Membrane proteins fulfill vital biological roles such as molecule/ion transport across membranes embedded within a lipid layer which needs to be extracted, solubilized and stabilized using membrane mimetics.
Among these, nanodiscs are increasingly drawing interest due to improved stability and ability to dilute to low concentrations without losing integrity or shape.
Mass Photometry and AQP4 Membrane Protein
Traditionally, mass photometry was used to examine the composition and quality of protein samples at different points during the purification process.
This process is usually followed by size exclusion chromatography (SEC) to separate the nanodisc-embedded AQP4 proteins from empty discs, aggregates and other impurities.
From the figure above, it can be observed that Fraction 3 has the greatest abundance of AQP4 singlets.
This fraction was selected to undergo a further step of affinity purification which resulted in a sample composed of mostly SMALP-embedded AQP4 singlets, smaller quantities of higher-order complexes and a minor amount of contaminant.
Identification of Desired Species
The presence of natively folded AQP4 singlets in the affinity-purified sample was confirmed by an anti-AQP4 antibody. A further analysis using mass photometry of the sample incubated with the anti-AQP4 antibody showed a new population at ≈449 kDa, corresponding to anti-AQP4 antibodies bound to nanodisc-embedded AQP4 singlets.
Following this, a negative control using a non-specific antibody showed a similar distribution to the untreated sample. The combination of both experiments as shown in figure below confirm that natively folded AQP4 is present in the sample.
Mass photometry addresses challenges met during the purification of membrane protein, as it requires a small amount of sample and is largely compatible with most membrane mimetics.
It is worth noting that SMALP’s are known to have variable sizes.
Nevertheless, the masses of the complexes identified here are consistent with theoretical predictions, and the SMALP and protein masses of AQP4 singlets match the results of other analytical methods like SEC and SMA-PAGE.
To conclude, it can be said that mass photometry provides valuable information about complex samples, which greatly helps in making decisions during membrane protein purification.
Systems for Research: Your Guide to Mass Photometry in Canada
Systems for Research is the exclusive representative for Refeyn in Canada. We work with Refeyn to make mass photometry instruments available to our customers all across Canada.
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