Evaluation of the Application of Sodium Deoxycholate to Proteomic Analysis of Rat Hippocampal Plasma Membrane

Abstract

Detergents have been widely used for the solubilization of membrane proteins and the improvement of their digestion. In this paper, we have evaluated the application of sodium deoxycholate (SDC) to the solubilization and digestion of rat hippocampal plasma membrane (PM) proteins. For in-solution digestion, rat hippocampal PM fraction from sucrose-density gradient centrifugation was solubilized by boiling in 1.0% SDC, and directly digested without dilution. During the in-gel digestion of the hippocampal PM proteins separated by SDS-PAGE, 0.1% SDC was added. Before analysis of peptide mixture by liquid chromatography and electrospray mass spectrometry, SDC in the tryptic digests was removed by centrifugation following acidification. Use of 1.0% SDC in solubilization and in-solution digestion of rat PM proteins had led to 77 PM or membrane-associated proteins identified, a more than 2-fold increase over that by use of SDS. The addition of 0.1% SDC to the in-gel digestion of SDS-PAGE-resolved membrane proteins remarkably enhanced the coverage of tryptic peptides and the number of hydrophobic membrane proteins identified. Being a cheaper and more tractable acid-insoluble detergent, SDC could be used at higher concentration in the solubilization and tryptic digestion of proteins including PM proteins with the purpose of enhancing the protein solubility and at the same time making no interference with trypsin activity and subsequent analyses.

Sample preparation for the analysis of membrane proteomes by mass spectrometry

“…Furthermore, because SDC precipitates at low pH, it can be removed from the digests by centrifugation following acidification prior to sample analysis, preventing any potential ion suppression Lin et al, 2010). In addition, Zhou et al (2006) also demonstrated that during in-gel digestion the addition of 0.1% SDC in the buffer could remarkably enhance the coverage of tryptic peptides and the number of hydrophobic membrane proteins identified.…”

“…However, sodium deoxycholate (SDC) was found to be compatible with trypsin activity, with enzyme activity not being significantly influences by concentrations up to 2% Lin et al, 2008). Zhou et al (2006) evaluated the use of SDC in place of 0.1% SDS for on-membrane digestion of rat hippocampal plasma membrane fraction as assessed by MALDI-TOF MS and CapLC-MS/MS. The use of SDC resulted in more total membrane protein identifications (71 vs. 31) and more integral membrane proteins (IMPs) (50 vs. 22) by CapLC-MS/MS.…”

Exploiting the multiplexing capabilities of tandem mass tags for high-throughput estimation of cellular protein abundances by mass spectrometry

“…The sequential double Lys--C/trypsin digestion has been recently shown to be more efficient in generating fully cleaved peptides than tryptic digest alone [30] and is therefore recommended for all protein samples analyzed by LC--MS. Sodium deoxycholate is one of a few LC--MS compatible detergents, since it precipitates after acidification [25,33,38] using 2M HCl to a final concentration of 50mM and can then be easily removed by centrifugation at 10,000g for 15min before LC--MS analysis. All peptide samples were then desalted by C18 reversed--phase spin columns according to the manufacturer's instructions (Macrospin, Harvard Apparatus), separated in aliquots of 25 ug peptides, dried under vacuum and stored at --80ºC until further use.…”

Properties of graphite-stainless steel composite in bipolar plates in simulated anode and cathode environments of PEM fuel cells

“…The most popular materials of bipolar plates, besides graphite, are metallic materials or composites based on carbon: carboncarbon composites [25,26], carbon-polymer composites [27], and carbon coated metal [27][28][29][30]. Metallic bipolar plates provide many advantages such as cheaper material cost, high thermal and electrical conductivity, variety of manufacturing processes and excellent mechanical properties.…”