Investigations of Parameters Impacting Transmembrane Electrophoresis for Improved SDS Depletion of Proteome Samples for Mass Spectrometry

Abstract

Sodium dodecyl sulfate (SDS) is a favoured reagent for proteomic sample preparation but detrimental to down-stream analysis by liquid chromatography and mass spectrometry (LC-MS). This group previously introduced a novel electrophoretic device termed transmembrane electrophoresis (TME), which depletes SDS to levels permitting LC-MS (≤100 ppm) while maintaining high protein yield (Kachuk et al. J. Proteome Res. 2016, p2634). Initially, TME required lengthy runs (1 hour), and frequent pausing/manual mixing to prevent overheating. As such, this thesis presents TME modifications to allow operation at higher currents, which increases the rate of SDS depletion. Changing different membrane parameters provides lower resistance thereby decreasing Joule heating. Operation at constant current (instead of constant voltage or power) is determined to be optimal by minimizing heat production. Increasing beyond 50 mA demands additional strategies for temperature management. Here, TME operation with a stir bar to agitate the sample cell is presented as a fully automated alternative to pausing/manual mixing. Constant stirring is shown to significantly reduce sample temperatures, allowing operation at higher constant current (70 mA). In doing so, the time required for SDS depletion below 100 ppm is reduced to ≤15 min. Reduced temperatures also translate to higher sample yields ≥95%. The stir bar also reduces concentration polarization and membrane fouling phenomena which reduces resistance and increases depletion rates. Finally, the application of the improved TME platform to bottom-up and top-down workflows is assessed in comparison to two popular depletion methods, acetone precipitation and filter-aided sample preparation (Wiśniewski et al. Nat. Methods 2009, p359). All methods successfully deplete SDS from a proteome mixture to ≤10 ppm, though TME provides consistently higher sample yields (≥95%). TME also gives higher peptide spectral matches per protein when analyzed by LC-MS, and favours the identification of low molecular weight proteins, which are often underrepresented in proteomics studies.