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    Principle of Peptide Sequencing

      Peptide sequencing refers to the process of determining the sequence of amino acid residues in peptides or protein molecules. This process is critical for the study of protein structure and function, as the sequence of amino acids directly determines the three-dimensional structure and biological function of the protein. Peptide sequencing mainly relies on Mass Spectrometry (MS), especially Tandem Mass Spectrometry (MS/MS). The principles of mass spectrometry sequencing are listed as follows.


      Ionization of Peptides

      In mass spectrometry analysis, the peptide sample needs to be ionized first. Ionization is the process of converting peptide molecules into charged particles, and this step is critical for subsequent mass spectrometry detection. Common ionization methods include Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption/Ionization (MALDI).


      Separation of Ions

      After ionization, the peptide ions are introduced into the mass spectrometer and separated by the mass analyzer. The mass analyzer separates them based on their mass-to-charge ratio (m/z). Common mass analyzers include Time-of-Flight (TOF), Ion Trap, and Fourier Transform Ion Cyclotron Resonance (FT-ICR).


      Fragmentation of Peptides

      In MS/MS analysis, selected precursor ions (i.e., peptide ions of a particular m/z ratio) are further fragmented. Fragmentation refers to the controlled breaking of peptide ions to produce a series of fragment ions that represent peptide sequence information. Common fragmentation techniques include Collision-Induced Dissociation (CID), Electron Transfer Dissociation (ETD), and Photon-Induced Dissociation (PID).


      Detection of Fragment Ions

      The fragmented ions are then introduced into a second mass analyzer for detection and analysis. By analyzing the m/z values of these fragment ions, information about the peptide amino acid sequence can be obtained.


      Sequence Analysis and Identification

      Finally, using bioinformatics software and database matching techniques, the original peptide amino acid sequence is inferred based on the mass-to-charge ratio and pattern of the fragment ions. This step usually involves comparison with a known protein database to identify the sequence of unknown peptides or proteins.


      The accuracy and success rate of peptide sequencing are influenced by many factors, including the purity of the sample, the efficiency of ionization, the fragmentation method, and the resolution and accuracy of the mass spectrometer. With the continuous development and optimization of mass spectrometry technology, peptide sequencing has become an indispensable tool in proteomics and biomolecular research.

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