Delicate Analysis of Interacting Proteins and Their Assemblies by Flow Field-Flow Fractionation Techniques

TL;DR

This paper evaluates different AF4 techniques to analyze wheat gluten proteins, revealing how buffer choice and device configuration affect protein aggregation and separation of complex assemblies.

Contribution

It introduces a comparative analysis of AF4 methods with different eluents and devices for studying wheat gluten protein assemblies.

Findings

Frit-inlet device improves fractionation of wheat proteins in SDS buffer.

Water/ethanol eluent effectively separates gluten proteins and their assemblies.

Conventional AF4 with SDS buffer can induce aggregation during focus steps.

Abstract

We study the efficiency of several Asymmetrical Flow Field-Flow Fractionation (AF4) techniques to investigate the self-associating wheat gluten proteins. We compare the use of a denaturing buffer including sodium dodecyl sulfate (SDS) and a mild chaotropic solvent, water/ethanol, as eluent, on a model gluten sample. Through a thorough analysis of the data obtained from coupled light scattering detectors, and with the identification of molecular composition of the eluted protein, we evidence co-elution events in several conditions. We show that the focus step used in conventional AF4 with the SDS buffer leads to the formation of aggregates that co-elute with monomeric proteins. By contrast, a frit-inlet device enables the fractionation of individual wheat proteins in the SDS buffer. Interestingly conventional AF4, using water/ethanol as eluent, is an effective method for fractionating gluten proteins and their complex dynamic assemblies which involve weak forces and are composed of both monomeric and polymeric proteins.