Principle of Peptide Analysis

Peptides are short chains of amino acids linked by peptide bonds, typically consisting of 2 to 50 amino acid residues. As subunits of proteins, peptides play crucial roles in biological processes, such as signal transduction, immune response, and metabolic regulation. Therefore, studying peptides is of great importance for understanding protein functions, disease mechanisms, and drug development. Peptide analysis, which involves the separation, identification, and quantification of peptides using advanced analytical techniques, is based on principles involving chemical reactions, separation techniques, and mass spectrometry analysis.

 

Separation Principles

The first step in peptide analysis is the separation of peptides from the sample, which is essential for subsequent identification and quantification. Commonly used separation methods include liquid chromatography (LC) and capillary electrophoresis (CE). Liquid chromatography separates peptides based on their affinity differences between a stationary phase and a mobile phase. Reversed-phase high-performance liquid chromatography (RP-HPLC) is the most commonly employed technique for peptide separation, which exploits the hydrophobicity differences among peptide molecules to achieve stepwise separation. Additionally, capillary electrophoresis relies on the differences in migration speed of charged peptides under an electric field in a capillary tube, providing high-resolution separation.

 

Identification Principles

After peptide separation, identifying the peptide sequence is a key task in peptide analysis. Mass spectrometry (MS) is the most widely used identification tool due to its high sensitivity and precision. The basic principle of mass spectrometry involves ionizing peptide molecules into charged ions, which are then separated and detected based on their mass-to-charge ratio (m/z) in an electric field. In peptide analysis, tandem mass spectrometry (MS/MS) is commonly employed, where peptides are first ionized using techniques such as electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI). The resulting ions are then subjected to collision-induced dissociation (CID) or high-energy collision dissociation (HCD) in a second mass spectrometer to generate characteristic fragment spectra. By comparing these spectra with known databases, peptide sequences can be determined.

 

Quantification Principles

Quantification of peptides is an indispensable part of peptide analysis. Common quantification methods include absolute quantification and relative quantification. Absolute quantification typically involves adding an internal standard peptide with a known concentration, often isotopically labeled, and comparing its signal to that of the target peptide to precisely measure the peptide concentration. Relative quantification methods often rely on labeling techniques, such as Isobaric Tags for Relative and Absolute Quantification (iTRAQ) or Tandem Mass Tags (TMT). These labels are chemically attached to the N-terminus or lysine residues of peptides, allowing the relative intensities of peptide signals from different samples to be measured by mass spectrometry, thus inferring peptide expression levels across samples.