Chirality Induction and Amplification in the 2,2,2‐Trifluoroethanol⋅⋅⋅Propylene Oxide Adduct

Chirality induction and amplification in a model system, that is, the 2,2,2‐trifluoroethanol (TFE)???propylene oxide (PO) adduct, were investigated using free‐space and cavity‐based Fourier transform microwave spectroscopy, complemented with high level ab initio calculations. Rotational spectra of four out of eight predicted TFE??PO adducts were assigned, and the remaining four were shown to relax to the geometries of the four observed in a jet expansion. The g+ TFE???S‐PO adduct was found to be favored over that of g? TFE???S‐PO by a factor of 2.8 at 60 K. This difference contrasts the TFE dimer for which an extreme case of chirality synchronization was previously reported. All TFE???PO conformers observed take on the open arrangement, in contrast to 2‐fluoroethanol???PO, which prefers the closed arrangement. Furthermore, perfluorination at CH₃ increases the hydrogen‐bonding energy by about 70 % over its ethanol counterpart.     

A proteomic approach to investigate potential biomarkers directed against membrane-associated breast cancer proteins

The identification of specific protein markers for breast cancer would provide the basis for early diagnosis. Particularly, membrane and membrane-associated proteins are rich in targets for antibodies that may constitute suitable biomarkers of carcinogenesis. However, membrane proteins separation using 2-DE remains difficult. In this work, the breast cancer cell line MCF7 was used as source of proteins for the screening of potential cell membrane-associated antigens recognized by autoantibodies in patients with breast cancer and healthy volunteers. The protein extract obtained using trifluoroethanol (TFE) as cosolvent was compared to a total cell lysate protein extract prepared by a current technique. After 2-DE separation of the two extracts, their protein patterns clearly differed. About 63% of the proteins identified in the TFE-extract were predicted to possess at least one transmembrane domain. 2-D blots probed with sera from cancer patients or from healthy volunteers showed that, as expected, additional antigens were provided in the TFE-extract. Thus, the method described here appeared well suited for proteomic investigation of potential biomarkers undetected by current techniques.     

Evaluation of the combinative application of SDS and sodium deoxycholate to the LC–MS‐based shotgun analysis of membrane proteomes

SDS and sodium deoxycholate (SDC) as two representative detergents have been widely used in LC–MS/MS‐based shotgun analysis of membrane proteomes. However, some inherent disadvantages limit their applications such as interference with MS analysis or their weak ability to disrupt membranes. To address this, the combinative application of SDS and SDC was developed and evaluated in our study, which comprehensively used the strong ability of SDS to lyse membranes and solubilize hydrophobic membrane proteins, and the high efficiencies of an optimized acetone precipitation method and SDC in sample clean‐up, protein recovery, and redissolution and digestion of precipitated proteins. The comparative study using a rat‐liver‐membrane‐enriched sample showed that, compared with other three commonly used methods including the filter‐aided sample preparation strategy, the combinative method not only increased the identified number of total proteins, membrane proteins, and integral membrane proteins by an average of 19.8, 23.9, and 24.8%, respectively, but also led to the identification of the highest number of matching peptides. All these results demonstrate that the method yielded better recovery and reliability in the identification of the proteins especially highly hydrophobic integral membrane proteins than the other three methods, and thereby has more potential in shotgun membrane proteomics.     

Proteomic study of the peripheral proteins from thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803

Thylakoid membranes of cyanobacteria and plants contain enzymes that function in diverse metabolic reactions. Many of these enzymes and regulatory proteins are associated with the membranes as peripheral proteins. To identify these proteins, we separated and identified the peripheral proteins of thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Trichloroacetic acid (TCA)-acetone extraction was used to enrich samples with peripheral proteins and to remove integral membrane proteins. The proteins were separated by two-dimensional electrophoresis (2-DE) and identified by peptide mass fingerprinting. More than 200 proteins were detected on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel that was stained with colloidal Coomassie blue. We analyzed 116 spots by peptide mass fingerprinting and identified 78 spots that were derived from 51 genes. Some proteins were found in multiple spots, indicating differential modifications resulting in charge differences. Therefore, a significant fraction of the peripheral proteins in thylakoid membranes is modified post-translationally. In our analysis, products of 17 hypothetical genes could be identified in the peripheral protein fraction. Therefore, proteomic analysis is a powerful tool to identify location of the products of hypothetical genes and to characterize complexity in gene expression due to post-translational modifications.     

Secreted proteome of the murine multipotent hematopoietic progenitor cell line DKmix

Administration of the multipotent hematopoietic progenitor cell (HPC) line DKmix improved cardiac function after myocardial infarction and accelerated dermal wound healing due to paracrine mechanisms. The aim of this study was to analyse the secreted proteins of DKmix cells in order to identify the responsible paracrine factors and assess their relevance to the wide spectrum of therapeutic effects. A mass spectrometry (MS)‐based approach was used to identify secreted proteins of DKmix cells. Serum free culture supernatants of DKmix‐conditioned medium were collected and the proteins present were separated, digested by trypsin and the resulting peptides were then analyzed by matrix‐assisted laser desorption/ionization tandem time‐of‐flight (MALDI‐TOF/TOF) MS. Overall 95 different proteins were identified. Among them, secretory proteins galectin‐3 and gelsolin were identified. These proteins are known to stimulate cell migration and influence wound healing and cardiac remodelling. The remaining proteins originate from intracellular compartments like cytoplasm (69%), nucleus (12%), mitochondria (4%), and cytoplasmic membrane (3%) indicating permeable or leaky DKmix cells in the conditioned medium. Additionally, a sandwich immunoassay was used to detect and quantify cytokines and chemokines. Interleukin‐6 (IL‐6), interleukin‐13 (IL‐13), monocyte‐chemoattractant protein‐1 (MCP‐1), monocyte‐chemoattractant protein‐3 (MCP‐3), monocyte‐chemoattractant protein‐1α (MIP‐1α) and monocyte‐chemoattractant protein‐1β (MIP‐1β) were detected in low concentrations. This study identified a subset of proteins present in the DKmix‐conditioned medium that act as paracrine modulators of tissue repair. Moreover, it suggests that DKmix‐derived conditioned medium might have therapeutic potency by promoting tissue regeneration. Copyright © 2010 John Wiley & Sons, Ltd.     

Amyloidogenic Protein–Membrane Interactions: Mechanistic Insight from Model Systems

The toxicity of amyloid‐forming proteins is correlated with their interactions with cell membranes. Binding events between amyloidogenic proteins and membranes result in mutally disruptive structural perturbations, which are associated with toxicity. Membrane surfaces promote the conversion of amyloid‐forming proteins into toxic aggregates, and amyloidogenic proteins, in turn, compromise the structural integrity of the cell membrane. Recent studies with artificial model membranes have highlighted the striking resemblance of the mechanisms of membrane permeabilization of amyloid‐forming proteins to those of pore‐forming toxins and antimicrobial peptides.     

Improved analysis of membrane protein by PVDF-aided, matrix-assisted laser desorption/ionization mass spectrometry

Characterization of membrane proteins remains an analytical challenge because of difficulties associated with tedious isolation and purification. This study presents the utility of the polyvinylidene difluoride (PVDF) membrane for direct sub-proteome profiling and membrane protein characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The hydrophobic adsorption of protein, particularly membrane proteins, on the PVDF surface enables efficient on-PVDF washing to remove high concentrations of detergents and salts, such as up to 5% sodium dodecyl sulfate (SDS). The enhanced spectrum quality for MALDI detection is particularly notable for high molecular weight proteins. By using on-PVDF washing prior to MALDI detection, we obtained protein profiles of the detergent-containing and detergent-insoluble membrane fractions from Methylococcus capsulatus (Bath). Similar improvements of signal-to-noise ratios were shown on the MALDI spectra for proteins electroblotted from SDS-polyacrylamide gel electrophoresis (SDS-PAGE) onto the PVDF membrane. We have applied this strategy to obtain intact molecular weights of the particulate methane monooxygenase (pMMO) composed of three intrinsic membrane-bound proteins, PmoA, PmoB, and PmoC. Together with peptide sequencing by tandem mass spectrometry, post-translational modifications including N-terminal acetylation of PmoA and PmoC and alternative C-terminal truncation of PmoB were identified. The above results show that PVDF-aided MALDI-MS can be an effective approach for profiling and characterization of membrane proteins.     

Membrane proteome analysis of the green-sulfur bacterium Chlorobium tepidum

An extensive proteomic approach relies on the possibility to visualize and analyze various types of proteins, including membrane proteins, which are rarely detectable on two-dimensional electrophoresis gels. In this study, different methods were employed for the enrichment of membrane proteins from Chlorobium tepidum prior to analysis with two-dimensional electrophoresis (2-DE). Isolated membranes were solubilized with Triton X-100 and from the supernatant we identified 58 unique proteins. The use of ionic sodium dodecyl sulfate (SDS) for protein solubilization, combined with acetone precipitation, resulted in an improved 2-DE pattern and the total number of the identified proteins was increased to 117. The use of acetone for protein precipitation improved the results by extracting compounds potentially deleterious to the resolution of 2-DE. However, the additional proteins detected by the use of SDS are in the majority more difficult to solubilize than less hydrophobic proteins. Further our attempts for selective extraction of the outer membrane proteins using the acid glycine method allowed the identification of 37 proteins of which 14 were predicted to have a signal sequence indicating their localization in the periplasmic space or in the outer membrane.     

Multiplexed quantification of proteins adsorbed to surface-modified and non-modified microdialysis membranes

A simple and straightforward method for discovery and quantification of proteins adsorbed onto delicate and sensitive membrane surfaces is presented. The adsorbed proteins were enzymatically cleaved while still adsorbed onto the membranes using an on-surface enzymatic digestion (oSED). This was followed by isobaric tagging, nanoliquid chromatography, and tandem mass spectrometry. Protein adsorption on tri-block copolymer Poloxamer 407 surface-modified microdialysis (MD) membranes were compared with protein adsorption on unmodified MD membranes. Ventricular cerebrospinal fluid (vCSF) kept at 37 °C was used as sample matrix. In total, 19 proteins were quantified in two biological replicates. The surface-modified membranes adsorbed 33% less proteins than control membranes and the most abundant proteins were subunits of hemoglobin and clusterin. The adsorption of clusterin on the modified membranes was on average 36% compared to control membranes. The most common protein in vCSF, Albumin, was not identified adsorbed to the surface at all. It was also experimentally verified that oSED, in conjunction with tandem mass spectrometry can be used to quantify femtomole amounts of proteins adsorbed on limited and delicate surfaces, such as MD membranes. The method has great potential and can be used to study much more complex protein adsorption systems than previously reported.     

纤维素亲和膜用于内毒素的去除

报道了用于内毒素去除的３种新型纤维素亲和膜的制备及应用,并对它们的性能进行了比较。结果表明,这３种亲和膜均能有效去除盐溶液中的内毒素,去除率在９０％以上。对人血清白蛋白溶液,季铵盐和壳聚糖亲和膜对内毒素的去除率较高,仍然在９０％以上,金属螯合物亲和膜的去除效果不如前两种,去除率仅为８３．３％。实验选择不同的亲和膜,考察了它们对内毒素的吸附容量、样品处理量以及再生效果,结果表明吸附量在 ２．４×１０６ＥＵ／ｇ以上,样品处理量较大,适合用于小批量医药制剂的生产过程;再生效果较好,可重复使用。     

Distance Measurement on an Endogenous Membrane Transporter in E. coli Cells and Native Membranes Using EPR Spectroscopy

Membrane proteins may be influenced by the environment, and they may be unstable in detergents or fail to crystallize. As a result, approaches to characterize structures in a native environment are highly desirable. Here, we report a novel general strategy for precise distance measurements on outer membrane proteins in whole Escherichia coli cells and isolated outer membranes. The cobalamin transporter BtuB was overexpressed and spin‐labeled in whole cells and outer membranes and interspin distances were measured to a spin‐labeled cobalamin using pulse EPR spectroscopy. A comparative analysis of the data reveals a similar interspin distance between whole cells, outer membranes, and synthetic vesicles. This approach provides an elegant way to study conformational changes or protein–protein/ligand interactions at surface‐exposed sites of membrane protein complexes in whole cells and native membranes, and provides a method to validate outer membrane protein structures in their native environment.     

Solubilization of cellular membrane proteins from Streptococcus mutans for two-dimensional gel electrophoresis

Membrane proteins are rarely identified in two-dimensional electrophoretic (2-DE) proteomics maps. This is due to low abundancy, poor solubility, and inherent hydrophobicity leading to self-aggregation during the first dimension. In this study, membrane proteins from the Gram-positive bacterium Streptococcus mutans were solubilized using three different methods and evaluated by 2-DE. In the first method, the extraction was performed using sodium dodecyl sulfate (SDS) followed by solubilization with a chaotropic buffer and precipitation with methanol/chloroform. The second method was based on temperature-dependent phase partitioning using Triton X-114 followed by purification using the ReadyPrep 2-D clean-up kit from Bio-Rad. The third method involved extraction using the organic solvents trifluoroethanol (TFE) and chloroform, which produced three separate phases. The upper aqueous phase, enriched with TFE, gave the highest overall protein yield and best 2-DE resolution. Protein spot identification by nanoelectrospray quadrupole time of flight (QTOF)-tandem mass spectrometry (MS/MS) revealed known membrane and surface-associated proteins. This is the first report describing the successful solubilization and 2-D electrophoresis of membrane proteins from a Gram-positive bacterium.     

Electrophoretic separation method for membrane pore‐forming proteins in multilayer lipid membranes

In this paper, we report on a novel electrophoretic separation and analysis method for membrane pore‐forming proteins in multilayer lipid membranes (MLMs) in order to overcome the problems related to current separation and analysis methods of membrane proteins, and to obtain a high‐performance separation method on the basis of specific properties of the lipid membranes. We constructed MLMs, and subsequently characterized membrane pore‐forming protein behavior in MLMs. Through the use of these MLMs, we were able to successfully separate and analyze membrane pore‐forming proteins in MLMs. To the best of our knowledge, this research is the first example of membrane pore‐forming protein separation in lipid membranes. Our method can be expected to be applied for the separation and analysis of other membrane proteins including intrinsic membrane proteins and to result in high‐performance by utilizing the specific properties of lipid membranes.     

Improvement of a sample preparation method assisted by sodium deoxycholate for mass‐spectrometry‐based shotgun membrane proteomics†

In current shotgun‐proteomics‐based biological discovery, the identification of membrane proteins is a challenge. This is especially true for integral membrane proteins due to their highly hydrophobic nature and low abundance. Thus, much effort has been directed at sample preparation strategies such as use of detergents, chaotropes, and organic solvents. We previously described a sample preparation method for shotgun membrane proteomics, the sodium deoxycholate assisted method, which cleverly circumvents many of the challenges associated with traditional sample preparation methods. However, the method is associated with significant sample loss due to the slightly weaker extraction/solubilization ability of sodium deoxycholate when it is used at relatively low concentrations such as 1%. Hence, we present an enhanced sodium deoxycholate sample preparation strategy that first uses a high concentration of sodium deoxycholate (5%) to lyse membranes and extract/solubilize hydrophobic membrane proteins, and then dilutes the detergent to 1% for a more efficient digestion. We then applied the improved method to shotgun analysis of proteins from rat liver membrane enriched fraction. Compared with other representative sample preparation strategies including our previous sodium deoxycholate assisted method, the enhanced sodium deoxycholate method exhibited superior sensitivity, coverage, and reliability for the identification of membrane proteins particularly those with high hydrophobicity and/or multiple transmembrane domains.     

Trifluoroacetic Acid in 2,2,2‐Trifluoroethanol Facilitates SNAr Reactions of Heterocycles with Arylamines

Small‐molecule drug discovery requires reliable synthetic methods for attaching amino compounds to heterocyclic scaffolds. Trifluoroacetic acid‐2,2,2‐trifluoroethanol (TFA‐TFE) is as an effective combination for achieving S_NAr reactions between anilines and heterocycles (e.g., purines and pyrimidines) substituted with a leaving group (fluoro‐, chloro‐, bromo‐ or alkylsulfonyl). This method provides a variety of compounds containing a “kinase‐privileged fragment” associated with potent inhibition of kinases. TFE is an advantageous solvent because of its low nucleophilicity, ease of removal and ability to solubilise polar substrates. Furthermore, TFE may assist the breakdown of the Meisenheimer–Jackson intermediate by solvating the leaving group. TFA is a necessary and effective acidic catalyst, which activates the heterocycle by N‐protonation without deactivating the aniline by conversion into an anilinium species. The TFA‐TFE methodology is compatible with a variety of functional groups and complements organometallic alternatives, which are often disadvantageous because of the expense of reagents, the frequent need to explore diverse sets of reaction conditions, and problems with product purification. In contrast, product isolation from TFA‐TFE reactions is straightforward: evaporation of the reaction mixture, basification and chromatography affords analytically pure material. A total of 45 examples are described with seven discrete heterocyclic scaffolds and 2‐, 3‐ and 4‐substituted anilines giving product yields that are normally in the range 50–90 %. Reactions can be performed with either conventional heating or microwave irradiation, with the latter often giving improved yields.     

Loss of epithelial polarity is accompanied by differential association of proteins with intracellular membranes

Cellular membranes play an important role in the formation and maintenance of epithelial polarity, which is lost early during carcinogenesis. We set out to identify membrane proteins which are altered during loss of cell polarity in mammary epithelium. As a model system we used murine mammary epithelial cells expressing the conditional oncoprotein c-JunER, which induces a reversible loss of polarity upon beta-estradiol-driven activation [1]. When grown either in the absence or presence of hormone, these cells exhibit a polarized or unpolarized phenotype, respectively. Different membrane fractions of polarized or unpolarized cells were analyzed by two-dimensional electrophoresis (2-DE) and differentially expressed membrane proteins were identified. To distinguish between transmembrane orientation and peripheral attachment of these proteins, were performed extractions with carbonate at high pH or with Triton X-114. In addition, cytosolic proteins of both states were analyzed to investigate their differential association with distinct membrane fractions. We found ten protein spots preferentially or exclusively in polarized cells and 17 other proteins as being upregulated during loss of polarity. Some of the peripheral membrane proteins were identified by microsequencing. The resident Golgi protein nucleobindin and fructose-bisphosphate aldolase were preferentially associated with membranes of polarized cells, whereas alphaB crystallin was detected exclusively and in high amounts in unpolarized cells.     

Small‐angle neutron scattering investigation of structural changes in nafion membranes induced by swelling with various solvents

The structure of Nafion‐117 perfluorosulfonate ionomer membranes was investigated with small‐angle neutron scattering techniques. Structural changes induced by the swelling of the membranes with water, alcohols, and dipolar, aprotic solvents were monitored at solvent‐swelling levels ranging from approximately 2 vol % to greater than 50 vol %. Membranes swollen up to approximately 50 vol % solvent exhibited two scattering maxima, one known to be associated with ionic regions of the membrane structure and one known to be associated with correlation distances between crystalline regions in the membrane structure. The positions of both maxima shifted toward lower scattering vector values as the solvent content in the membrane increased. The shift in the position of both maxima was linearly related to the solvent volume fraction in the membrane. The Bragg spacings corresponding to both the ionic‐feature scattering maximum and the crystalline‐feature scattering maximum were plotted versus the solvent volume fraction in the membranes, and the data fit with linear regression. The slopes associated with the curves of the spacing versus the solvent volume fraction were greater for the crystalline‐feature spacing than for the ionic‐feature spacing for all solvents other than water; this was indicative of preferential segregation of nonaqueous solvents into regions of the structure not directly associated with the ionic scattering maximum. © 2002 John Wiley & Sons, Inc. J Polym Sci Part B: Polym Phys 40: 387–400, 2002; DOI 10.1002/polb.10092     

Conversion of sodium lactate to lactic acid with water-splitting electrodialysis

The conversion of sodium lactate to lactic acid with water-splitting electrodialysis was investigated. One way of reducing the power consumption is to add a conductive layer to the acid compartment. Doing this reduced the power consumption by almost 50% in a two-compartment cell, whereas the electric current efficiency was not affected at all. Three different solutions were treated in the electrodialysis unit: a model solution with 70 g/L of sodium lactate and a fermentation broth that had been prefiltered two different ways. The fermentation broth was either filtered in an open ultrafiltration membrane (cut-off of 100,000 Dalton) in order to remove the microorganisms or first filtered in the open ultrafiltration membrane and then in an ultrafiltration membrane with a cut-off of 2000 Dalton to remove most of the proteins. The concentration of sodium lactate in the fermentation broth was 70 g/L, as well. Organic molecules present in the broth (peptides and similar organic material) fouled the membranes and, therefore, increased power consumption. Power consumption increased more when permeate from the more open ultrafiltration membrane was treated in the electrodialysis unit than when permeate from the membrane with the lower cut-off was treated, since there was a higher amount of foulants in the former permeate. However, the electrodialysis membranes could be cleaned efficiently with a 0.1 M sodium hydroxide solution.     

Murine fecal proteomics: A model system for the detection of potential biomarkers for colorectal cancer

Tumor related products shed into the feces offer a potential source of biomarkers for the detection of colorectal cancer (CRC). Using SDS-PAGE followed by nanoflow reversed-phased LC–MS/MS to analyse fecal samples from Apc^Min/+ mice (that develop spontaneous multiple intestinal neoplasia with age) we have identified 336 proteins (115 proteins of murine origin, 201 from fecal bacteria, 18 associated with food intake and 2 of apparent parasitic origin). 75% of the murine proteins identified in this study are predicted to be extracellular or associated with the cell plasma membrane. Of these proteins, a number of the murine homologues of colorectal cancer associated proteins (CCAP) such as hemoglobin, haptoglobin, hemopexin, alpha-2-macroglobulin and cadherin-17 have been identified, demonstrating the potential of fecal proteomics for detecting potential biomarkers and paving the way for subsequent MS/MS based biomarker studies on similar human samples.     

Extensive fractionation and identification of proteins within nasal lavage fluids from allergic rhinitis and asthmatic chronic rhinosinusitis patients

Allergic rhinitis (AR), chronic rhinosinusitis (CRS), and asthma are prevalent airway diseases that can have a substantial impact on a patient's quality of life. MS analyses of biological fluids can effectively screen for proteins associated with disease processes, however, initial detection of diagnostic proteins is difficult due to protein complexity and dynamic range. To enhance the detection of lower abundance proteins, intact nasal lavage fluid (NLF) proteins from nonpolypoid AR and from asthmatic CRS patients were extensively fractionated prior to LC/MS/MS analysis. Pooled NLF samples were processed to remove low molecular weight molecules and high abundance plasma proteins. Anion exchange (AX) chromatography followed by RP‐LC further separated the remaining intact NLF proteins. The resulting fractions were digested with trypsin and the peptides analyzed by LC/MS/MS. Spectra were searched with MASCOT, SEQUEST, and X!Tandem to obtain peptide identifications and subsequently analyzed by Scaffold software to identify parent proteins with at least 99% confidence. The 197 identified proteins are compared to those previously cited in the literature and the workflow evaluated to determine the usefulness for the detection of lower abundance proteins. This is the first extensive list of NLF proteins generated from CRS patients with coexisting asthma.