Mechanism of Peptide Sequencing by Mass Spectrometry

Mass spectrometry (MS) is a powerful technique in biological research, used to analyze the structure and function of proteins, peptides, nucleic acids, and other biological macromolecules. Through MS, we can determine peptide sequences, which is of great importance in proteomics research.

 

Basic Principles of Mass Spectrometry

The basic principle of MS is to ionize sample molecules to form charged ions, then separate and detect these ions based on their mass-to-charge ratio (m/z). A mass spectrometer mainly consists of three parts: an ionization source, a mass analyzer, and a detector.

 

1. Ionization Source

The ionization source converts sample molecules 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 analyzing large molecules in complex mixtures.

 

2. Mass Analyzer

The mass analyzer separates ions of different mass-to-charge ratios. Common mass analyzers include Quadrupole, Time-of-Flight (TOF), Ion Trap, and Fourier Transform Ion Cyclotron Resonance (FT-ICR). These analyzers separate ions based on their mass-to-charge ratios and send the results to the detector.

 

3. Detector

The detector records the ion signals passing through the mass analyzer, generating a mass spectrum. The horizontal axis of the mass spectrum represents the mass-to-charge ratio, and the vertical axis represents the ion signal intensity. By analyzing the mass spectrum, the mass-to-charge information of sample molecules can be obtained.

 

Mechanism of Peptide Sequencing

Peptide sequencing in MS involves multiple steps, including peptide generation, ionization and fragmentation of peptide segments, mass spectrometric analysis of fragment ions, and sequence inference.

 

1. Peptide Generation

Peptides are usually generated by enzymatic digestion using proteases like trypsin. Trypsin specifically cleaves protein chains at the carboxyl side of lysine and arginine residues, producing multiple peptide segments of varying lengths and compositions, providing rich information for mass spectrometric analysis.

 

2. Ionization and Fragmentation of Peptide Segments

The generated peptide segments are ionized by ESI or MALDI, forming charged ions. These ions are further selected and isolated in the mass analyzer, then enter the collision chamber for Collision-Induced Dissociation (CID). During CID, peptide ions fragment under the influence of collision gas (such as nitrogen or helium), producing a series of fragment ions.

 

3. Mass Spectrometric Analysis of Fragment Ions

The mass spectrometer analyzes the mass-to-charge ratios of the fragment ions, generating MS/MS (tandem mass spectrometry) spectra. In MS/MS spectra, b-ions and y-ions are the most common fragment types. b-ions are series ions starting from the amino end of the peptide chain, while y-ions start from the carboxyl end. By analyzing the mass-to-charge ratios of these fragment ions, the amino acid sequence of the peptide can be inferred.

 

4. Sequence Inference

Sequence inference is the final step in peptide sequencing. By analyzing the mass-to-charge ratios of b-ions and y-ions in the MS/MS spectra, the amino acid sequence of the peptide can be determined. Computer algorithms play a crucial role in sequence inference, with commonly used software including Sequest, Mascot, and X! Tandem. These programs automatically match and infer peptide sequences based on the mass-to-charge ratios and intensities of the fragment ions.

 

Applications and Prospects

Mass spectrometry has broad applications in proteomics, disease research, and drug development. With the advancement of technology, the sensitivity and accuracy of mass spectrometry are continuously improving, promising wider applications in the future.

 

Mass spectrometry determines peptide sequences through steps of ionization, mass analysis, fragmentation, and sequence inference, providing a powerful tool for biological research. MtoZ Biolabs provides integrate peptide sequencing service by mass spectrometry.