Mechanism of Mass Spectrometry in Protein Sequencing

Protein mass spectrometry sequencing is a powerful analytical tool used to determine the structure and function of proteins. Its applications are wide-ranging, from basic research to clinical diagnostics.

 

Basic Principles of Protein Mass Spectrometry Sequencing

The core of protein mass spectrometry sequencing lies in the mass spectrometer, which analyzes the molecular weight and sequence of ionized proteins or peptides by measuring their mass-to-charge ratio (m/z). Mass spectrometry sequencing typically involves four main steps: sample preparation, ionization, mass analysis, and data interpretation.

 

1. Sample Preparation

Sample preparation is the first step in mass spectrometry sequencing. Target proteins need to be purified, digested, and modified to meet the requirements of mass spectrometry analysis. Digestion usually involves specific enzymes such as trypsin, which cleave proteins into smaller peptides. These peptides are then further analyzed to determine their sequences.

 

2. Ionization

Ionization is the process of converting peptides into charged ions. Common ionization methods include Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption/Ionization (MALDI). ESI is suitable for coupling with liquid chromatography, while MALDI is used for simpler sample preparation and analysis.

 

3. Mass Analysis

The mass spectrometer determines the molecular weight of ionized peptides by analyzing their mass-to-charge ratio. Common mass analyzers include Time-of-Flight (TOF), quadrupole mass spectrometers, and ion trap mass spectrometers. Different analyzers offer varying resolutions and sensitivities, making them suitable for different types of samples and analytical needs.

 

4. Data Interpretation

Data interpretation is the process of converting mass spectrometry data into readable protein or peptide sequence information. Mass spectrometry data are typically presented as mass spectra, where each peak represents a charged ion. By comparing mass spectrometry data with known sequences in databases, the sequences of unknown proteins can be inferred.

 

Mechanisms of Mass Spectrometry Sequencing

1. Peptide Generation

Before mass spectrometry analysis, protein samples must be enzymatically digested into peptides. This step is fundamental to mass spectrometry sequencing, commonly employing trypsin, which specifically cleaves peptide bonds after lysine and arginine residues, generating relatively uniform peptide fragments.

 

2. Ionization Process

After enzymatic digestion, peptides need to be ionized to enter the mass spectrometer. ESI generates charged droplets from the sample solution, while MALDI uses laser pulses to evaporate the sample with the matrix, forming charged particles. The efficiency of ionization directly affects the sensitivity and accuracy of mass spectrometry analysis.

 

3. Mass-to-Charge Ratio Analysis

Ionized peptides enter the mass analyzer of the mass spectrometer, where they are separated based on their mass-to-charge ratio (m/z). Different types of mass spectrometers achieve this separation using various techniques. For example, TOF mass spectrometers determine the mass-to-charge ratio by measuring the time of flight of ions, while quadrupole mass spectrometers use electric and magnetic fields to separate ions.

 

4. Fragment Ion Generation

To obtain the sequence information of peptides, mass spectrometers often further fragment peptides into smaller ions. Common fragmentation methods include Collision-Induced Dissociation (CID) and Electron Transfer Dissociation (ETD). The mass-to-charge ratios of these fragment ions are measured, allowing the original peptide sequence to be inferred.

 

5. Data Interpretation and Sequence Inference

Finally, mass spectrometry data must be interpreted using bioinformatics tools. Each peak in a mass spectrum corresponds to a charged ion. By comparing these data with known protein sequences in databases, the sequences of unknown proteins can be inferred. This process involves complex algorithms and statistical analyses to ensure the accuracy of the results.

 

Applications and Prospects

Protein mass spectrometry sequencing has a wide range of applications in many fields. For instance, in proteomics research, mass spectrometry sequencing is used to comprehensively analyze the protein composition of biological samples; in clinical diagnostics, it can be used to discover disease-related biomarkers. With continuous technological advancements, the sensitivity and accuracy of mass spectrometry sequencing will further improve, providing stronger support for biological research and medical diagnostics.

 

MtoZ Biolabs provides integrate protein sequencing service by mass spectrometry.