Type Of Detergent Research Articles

The in vitro impact of toothpaste extracts on cell viability.

Toothpastes contain three main components: detergents, abrasives, and fluoride. Detergents, particularly sodium lauryl sulfate, have been proposed as components that enable toothpastes to produce cytotoxic effects in vitro. However, not all toothpastes contain sodium lauryl sulfate, and almost no studies have found an association between detergents and the in vitro cytotoxicity of toothpastes. The present study examined the in vitro cytotoxicity of nine commercially available toothpastes containing four different detergents. Toothpastes were diluted in serum-free medium, centrifuged, and filter sterilized. The half-lethal concentration of the toothpaste-conditioned medium (TCM) was calculated based on the formation of formazan by gingival fibroblasts, oral squamous cell carcinoma HSC-2 cells, and L929 cells. Cell proliferation was analyzed, and live-dead staining was performed, after exposure of cells to conditioned medium prepared with 1% toothpaste (1% TCM). It was found that toothpastes containing sodium lauryl sulfate and amine fluoride strongly inhibited cell viability with the half-lethal concentration being obtained with conditioned medium prepared with approximately 1% toothpaste (1% TCM). Toothpastes containing cocamidopropyl betaine and Steareth-20 showed higher half-lethal concentration values, with the half-lethal concentration being obtained with conditioned medium prepared with 10% (10% TCM) and 70% (70% TCM) toothpaste, respectively. Proliferation and live-dead data were consistent with the cell-viability analyses. These results demonstrate that the type of detergent in toothpastes can be associated with changes in in vitro cell toxicity.

불교전통 식생활방식에 따른 수질오염 저감 효과 연구

Abstract In this research traditional Buddhist dietary life style was studied as an alternative for both saving water and decreasing water pollution from household. A traditional dietary life style for the Buddhist monks, called Balwoo Gongyang, and its modernized one for citizens, called Dish Gongyang were examined with water use amount, wastewater quality and pollutant loading rate, and were compared with the kitchen wastewater from household. And several types of natural detergent were also examined to compare their effect on the wastewater quality and pollutant loading rate. This research was carried out using the wastewater from Balwoo and Dish Gongyang in J building located in Seoul. Wastewater generation from Balwoo Gongyang that was about 1.0 l/capita/day was very low compare to that from dish washing in normal household(32.6 l/capita/day). In case of Dish Gongyang, water generation was 1.8 times higher than that from Balwoo Gongyang, but it was just 1/19.2 of normal household.When the wastewater quality of Balwoo Gongyang was compared with the kitchen wastewater of normal household, SS was 1/16(15 mg/l), COD was 1/7(22.1 mg/l), BOD was 1/9(24.1 mg/l) and T-N was 1/16(1.7 mg/l). Pollutant loading rate from Balwoo Gongyang was very low, COD 18.0 mg/capita/day, SS 12.3 mg/capita/day and it was 1/290, 1/639 of that from dish washing wastewater from normal household, respectively. Pollutant loading rate from Dish Gongyang was also low 1/13(SS)∼1/144(TN) compared to dish washing wastewater from normal household.As a natural detergent, used water from washing rice showed very low pollutant loading rate. It was concluded that Dish Gongyang that was a modernized and simplified dietary life style of Balwoo Gongyang, was very eco-friendly. Especially when it was combined with natural detergents using by-product from cooking process, its effect on the water pollution was very low.

Sample preparation for phosphoproteomic analysis of circadian time series in Arabidopsis thaliana.

Systems biological approaches to study the Arabidopsis thaliana circadian clock have mainly focused on transcriptomics while little is known about the proteome, and even less about posttranslational modifications. Evidence has emerged that posttranslational protein modifications, in particular phosphorylation, play an important role for the clock and its output. Phosphoproteomics is the method of choice for a large-scale approach to gain more knowledge about rhythmic protein phosphorylation. Recent plant phosphoproteomics publications have identified several thousand phosphopeptides. However, the methods used in these studies are very labor-intensive and therefore not suitable to apply to a well-replicated circadian time series. To address this issue, we present and compare different strategies for sample preparation for phosphoproteomics that are compatible with large numbers of samples. Methods are compared regarding number of identifications, variability of quantitation, and functional categorization. We focus on the type of detergent used for protein extraction as well as methods for its removal. We also test a simple two-fraction separation of the protein extract.

Stigmatellin Probes the Electrostatic Potential in the QB Site of the Photosynthetic Reaction Center

The electrostatic potential in the secondary quinone (QB) binding site of the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides determines the rate and free energy change (driving force) of electron transfer to QB. It is controlled by the ionization states of residues in a strongly interacting cluster around the QB site. Reduction of the QB induces change of the ionization states of residues and binding of protons from the bulk. Stigmatellin, an inhibitor of the mitochondrial and photosynthetic respiratory chain, has been proven to be a unique voltage probe of the QB binding pocket. It binds to the QB site with high affinity, and the pK value of its phenolic group monitors the local electrostatic potential with high sensitivity. Investigations with different types of detergent as a model system of isolated RC revealed that the pK of stigmatellin was controlled overwhelmingly by electrostatic and slightly by hydrophobic interactions. Measurements showed a high pK value (>11) of stigmatellin in the QB pocket of the dark-state wild-type RC, indicating substantial negative potential. When the local electrostatics of the QB site was modulated by a single mutation, L213Asp→Ala, or double mutations, L213Asp-L212Glu→Ala-Ala (AA), the pK of stigmatellin dropped to 7.5 and 7.4, respectively, which corresponds to a >210 mV increase in the electrostatic potential relative to the wild-type RC. This significant pK drop (ΔpK > 3.5) decreased dramatically to (ΔpK > 0.75) in the RC of the compensatory mutant (AA+M44Asn→AA+M44Asp). Our results indicate that the L213Asp is the most important actor in the control of the electrostatic potential in the QB site of the dark-state wild-type RC, in good accordance with conclusions of former studies using theoretical calculations or light-induced charge recombination assay.

The Role of Annular Lipids in the Mechanism of Membrane Protein Solubilization and Formation of Functionally Competent Proteomicelles

The “solubilization” procedure in which membrane proteins are extracted from their native lipid membranes and transferred into detergent micelles (proteomicelles) is a critical step in the purification of the protein for subsequent structure/function studies. In some notable examples it has become clear, however, that the properties of the proteins in proteomicelles depend on the detergent type and its concentration relative to the residual membrane lipids originating from the preparation. To reveal the molecular mechanism of the solubilization process and the effect of various lipid-to-detergent (L-D) ratios on the properties of the system, we performed extensive atomistic molecular dynamics simulations. We previously explored the properties of micelles formed by dodecyl-β-maltoside (DDM) detergent around the leucine transporter (LeuT), a bacterial homolog of the neurotransmitter:sodium symporter family, and showed how the DDM concentration determines the extent of detergent penetration into the functionally relevant secondary substrate (S2) binding site in LeuT. Here we present computational studies of LeuT embedded in proteomicelles at different L-D ratios exploring configurations of LeuT proteomicelles at different stages of the protein solubilization process. Our results reveal that at a sparse lipid annulus around the transporter, consisting of no more than ∼10 lipids, DDM partitions into the LeuT, engaging with the residues in the S2 site. Higher lipid concentrations protect the S2 site from DDM penetration. In contrast to DDM, MNG-3 (lauryl maltose-neopentyl glycol) detergent, an amphiphile known to better stabilize membrane proteins, does not penetrate the transporter even in the absence of lipids. Parallel experimental measurements of ligand binding to LeuT in the presence of increasing MNG-3 concentrations revealed that the functionality of the S2 site was fully preserved even at the highest (0.5%) MNG-3 concentration probed.

Exploring the Arabidopsis proteome: influence of protein solubilization buffers on proteome coverage.

The study of proteomes provides new insights into stimulus-specific responses of protein synthesis and turnover, and the role of post-translational modifications at the systems level. Due to the diverse chemical nature of proteins and shortcomings in the analytical techniques used in their study, only a partial display of the proteome is achieved in any study, and this holds particularly true for plant proteomes. Here we show that different solubilization and separation methods have profound effects on the resulting proteome. In particular, we observed that the type of detergents employed in the solubilization buffer preferentially enriches proteins in different functional categories. These include proteins with a role in signaling, transport, response to temperature stimuli and metabolism. This data may offer a functional bias on comparative analysis studies. In order to obtain a broader coverage, we propose a two-step solubilization protocol with first a detergent-free buffer and then a second step utilizing a combination of two detergents to solubilize proteins.

The pool of preactivated Lck in the initiation of T-cell signaling: a critical re-evaluation of the Lck standby model.

The initiation of T-cell receptor (TCR) signaling, based on the cobinding of TCR and CD4-Lck heterodimer to a peptide-major histocompatibility complex II on antigen presenting cells, represents a classical model of T-cell signaling. What is less clear however, is the mechanism which translates TCR engagement to the phosphorylation of immunoreceptor tyrosine-based activation motifs on CD3 chains and how this event is coupled to the delivery of Lck function. Recently proposed 'standby model of Lck' posits that resting T-cells contain an abundant pool of constitutively active Lck (pY394(Lck)) required for TCR triggering, and this amount, upon TCR engagement, remains constant. Here, we show that although maintenance of the limited pool of pY394(Lck) is necessary for the generation of TCR proximal signals in a time-restricted fashion, the total amount of this pool, ~2%, is much smaller than previously reported (~40%). We provide evidence that this dramatic discrepancy in the content of pY394(Lck)is likely the consequence of spontaneous phosphorylation of Lck that occurred after cell solubilization. Additional discrepancies can be accounted for by the sensitivity of different pY394(Lck)-specific antibodies and the type of detergents used. These data suggest that reagents and conditions used for the quantification of signaling parameters must be carefully validated and interpreted. Thus, the limited size of pY394(Lck) pool in primary T-cells invites a discussion regarding the adjustment of the quantitative parameters of the standby model of Lck and reevaluation of the mechanism by which this pool contributes to the generation of proximal TCR signaling.

Biochemical characterization of the water‐soluble squalene synthase from Methylococcus capsulatus and the functional analyses of its two DXXD(E)D motifs and the highly conserved aromatic amino acid residues

Information regarding squalene synthases (SQSs) from prokaryotes is scarce. We aimed to characterize the SQS from Methylococcus capsulatus. We studied its reaction mechanism by kinetic analysis and evaluated the structure of the substrate/inhibitor-binding sites via homology modeling. The cloned M. capsulatus SQS was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid column chromatography. Interestingly, M. capsulatus SQS was water-soluble and did not require any detergent for its higher activity, unlike other SQSs studied previously; supplementation of any type of detergent inhibited enzyme activity. The specific activity and the kinetic values (Km and kcat ) for the substrate farnesyl diphosphate and NADPH are reported. The substrate analog farnesyl methylenediphosphonate showed potent inhibition toward the enzyme. We prepared the site-specific mutants directed at potential active-site residues (58) DXX(61) E(62) D (S1 site) and (213) DXX(216) D(217) D (S2 site), which were assumed to be involved in the binding of the substrate farnesyl diphosphate through the Mg(2+) ion. We first demonstrated that the S1 site and the two basic residues (R55 and K212) were responsible for the binding of farnesyl diphosphate. Furthermore, we examined the catalytic roles of the highly conserved aromatic residues and demonstrated that the Y164 residue abstracts the proton of cation 5, which is produced during the first half-reaction (Scheme 1), to afford presqualene diphosphate, and that the W224 residue stabilizes the intermediary cation 5 via the cation-π interaction. Furthermore, we confirm for the first time that the F32 and the Y51 residues also stabilize the carbocation intermediate(s) generated during the second half-reaction.

Impact of wash cycle time, temperature and detergent formulation on the hygiene effectiveness of domestic laundering.

Investigation of the effect of temperature and duration of the laundering process with and without activated oxygen bleach (AOB)-containing detergent on the hygienic effectiveness of laundering. Cotton test swatches were contaminated with Staphylococcus aureus, Enterococcus hirae, Pseudomonas aeruginosa, Candida albicans and Trichophyton mentagrophytes and were washed in a household washing machine using temperatures between 20 and 60°C and different wash cycle times. The logarithmic microbial reduction factor and cross-contamination (i.e. transfer from contaminated to sterile swatches) were used to indicate the hygienic effectiveness of the washing process. For all tested micro-organisms, the temperature needed for decontamination depended on washing time and detergent type. Hygiene effectiveness of laundering was enhanced by inclusion of AOB even at lowest temperatures, except for C.albicans, which was virtually unaffected by AOB. The use of AOB-containing detergents as well as high washing temperatures reduced cross-contamination to sterile swatches included in the load. Depending on the type of organism, longer wash cycle times or the use of AOB-containing detergents can be used to enhance the hygiene effectiveness of laundering. The study demonstrates that it is possible to compensate for the loss of hygiene effectiveness of laundering at lower temperatures using detergents with activated oxygen bleach or by extending the wash cycle time.

Bicelles Exhibiting Magnetic Alignment for a Broader Range of Temperatures: A Solid-State NMR Study

Bicelles are increasingly used as model membranes to suitably mimic the biological cell membrane for biophysical and biochemical studies by a variety of techniques including NMR and X-ray crystallography. Recent NMR studies have successfully utilized bicelles for atomic-resolution structural and dynamic studies of antimicrobial peptides, amyloid peptides, and membrane-bound proteins. Though bicelles composed with several different types of lipids and detergents have been reported, the NMR requirement of magnetic alignment of bicelles limits the temperature range in which they can be used and subsequently their composition. Because of this restriction, low-temperature experiments desirable for heat-sensitive membrane proteins have not been conducted because bicelles could not be aligned. In this study, we characterize the magnetic alignment of bicelles with various compositions for a broad range of temperatures using (31)P static NMR spectroscopy in search of temperature-resistant bicelles. Our systematic investigation identified a temperature range of magnetic alignment for bicelles composed of 4:1 DLPC:DHexPC, 4:1:0.2 DLPC:DHexPC:cholesterol, 4:1:0.13 DLPC:DHexPC:CTAB, 4:1:0.13:0.2 DLPC:DHexPC:CTAB:cholesterol, and 4:1:0.4 DLPC:DHexPC:cholesterol-3-sulfate. The amount of cholesterol-3-sulfate used was based on mole percent and was varied in order to determine the optimal amount. Our results indicate that the presence of 75 wt % or more water is essential to achieve maximum magnetic alignment, while the presence of cholesterol and cholesterol-3-sulfate stabilizes the alignment at extreme temperatures and the positively charged CTAB avoids the mixing of bicelles. We believe that the use of magnetically aligned 4:1:0.4 DLPC:DHexPC:cholesterol-3-sulfate bicelles at as low as -15 °C would pave avenues to study the structure, dynamics, and membrane orientation of heat-sensitive proteins such as cytochrome P450 and could also be useful to investigate protein-protein interactions in a membrane environment.

The Effect of Detergent on the Oligomerization of a 7-Transmembrane Protein

Membrane proteins serve a broad range of functions necessary for life; in fact, they are coded for by 30% of genes in the human genome. Unfortunately, the complex native membrane is irreproducible for many spectroscopic methods, and the necessary use of solubilizing detergents for biochemical and biophysical characterization can alter protein stability and function. We explore the effects of such environments on Proteorhodopsin (PR), a proton-pumping membrane protein from marine bacteria. PR has significant structural and dynamical commonalities to mammalian seven-transmembrane proteins, including the G-protein Coupled Receptors (GPCRs). Furthermore, PR associates with itself in the membrane to form oligomers similarly to GPCRs, and these different forms are thought to play a significant role in function. Using fast protein liquid chromatography and optical absorption experiments we show that the hexameric state of PR in the nonionic dodecylmaltoside (DDM) detergent has a lower pKa value for the primary proton-acceptor residue than both the monomeric and dimeric protein, suggesting that the hexamer is more optimized for proton transport. We have also observed this phenomenon in the zwitterionic detergent dodecylphosphocholine (DPC). Next, we studied the environmental effects on PR's oligomeric state, using crosslinking followed by SDS-PAGE, and found hexamers in DDM, DPC, and E. coli membranes expressing PR. Additionally, we examine structure and dynamics using continuous wave Electron Paramagnetic Resonance (EPR). Site-directed spin-labeling EPR elucidates site-specific dynamics and detects short distances (<2 nm), making it useful for studying these complex environments. We thereby determine that a change in detergent type can change side-chain dynamics. Our work lends insight into the study of more complex mammalian proteins by demonstrating the effect of detergent on function, dynamics, and protein-protein complexes.

가정용 세탁기, 세제 및 오염의 종류별 세탁 성능 비교 - 세탁성, 헹굼성, 섬유손상도, 엉킴도를 중심으로 -

The purpose of this research is to provide accurate information of household washers and detergents for consumers, so that help the producers who make washing machine and detergent to get basic material and also help consumers to choose washing machine. Experiment was proceed with two type of washers to compare energy consumption, washing performance, rinsing effectiveness further, damage caused by entanglement of laundry and fabric was assessed. Detergent P and T were used to compare the performance related to differences of ingredients of detergent. Soiled fabrics of EMPA 108 set were used to evaluate performance of washing by different types of contamination. A summary of experimental results are : First, for the consumption of water, drum-type washer consumed 53% less than pulsator-type washer. On the other hand, the washing time was almost similar for both these machines, but pulsator-type washer showed shorter progress, implying that power saving was more efficient in this case. Second, the drum-type washer showed better performance for contamination with all types of detergent, but the pulsator-type washer showed better rinsing performance. Third, the drum-type washer performed less data of tangle level and fabric damage. Fourth, detergent "P" exhibited better washing performance than did detergent "T", regardless of the type of soil. And with no limit of detergent variety, water-soluble protein soil showed high removal rate, liposoluble soil especially pigment was hardly removed.

Comparison of detergency effectiveness by the type of household washer, detergent and soil - Focused on detergency, rinsing, fabric damage and tanglement -

Comparison of detergency effectiveness by the type of household washer, detergent and soil - Focused on detergency, rinsing, fabric damage and tanglement -

Blue native electrophoresis as a tool for detection of PIP-containing protein complexes in the plasma membranes

Blue native electrophoresis (BN-PAGE) is presently considered as one of effective methods for the identification of membrane protein complexes. The choice of a nonionic detergent and the detergent to protein ratio are critically important. Our experiments with plasma membranes of etiolated pea (Pisum sativum L.) seedlings showed that various nonionic detergents—digitonin, dodecyl maltoside, and Triton X-100—solubilized similar assortments of protein complexes. Irrespective of the detergent type, PIP aquaporins were always observed in the 440-kD protein complex. Only in the case of dodecyl maltoside, the PIP aquaporins were also revealed in the complexes with the lower and higher molecular weights when the detergent/protein ratio increased.

RO membrane treatment of domestic grey-water containing different detergent types

Water reclamation from grey-water containing detergent and salinity (∼2,000 ppm) was carried out using indigenously developed brackish water TFC membrane module (4040 size) at 250 psi operating pressure. Reusable water with <300 ppm inorganic solutes and trace amounts of organic content of detergent origin was produced upon reverse osmosis (RO) treatment of grey-water. The RO module performance in terms of product flux was influenced by the detergent type present in the feed water. While the product flux was rather unchanged for the feed containing a commercial linear alkyl benzene-alfa olefin sulfonate-soda ash based detergent, a significant flux decline (∼25%) was observed for the feed containing a commercial C8–C24 primary/secondary ethoxylates based detergent. However, the membrane selectivity in terms of salt rejection was slightly higher for the feed with detergent than that of the detergent-free feed. This can be due to the surface-active agent of the detergent which alters the surface potential of the membrane. This is in agreement with the changes observed by atomic force microscopy (AFM) and the Zeta-potential measurements.

Polycarbonate baby bottles: study of the release of Bisphenol A

Recently, it has been demonstrated that the main Bisphenol A (BPA) release is a degradation process originated by the contact between detergent and polycarbonate (PC) at high temperatures. EU Laws for food contact materials authorize the use of BPA for food contact PC articles (some for the use of young children), and consequently, the degradation process should be taken into account when testing PC articles. In the present study, two types of samples were assayed, baby bottles that have previously undergone some migration assays and new baby bottles of a specific brand in order to test the degradation of not previously used articles. The tests were performed under different conditions and using two types of detergents. To quantify BPA, a high-performance liquid chromatographic system with fluorescence detector was used. The method was validated, and satisfactory results were achieved. Data obtained indicated that the trademark is a substantial factor in the level of BPA release from PC polymer; besides a great dispersion between samples of the same batch independently of the detergent and concentration employed used was found. On the other hand, for both types of samples, a previous food contact (infant formula) as well as the exposure procedure (immersion or article filling) has influenced on the PC degradation.

Interactions of Cymal-6 and Lipid Vesicles

The solubilization of biological or liposomal membranes, induced by detergents and detergent-like biomolecules, is important to many technical applications and biological phenomena. In fact, the interactions of classical detergents with lipid bilayers are described and understood quite well. Recently, new types of detergents with cyclohexyl groups or branches in their hydrophobic tails have been synthesized and proposed to be superior for membrane protein studies. Cymal-6 has, for example, been used for isolating membrane proteins such as CCR5 and HIV-1 corepressors. Here we provide a rather comprehensive description of the interactions of Cymal-6 with fluid membranes of POPC. This includes the temperature-dependent phase behavior (i.e., the onset and completion of solubilization), membrane partitioning, disordering, and permeabilization as seen using ITC, time-resolved fluorescence anisotropy of DPH, dynamic light scattering, and the lifetime-based vesicle leakage assay.

Secondary Structure and Topology of the Transmembrane Domain of LspA, from Mycobacterium Tuberculosis, using Solid State NMR Spectroscopy: Initial Study

Lipoprotein Signal Peptidase A (LspA) belongs to type II signal peptidases and it is an essential enzyme in one of the three distinct steps in lipoprotein maturation. After targeting the prolipoprotein, LspA cleaves the signal peptide to result in the signal peptide cleaved lipoprotein, which is the precursor for the mature lipoprotein.In Mycobacterium tuberculosis (Mtb), LspA is encoded by the gene Rv1539 resulting in a 202 amino acid residue peptide with a molecular weight of 21 kDa that is predicted to possess four transmembrane helices. It has been recognized in vivo that LspA is an essential protein for Mtb virulence and that it may also contribute to antibiotic resistance. As a result LspA has been identified as a potential drug target for the treatment of tuberculosis. Globomycin, a cyclic-peptide is known to inhibit LspA in a non-competitive manner.Solid-state NMR enables studies of membrane proteins in a lipid bilayer environment. Since the native-like environment is important for studying the structure and function of membrane proteins, ssNMR is increasingly useful. In Oriented Sample ssNMR used here, the protein is uniformly aligned with respect to the magnetic field and 15N spectra of isotopically labeled protein have provided initial structural insights.Here, all sample preparation steps and initial spectroscopy will be emphasized. Expression of LspA in E.coli, purification using IMAC, reconstitution of LspA into proteoliposomes via dialysis and the preparation of bicelles as an alignment media with different types of lipids and detergents will be presented. 1D NMR 31P and 15N spectra and 2D separated local field NMR spectra of uniformly and selectively labeled LspA will be shown.

Examining kinetic parameters of fermentation process in batch cultures on the production of alkaline protease

Alkaline proteases are important group of enzymes because of their use in many industrial applications such as in detergent materials for the removal of the protein stains on clothes. The aim of this study is to investigate the kinetic parameters of the fermentation process inspected for alkaline protease production by using a localBacillus clausii. In this way, it is carried out under fed-batch operations of fermentation in erlyne. Designing full factorial is used for optimizing appetite composition for the three factors of sponge carbon glucose, yeast extract (organic nitrogen), salt solution (including K2HPO4, MgSO4.7H2O), and trace element solution (TES) each at two levels and designed by eight tests in total. All experiments were carried out in duplicates to decrease the possibility of errors occurring in the research. Analysis of variance (ANOVA) showed that the high coefficient of the determination (R2) value is 0.99, thus ensuring a significant adjustment of the suggested polynomial model with the experimental data. The results showed that protease production was controlled because of catabolic inhibition in the initial glucose concentration and the amount of enzyme activity which is important for protease production which increased in the end of the stationary phase that was often accompanied by sporulation phenomena. The results on comparison and inspection in batch cultures with glucose demonstrated that different values revealed that the alkaline protease was highly produced in a few hours after complete consumption of glucose. Also, mineral salts and yeast extract induce the protease production as increasing protease is a changing approach of bacteria catabolitic from development to extra cell enzyme production. Meanwhile, results showed that the detergent type do not have any effect on protease activity. Therefore, the production enzyme can be used as detergent powders. Key words: Alkaline protease, Bacillus clausii, fed-batch fermentation, sporulation, kinetic parameters, full factorial, detergent.

Perfusion reversed-phase high-performance liquid chromatography for protein separation from detergent-containing solutions: An alternative to gel-based approaches

Detergents are frequently used for the solubilization of membrane proteins during and after purification steps. Unfortunately some of these detergents impair chromatographic separations and mass spectrometry (MS) analysis. Perfusion reversed-phase high-performance liquid chromatography (RP–HPLC) using POROS materials is suited for separating intact proteins solubilized by detergents due to the particles’ highly diffusive pores and chemical stability. In this article, the use of perfusive reversed-phase material packed into small inner diameter capillary columns is presented as a cheap, rapid, and efficient method for the removal of different types of detergents from protein solutions. The ability to purify and separate the subunits of membrane protein complexes with self-packed capillary columns is exemplified for bovine cytochrome bc1 complex. Even highly hydrophobic subunits can be detected in collected fractions by intact mass measurements and identified after proteolytic digestion and matrix-assisted laser desorption/ionization tandem MS (MALDI MS/MS). The comparison with a gel-based approach shows that this method is a valuable alternative for purification and separation of intact proteins with subsequent MS analysis and that hydrophobic proteins are even better represented in the LC-based approach.