Peptide analysis by rapid, orthogonal technologies with high separation selectivities and sensitivities

Area of Science:

• Analytical Chemistry
• Biochemistry
• Separation Science

Background:

• Peptide and protein analysis is crucial for biological product standardization.
• Existing methods face challenges in sensitivity and resolution for complex mixtures.
• Advancements in separation technologies are needed for precise biomacromolecule characterization.

Purpose of the Study:

• To review the current status of peptide analysis using orthogonal micro-/nano-separation strategies.
• To highlight the synergistic benefits of combining different high-resolution separation techniques.
• To assess the potential of these methods for robust and sensitive biological product standardization.

Main Methods:

• High-performance capillary liquid chromatography (micro-HPLC)
• Capillary zonal electrophoresis (HPCZE)
• Open tubular capillary electrochromatography (OT-CEC)
• Packed capillary electrochromatography (P-CEC)
• Mass Spectrometry (MS) interfacing

Main Results:

• Orthogonal separation techniques offer complementary selectivity for complex peptide and protein mixtures.
• Interfacing separation methods with MS enables analysis of minute sample quantities.
• Staged application of these techniques facilitates robust and sensitive protocols for standardization.
• Recent theoretical advances improve prediction and optimization of separation procedures.

Conclusions:

• The combination of micro-HPLC, HPCZE, and HPCEC provides unprecedented opportunities for rapid, orthogonal, and sensitive peptide and protein separation.
• These sophisticated techniques are essential for the standardization of biological products.
• Tandem use of these technologies offers significant advantages in biomacromolecule analysis.