Kachuk et al. (2016) present an automated solution for sodium dodecyl sulfate (SDS) depletion prior to mass spectrometric (MS) proteomic analysis that works efficiently for characterizing the membrane proteome.1 Not only is the depletion step complete in an hour, but the workflow is also suitable for evaluation of both intact proteins and peptide samples post-digestion.
Researchers commonly use SDS as a denaturing agent in sample preparation for MS proteomic evaluation. However, since SDS interferes with generation of MS spectra, scientists must run samples through a depletion step, such as SDS polyacrylamide gel electrophoresis (SDS-PAGE), dialysis or column separation, to remove as much free and protein-bound agent as possible. This often results in protein loss. The additional complication for membrane proteome characterization is that this SDS depletion step frequently removes the low-abundance membrane proteins too.
Kachuk and co-authors constructed an electrophoretic device that would apply a constant electric current to separate SDS from protein across a molecular weight cut-off (MWCO) filter in solution. They built the chamber from a Teflon plate, sandwiching a membrane comprising a regenerated cellulose dialysis filter with MWCO of 3.5 kDa (Thermo Scientific). Chambers on either side were connected to buffer wells in contact with cathode or anode elements. The team ran the transmembrane electrophoresis (TME) device using constant current driven by a basic laboratory power supply. During operation, the electrical current draws the anionic surfactant away and retains purified proteins in the MWCO filter. In this way, the device does not become clogged while operating, and the electric current removes impurities.
First, the team examined the operating parameters of the device using protein solutions containing commercial bovine serum albumin (BSA) and myoglobin. They ran the device at different constant currents, and then measured SDS depletion and protein recoveries. At a constant current of 30 mA for one hour, the researchers obtained depletion to less than 100 parts per million (ppm) of SDS. They improved on this with currents of 40 mA (4.7 ± 3 ppm) and 50 mA (0.8 ± 1 ppm). However, when considering recoveries, they found that the high temperatures generated in the device during operation resulted in a reduction at 50 mA reduced recovery to 60%, whereas operating the TME device at 40 mA recovered more than 90% of the BSA stock solutions.
Their next step was to optimize the device using varying starting concentrations of both SDS and protein, which showed that the method was stable, with better operation at low protein levels.
Kachuk et al. then turned to MS analysis of intact proteins, running myoglobin samples through liquid chromatography–tandem MS (LC-MS/MS) analysis on an LTQ Linear Ion Trap mass spectrometer (Thermo Scientific) in addition to time-of-flight (TOF) MS evaluation. They found that TME completely removed SDS adducts from the sample. As a further validation, the research team examined Escherichia coli proteome extracts spiked with 0.5% SDS and found sufficient depletion for bottom-up MS analysis.
The scientists then examined performance of the depletion protocol for membrane proteomics using a standard enrichment ultracentrifugation protocol for E. coli samples. One problem arising from SDS depletion is reduced solubility for membrane proteins. Kachuk et al. overcame this obstacle by using a wash step in ice cold (−20°C) formic acid to increase protein recovery from the TME cell post-electrophoresis. Once the team confirmed suitable protein recovery, they compared LC-MS/MS data obtained from TME preparation with results from a standard chloroform/methanol/water (CMW) precipitation protocol. They found that the results from TME treatment agreed with those from the traditional method.
Kachuk et al. propose that the automated TME workflow is an efficient method for depleting SDS prior to MS evaluation of intact proteins and peptide digests. Moreover, the tool also purifies proteins and shows good recovery levels. With further development and device automation, researchers could eventually process multiple samples at once.
1. Kachuk, C., et al. (2016) “Automated SDS depletion for mass spectrometry of intact membrane proteins though transmembrane electrophoresis,” Journal of Proteome Research, 15(8) (pp. 2634–2642), doi:10.1021/acs.jproteome.6b00199.