Analysis of Peptides using Asymmetrical Flow Field-flow Fractionation (AF4)

Area of Science:

• Analytical Chemistry
• Biochemistry
• Separation Science

Background:

• Asymmetrical flow field-flow fractionation (AF4) is typically used for high molecular weight species.
• The resolution of lower molecular weight species by AF4 remains underexplored.

Purpose of the Study:

• To investigate the capability of AF4 for separating low molecular weight peptides.
• To evaluate AF4 as a method for monitoring peptide aggregation.

Main Methods:

• Asymmetrical flow field-flow fractionation (AF4) was employed.
• Multi-angle laser light scattering (MALLS) detection was utilized for mass determination.
• Peptides as small as 2 kD were analyzed.

Main Results:

• AF4 demonstrated effectiveness in separating peptides from oligomers and higher molecular weight aggregates.
• Accurate parent peptide masses were obtained using AF4-MALLS with appropriate extinction coefficients.
• AF4 was shown to be a stability-indicating method.

Conclusions:

• AF4 is a viable analytical technique for resolving low molecular weight peptides.
• AF4-MALLS presents a potential alternative to size exclusion chromatography (SEC) for tracking peptide aggregation and stability.