Procedure for Protein Identification Using Tandem MS

Proteins are fundamental molecular units of life, and understanding their structure and function is crucial for biological research. Mass spectrometry (MS) is an efficient analytical tool widely used in proteomics. Specifically, tandem mass spectrometry (MS/MS) provides detailed information about proteins and their peptides, significantly advancing the process of protein identification. This article details the procedure of protein identification using tandem mass spectrometry.

 

Tandem mass spectrometry achieves high sensitivity and specificity in identifying proteins from complex biological samples through two or more rounds of mass spectrometry analysis. The basic workflow includes sample preparation, mass spectrometry analysis, and data processing.

 

Sample Preparation

Proper sample preparation is required before mass spectrometry analysis. The steps for protein sample preparation include:

 

1. Protein Extraction

Proteins are extracted from biological samples such as cells, tissues, or blood using methods such as solvent extraction, ultrasonic disruption, and chemical lysis.

 

2. Protein Digestion

Specific proteases (e.g., trypsin) hydrolyze proteins into smaller peptides. This step is crucial for mass spectrometry analysis because the mass and sequence of peptides are key for protein identification.

 

3. Sample Purification

The digested peptides are purified using liquid chromatography (e.g., reverse-phase high-performance liquid chromatography, RP-HPLC) to remove impurities and interfering substances.

 

Mass Spectrometry Analysis

Mass spectrometry analysis is the core of the identification process and includes the following steps:

 

1. Ionization

Purified peptides are converted into gas-phase ions using methods such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI).

 

2. Primary Mass Spectrometry (MS1) Analysis

In the first stage of the mass spectrometer (MS1), the mass-to-charge ratio (m/z) of ionized peptides is analyzed. The MS1 spectrum provides mass information for all peptides in the sample.

 

3. Peptide Selection and Fragmentation

Specific peptide ions are selected for fragmentation, usually through collision-induced dissociation (CID), generating a series of fragment ions. This process is known as MS/MS or MS2.

 

4. Secondary Mass Spectrometry (MS2) Analysis

In the second stage of the mass spectrometer (MS2), the m/z values of the fragment ions are analyzed. The mass information of these fragment ions is used to deduce peptide sequences.

 

Data Processing and Protein Identification

Data processing is the final step of protein identification, involving complex algorithms and database matching.

 

1. Spectrum Matching

The fragment ion information from MS/MS spectra is matched with theoretical spectra in known protein databases, such as SwissProt and NCBI.

 

2. Peptide Identification

Specific peptide sequences are identified based on spectrum matching results. The reliability of the matches is assessed using statistical methods such as the false discovery rate (FDR) to ensure accurate identification.

 

3. Protein Assembly

The original protein sequences and structures are reconstructed based on the identified peptide sequences. Integrating information from multiple peptides enhances the accuracy of protein identification.

 

Tandem mass spectrometry significantly enhances the efficiency and accuracy of protein identification through its high sensitivity and specificity. With continuous technological advancements, tandem mass spectrometry will play an increasingly important role in biological research.