MSACL 2023 Abstract

Introduction:
Membrane proteins have multiple critical physiological roles including signal transduction, membrane trafficking, subcellular compartmentalization, and protein secretion. These functions lend membrane proteins as attractive targets for drug therapeutics. Despite their clinical significance, membrane proteins are notoriously underrepresented in proteomics workflows due to their poor solubility.
To improve the solubility of membrane proteins, previous groups have employed mass spectrometry (MS) compatible surfactant, as well as methanol (60%). Despite these approaches, sodium dodecyl sulfate (SDS) is still favored for the solubilization of membrane proteins. The addition of SDS however is known to impose challenges in downstream processing, as it deteriorates reverse-phase separation and suppresses MS ionization. Our group has developed an electrophoretic approach known as transmembrane electrophoresis (TME) to deplete SDS while maintaining a high protein yield (>95%) in 5 minutes 1,2.

Objectives:
The current project employs a solvent system tailored to membrane proteins to accelerate SDS depletion while maintaining the higher solubility of hydrophobic proteins.

Method:
Methanol and SDS were added to an S. cerevisiae membrane proteome extract in varying quantities. Samples were then depleted of SDS in a custom-built TME device 2. BCA assay was used to quantify proteins recovered after a TME run, while MBAS assay was used to quantify the residual SDS in the sample. SDS PAGE was also used to further verify the quantity of proteins recovered. Samples were then subjected to an orbitrap MS for profiling. The resultant spectrum was searched against a uniport database for protein count and identification.

Results:
BCA assay, SDS PAGE, and MS results have suggested that the inclusion of 40% methanol improved protein recovery. Increasing the methanol concentration beyond 40% caused a reduction in the recovery. Measurement of residual SDS in the sample using an MBAS assay after a TME run showed that the higher the methanol concentration the higher the rate of SDS depletion. Lastly, 223 hydrophobic proteins have been identified in the 40% methanol that was not present in 0% methanol, 192 of which are membrane proteins of the cell and mitochondria.

Conclusion:
The inclusion of 40% methanol has increased the SDS depletion rate in the TME along with higher recovery of membrane proteins.

References
1- Kachuk, C.; Doucette, A. A. J. Proteomics 2018, 175, 75−86.
2- Jakubec, P.; Doucette, A. Automated Electrokinetic Platform for High-Throughput Sodium Dodecyl Sulfate Depletion Ahead of Proteome Analysis by Mass Spectrometry. Anal. Chem. 2021, 93(42), 14042–14047.