Automated Electrokinetic Platform for High-Throughput Sodium Dodecyl Sulfate Depletion Ahead of Proteome Analysis by Mass Spectrometry

Abstract

Sodium dodecyl sulfate (SDS) provides numerous benefits for proteome sample preparation. However, the surfactant can be detrimental to downstream mass spectrometry analysis. Although strategies are available to deplete SDS from proteins, each is plagued by unique deficiencies that challenge their utility for highthroughput proteomics. An optimal approach would rapidly and reproducibly achieve less than 10 ppm residual SDS while simultaneously maximizing analyte recovery. Here, we describe improvements to a simple electrokinetic device termed transmembrane electrophoresis, which we previously reported for automated, rapid SDS depletion of proteome samples. Voltagedriven transport of SDS across a molecular weight cutoff membrane is enhanced at higher electric fields, which is herein achieved by integrating an active cooling mechanism to mitigate the impacts of Joule heating. We report 99.9% reduction of SDS (final concentration < 5 ppm) in 5 min. The device is employed in a detergent-based proteomic workflow for analysis of an enriched yeast membrane proteome extract, demonstrating quantitative protein recovery (>98%) and increasing the number of identifications by liquid chromatography-tandem mass spectrometry.

Enhanced Electrophoretic Depletion of Sodium Dodecyl Sulfate with Methanol for Membrane Proteome Analysis by Mass Spectrometry

“…The custom-built TME device, shown in Supplemental Figure S2, was assembled as described previously [33]. The device uses a 3.5 kDa regenerated cellulose dialysis membrane (Thermo Fisher Scientific) and is operated with Tris/Tricine buffer (63 mM Tris, 100 mM Tricine).…”

“…The device supplies 5 sample cells, each 3 mm in thickness and 1 cm in diameter, and can accommodate up to 250 µL of sample per cell. Unless otherwise stated, TME was operated in constant current mode (250 mA) with active cooling (AC) achieved by circulating cold water through glass channels that cool the TME buffer within the cathode and anode chambers [33]. The membrane-enriched proteome extracts, initially containing 0.5% SDS prepared in water, were loaded into each of the 5 sample cells of the TME device and subjected to SDS depletion at 250 mA for 9 min, at which point the still-soluble proteins were recovered from the device by pipetting the solution from the sample well.…”

“…FASP, for example, has proven to be highly effective in depleting SDS but suffers highly variable proteome recovery, with sample losses of 50% or more [30]. As an alternative, we introduced a fully automated approach to SDS depletion known as transmembrane electrophoresis (TME) [31][32][33]. TME employs an electric field applied perpendicular to a molecular weight cut-off (MWCO) membrane, which retains the protein while allowing dodecyl sulfate anions to migrate through the membrane and towards the anode (sodium in turn migrates towards the cathode, though this is less of a concern as a protein interference) [32].…”

“…Nonetheless, Joule heating presents a significant risk to protein loss, particularly following SDS depletion, where elevated temperatures can induce protein aggregation [31,32]. Joule heating also challenges the process of accelerated SDS removal; increasing the magnitude of the applied voltage would theoretically allow faster SDS depletion, though the resulting temperature increase would compromise protein recovery and eventually lead to the boiling of the sample solution [33]. The fastest TME depletion experiments reported to date employ an active cooling system to better manage Joule heating and maintain high protein recovery, but still require 5 min for SDS removal [33].…”