A simple apparatus for electrokinetic removal of sodium dodecyl sulfate from protein digests.

Sodium dodecyl sulfate (SDS) in proteomics samples needs to be removed and estimated prior to mass spectrometry (MS)-based analysis and to avoid MS ion-source contamination. Here, we describe an organic solvent free method to remove SDS using a simple apparatus that mainly consists of an agarose gel inside a 1 mL plastic micropipette tip and a voltage power supply with electrodes. A small volume of sample (e.g., 50 µL) is loaded on top of the gel and then voltage (cathode at the sample side) is applied with an acidic solution at the other end of the micropipette tip. Within 25 min, SDS was removed (e.g., ≥99% SDS in 3.5 mM SDS) and the peptides were retained in the sample solution. The strategy was compared to the commercially available and expensive Pierce spin column for the removal of SDS and recovery of peptides from a digested bovine serum albumin sample.

Protein Extraction from Gels: A Brief Review.

Protein gel electrophoresis is an important procedure carried out in protein studies. Elution and recovery of proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are often necessary for further downstream analyses. The process involves localizing the protein of interest on the gel following SDS-PAGE, eluting the protein from the gel, removing SDS from the eluted sample, and finally renaturing the protein (e.g., enzymes) for subsequent analyses. Investigators have extracted proteins from gels by a variety of techniques. These include dissolution of the gel matrix, passive diffusion, and electrophoretic elution. Proteins eluted from gels have been used successfully in a variety of downstream applications, including protein chemistry, proteolytic cleavage, determination of amino acid composition, polypeptide identification by trypsin digestion and matrix-assisted laser desorption ionization-time of flight mass spectroscopy, as antigens for antibody production, identifying a polypeptide corresponding to an enzyme activity, and other purposes. Protein yields ranging from nanogram levels to 100 µg have been obtained. Here, we review some of the methods that have been used to elute proteins from gels.

Membrane-Free Electrokinetic Device Integrated to Electrospray-Ionization Mass Spectrometry for the Simultaneous Removal of Sodium Dodecyl Sulfate and Enrichment of Peptides.

The removal of sodium dodecyl sulfate (SDS) in SDS-assisted proteomics with electrospray-ionization-mass-spectrometric (ESI-MS) analysis is an essential step in the analysis. Off-line state-of-the-art sample-preparation strategies can allow 100% removal of DS- and up to 100% peptide recoveries. These strategies, however, are typically laborious and require long analysis times and a complex experimental setup. Here, we developed a simple, membrane-free, electrokinetic, on-line, integrated SDS removal-ESI-MS device that was able to enhance ESI-MS signals of bradykinin and peptides from trypsin-digested bovine serum albumin (BSA) in samples that contained SDS micelles. The significant peptide-signal improvements were contributed by the complete removal of DS- and the enrichment of the peptides in the presence of an electric field. Enrichment was via micelle-to-solvent stacking, initially developed in capillary electrophoresis. Bradykinin percent recovery was 800%, and BSA peptide percent recovery was 87%. Enhancement factors in ESI-MS signals (after and before removal) for selected m/ z values of peptides from the BSA digest were 535-693.

Online Removal of Sodium Dodecyl Sulfate via Weak Cation Exchange in Liquid Chromatography-Mass Spectrometry Based Proteomics.

Biological research often requires the use of sodium dodecyl sulfate (SDS) to solubilize protein samples; however, this detergent is not compatible with direct mass spectrometry (MS) analysis. Here, we report an online high-throughput proteomics method that permits standard in-solution digestion of SDS-containing samples followed by direct liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis using weak cation-exchange chromatography (WCX). This approach, called the online removal of sodium dodecyl sulfate (Online reSDS), exploits the properties of WCX in a highly organic and mildly acidic medium to retain positively charged peptides by both hydrophilic interaction and electrostatic attraction while simultaneously repelling negative SDS molecules. This method was optimized to successfully analyze complex samples that contain up to 1% of SDS. Furthermore, online reSDS improves the identification of peptides with post-translational modifications (PTMs), such as deamidation and phosphorylation, without preliminary enrichment. In conclusion, we show that reSDS can facilitate research in proteomics by allowing the use of SDS in a wide range of LC-MS/MS applications with simplified sample-processing procedures.

Sodium dodecyl sulfate removal during electrospray ionization using cyclodextrins as simple sample solution additive for improved mass spectrometric detection of peptides.

Sodium dodecyl sulfate (SDS) removal is a vital procedure in SDS-assisted bottom-up proteomics because SDS affects the quality of the data in electrospray ionization mass spectrometry (ESI-MS). SDS removal methods provide efficient removal of SDS and improved peptide analysis, but would usually require time, specialised devices, and experienced analysts. Here, by simple addition of γ-cyclodextrin (γ-CD) to the solution at concentrations 1 to 2x the SDS in the sample, the SDS related signals in positive ionization ESI-MS can be significantly removed (70-99% reduction), without an additional sample manipulation step of extraction or purification. The mechanism for removal is based on the formation of tightly bound CD-SDS inclusion complexes, which hampered the generation of positively charged SDS multimers during ESI. For a sample with peptides (glu-val-phe, tyr-tyr-tyr, and bradykinin) and 3 mM SDS where 6 mM γ-CD was added, the %signal recoveries of peptides calculated by comparison with signals from standard samples without SDS were 49-59%. The space charge effect by SDS on bradykinin was also reduced, increasing the signal for bradykinin 12x in the presence of γ-CD. For a protein (bovine serum albumin, BSA) digest with 3 mM SDS, which is an expected concentration in trypsin treated samples, a noticeable 7-fold improvement in the peptide to SDS signal ratio and a 91% reduction of SDS signals were observed upon addition of 6 mM γ-CD. However, there were only small changes in the ESI-MS intensities for the BSA peptides (compared to without addition of γ-CD). This new approach to SDS signal removal using CDs in ESI-MS may find use in proteomic studies.

Electrokinetic Removal of Dodecyl Sulfate Micelles from Digested Protein Samples Prior to Electrospray-Ionization Mass Spectrometry.

In proteomics, dodecyl sulfate (DS-) as sodium salt is commonly used in protein solubilization prior to tryptic digestion, but the presence of the DS- hampers the electrospray ionization mass spectrometric (ESI-MS) analysis. The development of DS- depletion techniques is therefore important especially when dealing with small samples where there could be poor sensitivity due to sample loss or dilution during sample preparation. Here, we present a simple and fast electrokinetic removal method of DS- from small volumes of peptide and digested protein samples prior to ESI-MS. The selective removal was accomplished using an acidic extraction solution (ES) containing acetonitrile (ACN) inside a fused-silica capillary that was dipped into the sample. The use of acidic ES suppressed the electroosmotic flow; allowing the electrokinetic movement of DS- monomers and micelles into the capillary. The high amount of ACN present at the tip of the capillary served to collapse the micelles migrating into the capillary, thereby releasing the peptides that were bound to these micelles, facilitating peptide retention in the sample and efficient DS- removal. Increased % MS signal intensity (SI) restoration of the peptide was observed, while DS- removal was unaffected when the amount of ACN in the ES was increased. This is because of the micelle to solvent stacking mechanism (effective electrophoretic mobility reversal) working at high concentration of ACN for the improved recovery of the peptides. % MS SI restoration for the Z-Gly-Gly-Val and bradykinin peptides were 75-83% while % MS SI reduction of DS- was up to 99% under optimal conditions, that is, 40% ACN in the ES. Higher % peptide recoveries from digested protein samples were obtained using the proposed method compared to the conventional cold acetone precipitation method.

Removal of sodium dodecyl sulfate from protein and peptide samples with cross-linked [Os(dmebpy)2 Cl](+/2+) -derivatized acrylamide and vinylimidazole copolymer.

RATIONALE: Sodium dodecyl sulfate (SDS) is widely used for the solubilization and denaturation of proteins, but it interferes with liquid chromatography/mass spectrometry (LC/MS), suppressing protein signals or forming adduct ions. A quick and effective clean-up technique of SDS is essential for MS analysis of proteins. Ion-exchange spin columns are commonly used for SDS removal in protein samples. METHODS: A bulk sample of insoluble, cross-linked [Os(dimethylbipyridine)2 Cl](+/2+) -derivatized poly(acrylamide)-poly(vinylimidazole) copolymer was synthesized and broken into small particles. The polymer was activated by washing with 1:1 ACN/water 50 mM triethylammonium phosphate 0.05% TFA, 0.1% TFA ACN and then 0.1% TFA water. Under acidic aqueous conditions, SDS adsorbs on the activated surfaces of the Os-complexed copolymer particles, but not the proteins and peptides in the same mixtures. Thus, the copolymer can be used to remove SDS from protein and peptide samples. The copolymer-adsorbed SDS is removed by washing with 0.1% TFA ACN, permitting re-use of the copolymer. RESULTS: Standard myoglobin and some practical protein samples from a biochemistry lab spiked with different concentrations of SDS were successfully cleaned up using this Os-copolymer for LC/MS analyses. Up to 0.2% (w/v %) of SDS can be successfully removed from those protein samples. CONCLUSIONS: This Os-complexed copolymer provides a new alternative for quick cleanup of SDS from protein samples, and can serve as a new class of metal complex based anion exchanger for protein purification.

Simple sodium dodecyl sulfate-assisted sample preparation method for LC-MS-based proteomics applications.

Sodium dodecyl sulfate (SDS) is one of the most popular laboratory reagents used for biological sample extraction; however, the presence of this reagent in samples challenges LC-MS-based proteomics analyses because it can interfere with reversed-phase LC separations and electrospray ionization. This study reports a simple SDS-assisted proteomics sample preparation method facilitated by a novel peptide-level SDS removal step. In an initial demonstration, SDS was effectively (>99.9%) removed from peptide samples through ion substitution-mediated DS(-) precipitation using potassium chloride (KCl), and excellent peptide recovery (>95%) was observed for <20 µg of peptides. Further experiments demonstrated the compatibility of this protocol with LC-MS/MS analyses. The resulting proteome coverage obtained for both mammalian tissues and bacterial samples was comparable to or better than that obtained for the same sample types prepared using standard proteomics preparation methods and analyzed using LC-MS/MS. These results suggest the SDS-assisted protocol is a practical, simple, and broadly applicable proteomics sample processing method, which can be particularly useful when dealing with samples difficult to solubilize by other methods.

Integrated SDS removal and peptide separation by strong-cation exchange liquid chromatography for SDS-assisted shotgun proteome analysis.

We report an improved shotgun method for analyzing proteomic samples containing sodium dodecyl sulfate (SDS). This method is based on the use of strong-cation exchange (SCX) liquid chromatography (LC) for SDS removal that can be integrated with peptide separation as the first dimension of the two-dimensional LC tandem mass spectrometry workflow. To optimize the performance of SDS removal, various experimental conditions, including the concentrations of chemical reagents and salts in the sample, the SDS concentration, and the SCX mobile phase composition, were investigated. It was found that a peptide recovery rate of about 90% could be achieved while removing SDS efficiently. One key finding was that, by increasing the SDS concentration to a certain level (0.5%) in the digested peptide sample, the sample recovery rate could be increased. The peptide recovery rate of BSA digests was found to be 90.6 ± 1.0% (n = 3), and SDS in the SCX fractions collected was not detectable by pyrolysis GC-MS, i.e., below the detection limit of 0.00006% for the undesalted SCX fractions. The peptide recovery rates were found to be 90.9% ± 2.7 (n = 3) and 89.5% ± 0.5% (n = 3) for the digests of the membrane-protein-enriched fractions of E. coli cell lysates and the MCF-7 breast cancer cell line, respectively. Compared to the methods that use acid-labile surfactants, such as RapiGest and PPS, for the MCF-7 membrane fraction sample, the SDS method identified, on average (n = 3), more peptides (∼5%) and proteins (∼16%) than the RapiGest method, while the RapiGest method identified more peptides (∼21%) and proteins (∼7%) from the E. coli membrane fraction than the SDS method. In both cases, the two methods identified more peptides and proteins than the PPS method. Since SCX is widely used as the first dimension of 2D-LC MS/MS, integration of SDS removal with peptide separation in SCX does not add any extra steps to the sample handling process. We demonstrated the application of this method for 2D-LC MS/MS profiling of the MCF-7 membrane protein fraction and identified 6889 unique peptides, corresponding to 2258 unique proteins or protein groups from two replicate experiments with a false peptide discovery rate of ∼0.8%, compared to 5172 unique peptides and 1847 unique proteins identified by the RapiGest method.

Simultaneous treatment of graywater and waste gas in a biological trickling filter.

Biological processors are typically used in liquid- and gas-phase remediation as separately staged systems. This research presents a novel application of a biotrickling filter operated for simultaneous treatment of contaminants present in graywater and waste gas (ammonia and hydrogen sulfide). Liquid- and gas-phase contaminants were monitored via bioreactor influent/effluent samples over the course of a 300-day study. An oxygen-based bioassay was used to determine spatial location of the functional groups involved in the biodegradation of surfactants, dissolved hydrogen sulfide, and ammonium. Results indicated that a biotrickling filter is able to support the wide range of microbial species required to degrade the compounds found in graywater and waste gas, maintaining conversion efficiencies greater than 90% for parent surfactant compounds and waste gas constituents. These results provide evidence of an operational scheme that potentially reduces footprint size and cost of graywater/waste gas biotreatment.

Automated on-line ionic detergent removal from minute protein/peptide samples prior to liquid chromatography-electrospray mass spectrometry.

An automated on-line ionic detergent removal pre-column system coupled to capillary liquid chromatography-electrospray mass spectrometry is described. The system involves two micro precolumns, composed of a specific ionic detergent trapping column and a preconcentration column, respectively, and a packed 300 microns I.D. analytical column. Sample loading to the micro precolumns and regeneration of the detergent trapping column were performed at a flow-rate of 50 microliters/min, while the flow-rate through the analytical column was set at 5.0 microliters/min. Ionic detergent-containing tryptic-digested protein samples were directly applied to the micro precolumns without sample pretreatment and were analysed by UV absorption detection and electrospray mass spectrometry. The presented system allows for the fully automated removal of SDS with virtually no loss in protein/peptides. Maximum SDS load and breakthrough have been determined. Excellent protein recovery and complete removal of SDS is found. The chromatographic separation after SDS removal was completely restored and equalled the reference chromatogram. Mass spectral data confirm these findings. Finally, this technique allows for SDS removal from minute protein samples without the need for any sample handling.

A new two-step precipitation method removes free-SDS and thiol reagents from diluted solutions, and then allows recovery and quantitation of proteins.

Several polypeptides are present in a few copies per cell (i.e., membrane receptors or transcription factors), and therefore their concentration and quantitation from highly diluted solution after detergent solubilization is often an essential and difficult step in their purification by gel electrophoresis. A solution (optimized to Tris-glycine, Tris-borate, and Na-phosphate buffers) to all of these problems is here described, detailing a two-step procedure which takes advantage of the lower solubility of free potassium dodecyl sulfate (KDS) vs micellar KDS even at neutral pH. In the first step, more than 90% of free DS precipitates out from protein solution, the method being poorly sensitive to dodecyl sulfate/KCl ratio from almost no SDS to 10%. Indeed to obtain a residual 0.01% of SDS in the solution no adjustment of KCl concentration is needed from 0.1 to 2.3% SDS. When the supernatant is added with ice-cold TCA and K+, protein solubility is severely affected. Excellent protein recovery, at least 90%, is obtained with hydrophilic proteins. On the other hand, hydrophobic or low-ionic-strength-insoluble proteins are partially lost in step 1 precipitate, so that protein yield decreases, still remaining to about 80%. Furthermore our procedure simplifies determination of protein contents of diluted mercaptoethanol-SDS-solubilized proteins: since thiol reagents are discarded with the final supernatant, proteins can be quantitated in the final pellet by standard colorimetric methods.

Selective removal of free dodecyl sulfate from 2-mercaptoethanol-SDS-solubilized proteins before KDS-protein precipitation.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the discontinuous system of Laemmli is used world-wide for analytical and preparative gel electrophoresis of polypeptides. A minor but disturbing problem is the difficulty of concentrating highly diluted solutions and determining their protein content after 2-mercaptoethanol-SDS solubilization. We describe a solution to both of these problems, detailing a two-step procedure which takes advantage of the low solubility of potassium dodecyl sulfate (KDS). Removal of excess of SDS and 2-mercaptoethanol, and concentration of proteins from even a nanomolar solution, is achieved by a two-step KDS precipitation. Free dodecyl sulfate is precipitated in step one, while KDS-proteins are pelleted in the second step, allowing the thiol agents to be discarded with the supernatant. The effects of changing [SDS] and [KC1], temperature, and pH were studied to optimize the separation of free SDS from proteins. After final precipitation, the hundred- or thousandfold concentrated proteins can be suspended in a small volume of any required medium. The procedure allows protein determination by the Lowry method, peptide mapping of 2-mercaptoethanol-SDS-solubilized polypeptides, and all other analyses which are otherwise hampered by excesses of SDS and/or thiol reagents.