Method for Determining the Sequence of Cyclic Peptides
Cyclic peptides are peptides with a circular structure, and their sequence determination is of significant importance in drug development and protein engineering. The methods for sequencing cyclic peptides are relatively complex, with common techniques including chemical degradation, mass spectrometry, nuclear magnetic resonance (NMR), and enzymatic digestion. Below is a detailed introduction:
1. Chemical Degradation Method
Edman Degradation
(1) Steps:
① Ring Opening: Use acidic hydrolysis or alkaline conditions to open the cyclic peptide and convert it into a linear peptide.
② Edman Degradation: Use phenyl isothiocyanate (PITC) to sequentially degrade the peptide chain, releasing one amino acid at a time from the N-terminus, and determine the amino acid released at each step.
(2) Advantages: Suitable for determining short peptide sequences.
(3) Disadvantages: The accuracy and efficiency of Edman degradation are lower for long-chain peptides and peptides with numerous modifications.
2. Mass Spectrometry (MS)
1. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)
(1) Steps:
① Sample Preparation: Mix the cyclic peptide sample with a matrix.
② Laser Desorption/Ionization: Use a laser to excite the sample, causing ionization.
③ Mass Spectrometry: Measure the mass-to-charge ratio of ions using a time-of-flight mass spectrometer.
④ Data Analysis: Infer the sequence of the cyclic peptide based on the mass spectrum, using databases or sequence inference software.
(2) Advantages: High sensitivity, suitable for complex mixtures.
(3) Disadvantages: Requires complex data analysis, and multiple ionization forms may exist.
2. Liquid Chromatography-Mass Spectrometry (LC-MS/MS)
(1) Steps:
① Sample Separation: Use liquid chromatography (LC) to separate the cyclic peptide sample.
② Mass Spectrometry: Measure the separated peptide fragments using tandem mass spectrometry (MS/MS).
③ Data Analysis: Determine the peptide fragment sequences through database matching or sequence inference software.
(2) Advantages: Can determine the sequences of multiple cyclic peptides simultaneously, suitable for complex samples.
(3) Disadvantages: Complex data processing, requires high instrument sensitivity.
3. Nuclear Magnetic Resonance (NMR)
Two-Dimensional Nuclear Magnetic Resonance (2D NMR)
(1) Steps:
① Sample Preparation: Dissolve the cyclic peptide in an appropriate solvent.
② NMR Measurement: Conduct two-dimensional NMR experiments (such as COSY, NOESY, TOCSY, etc.).
③ Data Analysis: Analyze the 2D NMR spectra to determine interactions between amino acid residues, thereby inferring the peptide sequence.
(2) Advantages: No need for sample degradation, suitable for structurally complex cyclic peptides.
(3) Disadvantages: Requires high-purity samples and high-resolution NMR instruments, data analysis is complex.
4. Enzymatic Digestion Method
Enzymatic Digestion Combined with Mass Spectrometry
(1) Steps:
① Sample Digestion: Use specific enzymes (such as trypsin, chymotrypsin, etc.) to digest the cyclic peptide, generating small peptide fragments.
② Mass Spectrometry: Measure the generated small peptide fragments using a mass spectrometer.
③ Data Analysis: Infer the sequence of the cyclic peptide based on mass spectrometry data and enzyme specificity.
(2) Advantages: Can handle larger cyclic peptides, simple enzymatic digestion steps.
(3) Disadvantages: Requires selecting appropriate enzymes, and the complexity of digestion products may increase the difficulty of data analysis.
The sequencing of cyclic peptides usually requires a combination of multiple methods, selecting the most suitable technique based on the sample's characteristics and experimental needs. Chemical degradation is suitable for shorter cyclic peptides, mass spectrometry is suitable for complex samples, NMR is suitable for high-purity samples, and enzymatic digestion is suitable for larger cyclic peptides. Through reasonable experimental design and data analysis, the sequence of cyclic peptides can be accurately determined, providing fundamental data for their application in the biomedical field.
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