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Analysis of αB-crystallin polydispersity in solution through native microfluidic electrophoresis

Authors: Maya A. Wright, Francesco Simone Ruggeri, Kadi L. Saar, Pavan K. Challa, Justin L. P. Benesch and Tuomas P. J. Knowles

Analyst, 2019,144, 4413-4424. DOI: 10.1039/C9AN00382G

 

Abstract

In recent years, significant advancements have been made in the understanding of the population distributions and dynamic oligomeric states of the molecular chaperone αB-crystallin and its core domain variants. In this work, Wright et al.,  provide solution-phase evidence of the polydispersity of αB-crystallin using microfluidic methods, used for separating the oligomeric species present in solution according to their different electrophoretic mobilities on-chip in a matter of seconds.

They in particular demonstrate that microfluidic high-field electrophoresis and diffusion can detect the oligomerisation of these highly dynamic molecular chaperones and characterise the dominant oligomeric species present. They thereby provide a robust microfluidic method for characterising the individual species within complex protein mixtures of biological relevance.

In this paper, the authors utilise microfluidic diffusional sizing (MDS) and electrophoresis to characterise the oligomeric states of the chaperone protein alphaB-crystallin. The MDS and electrophoresis measurements confirm that both wild-type and truncated variants of alphaB-crystallin have different degrees of oligomerisation in the solution phase. In addition, insights into the distribution and specific species present in heterogeneous alphaB-crystallin mixtures could be obtained by combining electrophoresis data with spectroscopic analysis techniques.

wright et al fig 2

Figure 1: The high-field electrophoresis device. Schematic of the device. Electrolyte solution (yellow) is infused into the device via syringes and pumps, while sample and carrier medium stored in gel loading tips are introduced by withdrawing from the outlets using a flow splitter. A voltage is applied at the outlets and is carried into the device via the electrolyte solution (yellow), resulting in an electric field being applied across the main channel. Deflection of the analyte is imaged at the position highlighted by the orange square. The image in the orange square corresponds to BSA at 1 mg mL−1. The image in the green square is a close up the electrolyte channels filled with 3 M KCl plus BSA 2 mg mL−1 for visualisation.

 

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Instrument
Therapeutic area: protein oligomerization

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