation

For important findings, further verification may be necessary. This can be achieved through repeated experiments, the use of different mass spectrometry techniques, or biological validation.

 

Report and Share Data

Generate detailed experimental reports, and possibly upload data to public mass spectrometry databases (such as ProteomeXchange).

 

Mass spectrometry analysis provides a wealth of information. In addition to the identity and quantity of proteins, it can also be used to study post-translational modifications, protein-protein interactions, etc.