How to Verify a Peptide After Synthesis and Function Prediction
Verification following synthesis and functional prediction can be conducted from the following perspectives:
Peptide Identification
1. Mass Spectrometry (MS): Applied to determine the molecular weight of the peptide and confirm whether the synthesized peptide corresponds to the predicted sequence.
2. Amino Acid Sequence Analysis: Tandem mass spectrometry (MS/MS) is used to analyze the amino acid sequence of the peptide, ensuring the sequence is accurate and complete.
3. High-Performance Liquid Chromatography (HPLC): Utilized to assess the purity level of the synthesized peptide.
Peptide Function Verification
1. In Vitro Assays
Cell-based experiments can be conducted to evaluate peptide bioactivity, including assays for cell proliferation, apoptosis, and activation of signaling pathways.
(1) Enzyme Activity Assays: If the peptide is predicted to possess enzymatic inhibitory or activating functions, substrate conversion assays can be employed to quantify its activity.
(2) Binding Assays: In cases where the predicted function involves interaction with proteins or nucleic acids (DNA/RNA), techniques such as co-immunoprecipitation, yeast two-hybrid analysis, and electrophoretic mobility shift assays (EMSA) can be used to examine binding affinity and specificity.
2. In Vivo Assays (Optional)
If the peptide is predicted to exert biological activity in vivo, its function may be further validated using appropriate animal models. This includes pharmacokinetic evaluations (e.g., absorption, distribution, metabolism, excretion) and pharmacodynamic studies (e.g., dose-response profiles, therapeutic efficacy, and potential side effects).
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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