Peptide purity analysis is typically performed using High-Performance Liquid Chromatography (HPLC). The primary objective of mass spectrometry analysis is to confirm the molecular weight; currently, the main techniques employed for peptide analysis are Electrospray Ionization Mass Spectrometry (ESI-MS) and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS).
Amino acid composition analysis reveals the types and quantities of amino acids present in a peptide, while the fundamental principle underlying amino acid sequence analysis is Edman degradation.
Methods for identifying peptide secondary structures include Circular Dichroism (CD), Nuclear Magnetic Resonance (NMR), X-ray diffraction, among others.
During the solid-phase synthesis process, various impurities-such as deletion sequences, insertion sequences, mis-coupled peptides, epimerized peptides, and oxidized peptides-may be generated. The identification of such peptide-related impurities is typically accomplished using Liquid Chromatography-Tandem Mass Spectrometry (LC/MS/MS); LC-MS enables the accurate determination of a peptide's mass, while MS/MS is utilized to elucidate its sequence. Additionally, HPLC chromatographic techniques can be employed to achieve effective separation and detection.
In solid-phase peptide synthesis, coupling efficiency is primarily assessed by detecting the presence of free amino groups on the resin; this detection method is known as the Kaiser test (or Ninhydrin test). If free amino groups are present, a blue or reddish-brown color develops.