Principle of Peptide Sequencing by Mass Spectrometry

Mass spectrometry (MS) is a critical analytical technique in contemporary biological research, extensively utilized in proteomics, metabolomics, and clinical diagnostics. Peptide sequencing by mass spectrometry involves detecting and analyzing protein samples with a mass spectrometer to determine their peptide sequences.

 

Basic Components of a Mass Spectrometer

A mass spectrometer consists of three main parts: the ion source, the mass analyzer, and the detector.

 

1. Ion Source

This component ionizes sample molecules to produce charged ions. Common ion sources include Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption/Ionization (MALDI).

 

2. Mass Analyzer

It separates ions based on their mass-to-charge ratio (m/z). Common mass analyzers include quadrupoles, time-of-flight (TOF), and ion traps.

 

3. Detector

This part detects the separated ions and produces a mass spectrum.

 

Ionization and Fragmentation of Peptides

In mass spectrometry sequencing, proteins are first digested into smaller peptides, typically using enzymatic digestion. These peptides are then ionized in the mass spectrometer's ion source to create charged peptide ions. Common ionization methods include:

 

1. Electrospray Ionization (ESI)

Liquid samples are nebulized under high voltage, ionizing the peptides during the spray process.

 

2. Matrix-Assisted Laser Desorption/Ionization (MALDI)

The matrix is excited by a laser, transferring energy to the peptides and ionizing them.

 

The ionized peptides are separated in the mass analyzer and further fragmented into smaller ion fragments using methods like Collision-Induced Dissociation (CID). These fragmentation methods produce different types of ions, such as b-ions and y-ions, which carry the peptide sequence information.

 

Collection and Analysis of Mass Spectrometry Data

Once the ions are detected by the mass spectrometer, a mass spectrum is generated, with each peak representing an ion with a specific m/z ratio. Determining the peptide sequence involves several steps:

 

1. Peak Identification in the Mass Spectrum

Identifying all peaks and recording their m/z ratios and intensities.

 

2. Peptide Sequence Inference

Inferring the peptide sequence by matching the peaks to known fragmentation patterns using software tools like Mascot or Sequest.

 

3. Data Validation and Result Interpretation

Validating the inferred peptide sequences to ensure accuracy and applying the results to subsequent biological research.

 

Advances in mass spectrometry technology have greatly enhanced the accuracy and sensitivity of peptide sequence analysis, providing a robust tool for proteomics research. MtoZ Biolabs provides integrate peptide sequencing service by mass spectrometry.