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PubMed2000

Separation of closely related peptide substrates of human proteinases by micellar electrokinetic chromatography with anionic and nonionic surfactants.

In order to use micellar electrokinetic chromatography to determine the proteolytic activity of different proteinases simultaneously present in physiological fluids, the technique must be able to separate mixtures of substrates with closely related structures. In an attempt to determine the best electrophoretic conditions for resolving six p-nitroanilide peptides used as synthetic substrates of the elastolytic enzymes (human neutrophil elastase, cathepsin G, Pseudomonas aeruginosa elastase) most commonly involved in pulmonary diseases, we investigated the efficiency of ionic and nonionic surfactants in achieving the separation of this complex mixture. The results presented here show that, of all the electrophoretic systems tested, 30 mM sodium tetraborate, pH 9.3, containing 25 mM Brij 35 as micellar agent offered the best performance; the separation efficiency of peptides is greater than that obtained with other reagents and all peaks are baseline resolved and unambiguously identifiable. Analysis of the micelle-solute interaction with the surfactants investigated allowed better definition of the mechanism involved in the distribution of these peptides to the micelles and identification of some structural features that determined the magnitude of the micelle peptide complex formation.

Cathepsin G↗
PubMed1999

Determination of surfactants by capillary electrophoresis.

Capillary electrophoresis has been increasingly used during the past few years for the separation and determination of surfactants. These substances are applied in many household and industrial products such as laundry detergents, cosmetics and pharmaceuticals, often as homologous and isomeric mixtures. Product development and control as well as toxicological and environmental analyses require selective and sensitive analytical methods. This review presents capillary electrophoretic techniques to determine important representatives of cationic, anionic, and neutral surfactants. The application of different buffer additives such as organic solvents, cyclodextrins or micelles to enhance the resolution of complex mixtures is discussed. Besides direct and indirect UV and fluorescence detection, examples for conductivity and mass spectrometric detection are also given. Derivatization procedures to improve the detectability and implement charge in neutral analytes are described. The successful use of capillary electrophoresis for surfactant determinations has proven that it can serve as a routine technique in many real-world applications. Robust, validated methods for the quantitation of single compounds, such as alkylbenzene sulfonates, sodium dodecyl sulfate and benzalkonium salts, are now available. Characteristic peak patterns (fingerprint analysis) can be used for the identification of surfactants in multicomponent formulations (e.g. ethoxylates and phosphonates).

Anions↗
PubMed2005

Separation of lactose from human milk oligosaccharides with simulated moving bed chromatography.

The successful separation of the disaccharide lactose from a complex mixture of human milk oligosaccharides (HMOS) with the continuous chromatography of simulated moving bed (SMB) technique is described. Since lactose is the main carbohydrate in human milk with well-known functions for the infant, it is necessary to separate it from the rest of the oligosaccharides to divide them into less complex fractions and analyse their partial unknown functions. For separation of lactose from HMOS two different stationary phases (size-exclusion gel as well as ion-exchange gel) were used. As the main result, it is shown that a size-exclusion gel with the particle size of 50-100 microm and porosity of 50 A was the preferred stationary phase for our separation process with almost complete lactose separation and stable conditions.

Chromatography, Gel↗
PubMed2006

Characterization of moenomycin antibiotic complex by multistage MALDI-IT/RTOF-MS and ESI-IT-MS.

Flavomycin is a commercially available antimicrobial growth promoter and an authorized additive for feeding stuffs in the EU and in the USA. As most antibiotically active products biosynthesized by microorganisms, it contains not only a single active compound but is a complex mixture of structurally closely related substances. Multistage matrix-assisted laser desorption/ionization-ion trap/reflectron time-of-flight mass spectrometry (MALDI-IT/RTOF-MS) and liquid chromatography-electrospray ionization-ion trap-mass spectrometry (LC-ESI-IT-MS) were utilized for a detailed analysis of the constituents of the Flavomycin complex based on low-energy collision induced dissociation (CID). An optimal sample preparation for negative ion vacuum MALDI-MS for this compound class was developed. The MALDI-IT/RTOF-MS2 and -MS3 analysis starting with the precursor [M - H]- ions of these interesting phosphoglycolipids, named moenomycins, yielded a large variety of product ions that facilitated the structural characterization of this class of compounds. Based on the derived CID fragmentation pathway of the five known major constituents, namely moenomycin A, moenomycin A12, moenomycin C4, moenomycin C3. and moenomycin C1, four not yet described moenomycin-type constituents could be characterized. They were assigned as 4F-demethyl-6E-O-de-beta-D-glucopyranosyl-moenomycin A, 6B-N-de(2-hydroxy-5-oxo-1-cyclopenten-1-yl)-moenomycin A, 6B-hydroxy-6B-de[N-(2-hydroxy-5-oxo-1-cyclopenten-1-yl)amino]-moenomycin A, and 6C-hydroxy-moenomycin A. In addition, a moenomycin A carrying an oxygen in the moenocinol-group was found, which is most probably a chemical degradation product. These new compounds were verified by LC-ESI-IT-MS.

Anti-Bacterial Agents↗
PubMed2005

The first cobalt metallacrowns: preparation and characterization of mixed-valence cobalt(II/III), inverse 12-metallacrown-4 complexes.

Aerobic reactions of Co(O(2)CMe)(2).4H(2)O with di-2-pyridyl ketone oxime (Hpko) in the presence of counterions (ClO(4)(-), PF(6-)) give the tetranuclear, mixed-valence cobalt(II/III) clusters [Co(II)(2)Co(III)(2)(OR)(2)(O(2)CMe)(2)(pko)(4)S(2)]X(2) [R = H, S = MeOH, X = ClO(4) (1); R = Me, S = EtOH, X = PF(6) (2)] depending on the solvent mixture. Complexes 1 and 2 are the first Co members in the family of metallacrowns adopting the extremely rare inverse 12-metallacrown-4 motif.

Journal Article↗
PubMed2004

Coated microwell plate-based affinity purification of antigens.

Antibody-based affinity capture of antigens is widely used in the isolation of antigens from complex mixtures. Antibody and the corresponding antigen are allowed to interact with each other to form immunocomplexes which are then typically captured by protein A or protein G immobilized on beaded support. Antigen capture performed using this method generally requires multiple centrifugation steps and careful pipetting to avoid loss of the bead-bound complexes. This traditional procedure is tedious and not easily reproducible, especially when working with multiple samples. To address these issues we have demonstrated that antigens can be captured with protein A/G, protein G, and high binding-capacity streptavidin 96-well strip-coated plates. The coated plate method of antigen purification is reproducible, within the same experiment and between experiments, due to the uniform binding capacity of the plates and wells. Here we report the use of coated microwell plates for antigen capture and for protein-protein interaction studies with the well-characterized BIR2-SMAC, transferrin receptor/ transferrin, and other systems.

Antibody Affinity↗
PubMed1998

Gas-liquid chromatography-mass spectrometry of hydroxy fatty acids as their methyl esters tert.-butyldimethylsilyl ethers.

tert.-Butyldimethylsilyl ethers of secondary hydroxy fatty acid methyl esters (tBDMS-O-FAMEs) produce stable derivatives amenable to gas-liquid chromatography (GLC) and mass spectrometry (MS). Derivatives produce prominent molecular mass minus 57 [M-57]+ fragment ions and unique marker fragment ions indicating the location of the secondary hydroxyl groups along the aliphatic chain from the omega-2 carbon to carbon numbers 5 from the carboxylic terminus, in addition to yielding information regarding carbon chain length, and degree of unsaturation. The tBDMS-derivatives of C-2, C-3 hydroxy fatty acids and the unique GLC-MS data of gamma- and delta-lactones are also presented. Though several tBDMS-O-FAMEs with centrally located hydroxyl groups were not chromatographically resolved, the combination of GLC retention times and monitoring of key diagnostic fragment ions of each tBDMS-derivative, when applied to mixtures containing all hydroxy isomers of palmitic through arachidic acid methyl esters, and to several monounsaturated, monohydroxylated fatty acid methyl esters, allowed for their unambiguous identification. Coupled with derivative stability, permitting their purification and concentration, this method was applied to the identification of trace lipids isolated from bovine skim milk which contained a complex mixture of hydroxy fatty acids of which 19 were newly identified.

Animals↗
PubMed2003

Pyridostigmine bromide modulates topical irritant-induced cytokine release from human epidermal keratinocytes and isolated perfused porcine skin.

Gulf War personnel were given pyridostigmine bromide (PB) as a prophylactic treatment against organophosphate nerve agent exposure, and were exposed to the insecticide permethrin and the insect repellent N,N-diethyl-m-toluamide (DEET). The purpose of this study was to assess the effects of PB to modulate release of inflammatory biomarkers after topical chemical exposure to chemical mixtures containing permethrin and DEET applied in ethanol or water vehicles. Treatments were topically applied to isolated perfused porcine skin flaps (IPPSFs). Concentrations of interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E(2) (PGE(2)) were assayed in perfusate to probe for potential inflammatory effects after complex mixture application. IPPSFs (n=4/treatment) were topically dosed with mixtures of permethrin, DEET, and permethrin/DEET, in ethanol. Each treatment was repeated with perfusate spiked with 50 ng/ml of PB. Perfusate was also spiked with 30 ng/ml diisopropylfluorophosphate to simulate low level organophosphate nerve agent exposure. Timed IPPSF venous effluent samples (0.5,1,2,4, and 8 h) were assayed by ELISA for IL-8 and TNF-alpha and by EIA for PGE(2). Overall, PB infusion caused a decrease or IL-8 and PGE(2) release. Effects on TNF-alpha were vehicle dependent. To probe the potential mechanism of this PB effect, human epidermal keratinocyte HEK cell cultures were exposed to permethrin DEET permethrin/DEET, with and without PB in DMSO. IL-8 was assayed at 1, 2, 4, 8, 12 and 24 h. PB suppressed IL-8 in permethrin and ethanol treatment from 4 to 24 h confirming the IPPSF results. In conclusion, these studies suggest that systemic exposure to PB suppressed IL-8 release at multiple time points in two skin model systems. This interaction merits further study.

Administration, Cutaneous↗
PubMed1995

High-performance capillary electrophoretic separation of proteins and peptides using a bonded hydrophilic phase capillary.

High performance capillary electrophoresis (HPCE) was applied to the separation of protein and peptide mixtures with molecular masses ranging from 1300 to 96000 Da using a new bonded hydrophilic phase capillary, CElect-P150. This coated capillary reduces the interaction between proteins and silanol groups in capillary walls, allowing a complete recovery of the proteins and peptides of interest. HPCE was also used for the analysis of a complex mixture of tryptic fragments and to monitor the process of enzymatic digestion. Moreover, using a CElect-P150 capillary, highly reproducible analysis was possible without preconditioning the capillary with acid or basic solutions before each new analysis.

Animals↗
PubMed2005

Proteasome-inhibited dendritic cells demonstrate improved presentation of exogenous synthetic and natural HLA-class I peptide epitopes.

The design and successful clinical implementation of cancer vaccines targeting the induction of T-cell mediated immunity is a rapidly evolving field that is hampered by an empirical selection of antigen and adjuvant. In particular, vaccines using defined tumor-associated peptide epitopes elicit only a restricted T-cell repertoire in a minority of patients. In this regard, vaccines comprising the whole spectrum of antigens presented by individual autologous tumors would be advantageous. In an in vitro model, we evaluated the capacity of naturally processed Epstein-Barr virus-transformed B-lymphoblastoid-cell line (LCL)-derived peptides to activate virus-specific CD8+ T cells of seropositive healthy individuals. While bulk peptides obtained by mild acid elution from LCL contained multiple T-cell epitopes, this complex mixture of peptides was poorly immunogenic, even when presented by mature dendritic cells (DC). Pretreatment of DC with proteasome inhibitors strongly enhanced the immunogenicity of single viral synthetic as well as bulk LCL peptides. This was most likely achieved by facilitating the loading of exogenous epitopes onto DC-associated HLA-class I complexes in the face of significant inter-peptide competition for such loading. Our results suggest that proteasome inhibitors may be used to increase the antigenicity of mature DC pulsed with exogenous synthetic or naturally processed peptide epitopes in vaccination trials.

Antigen Presentation↗
PubMed1980

A general and sensitive chemical method for sequencing the glycosyl residues of complex carbohydrates.

This paper describes a new glycosyl-sequencing method. This method was made possible by the ability to fractionate complex mixtures of peralkylated oligosaccharides by reversed-phase, high-pressure liquid chromatography. The fractionation ability of the reversed-phase system allows the isolation and subsequent unambiguous identification by g.l.c.-m.s. of disaccharides, almost all trisaccharides, and, in some cases, tetrasaccharides generated by successive partial acid hydrolysis, reduction, and ethylation of a permethylated, complex carbohydrate. As these small oligosaccharides overlap within the unhydrolyzed, complex carbohydrate, the oligosaccharide sequences may be pieced together, and, with the glycosyl-linkage composition of the intact complex carbohydrate, can be used to determine the glycosyl sequence of the complex carbohydrate. The details of the sequencing method are illustrated by the elucidation of the glycosyl sequences of three complex carbohydrates. These examples demonstrate the wide variety of complex carbohydrates whose structures can be ascertained by the new sequencing technique. Two of the examples are the commercially available polysaccharides, lichenan and xanthan, whose structures have already been reported. The other example is a nonasaccharide derived from xyloglucan, a structural polymer of plant cell-walls. The glycosyl residues of the complex carbohydrates studied include hexosyl, deoxyhexosyl, pentosyl, glycosyluronic, and pyruvic acetal-substituted hexosyl residues. It will be demonstrated that the new glycosyl-sequencing technique is not compromised by the presence, in the carbohydrate to be analyzed, of glycosyl linkages possessing very different acid labilities. Two major advantages of this sequencing technique are that it is relatively rapid and that it requires only milligram quantities of carbohydrate.

Carbohydrate Sequence↗
PubMed1997

Comprehensive on-line LC/LC/MS of proteins.

This is a description of a comprehensive two-dimensional liquid chromatography (LC) system for the separation of protein mixtures. This system uses cation-exchange chromatography followed by reversed-phase chromatography (RPLC). The two LC systems are coupled by an eight-port valve equipped with two storage loops and under computer control. The RPLC effluent is sampled by both a UV detector and an electrospray mass spectrometer. In this way, complex mixtures of large biomolecules can be rapidly separated, desalted, and analyzed for molecular weight in less than 2 h. The system's utility is demonstrated with a mixture of standards and an Escherichia coli cell lysate.

Chromatography, Ion Exchange↗
PubMed1999

Effect of welding fume solubility on lung macrophage viability and function in vitro.

It was shown previously that fumes generated from stainless steel (SS) welding induced more pneumotoxicity and were cleared from the lungs at a slower rate than fumes collected from mild steel (MS) welding. These differences in response may be attributed to the metal composition of SS and MS welding fumes. In this study, fumes with vastly different metal profiles were collected during gas metal arc (GMA) or flux-covered manual metal arc (MMA) welding using two different consumable electrodes, SS or MS. The collected samples were suspended in saline, incubated for 24 h at 37 degrees C, and centrifuged. The supernatant (soluble components) and pellets (insoluble particulates) were separated, and their effects on lung macrophage viability and the release of reactive oxygen species (ROS) by macrophages were examined in vitro. The soluble MMA-SS sample was shown to be the most cytotoxic to macrophages and to have the greatest effect on their function as compared to the GMA-SS and GMA-MS fumes. Neither the soluble nor insoluble forms of the GMA-MS sample had any marked effect on macrophage viability. The flux-covered MMA-SS fume was found to be much more water soluble as compared to either the GMA-SS or the GMA-MS fumes. The soluble fraction of the MMA-SS samples was comprised almost entirely of Cr. The small fraction of the GMA-MS sample that was soluble contained Mn with little Fe, while a more complex mixture was observed in the soluble portion of the GMA-SS sample, which contained Mn, Ni, Fe, Cr, and Cu. Data show that differences in the solubility of welding fumes influence the viability and ROS production of macrophages. The presence of soluble metals, such as Fe, Cr, Ni, Cu, and Mn, and the complexes formed by these different metals are likely important in the pulmonary responses observed after welding fume exposure.

Air Pollutants, Occupational↗
PubMed1988

Derivatization of prostaglandins and related compounds to (methoxime) alkyl ester alkyl ether derivatives for gas chromatographic analysis.

Rapid and convenient methods are described for the exhaustive derivatization of carbonyl, carboxyl and hydroxyl groups of prostaglandins and related compounds to methoxime, alkyl ester and alkyl ether compounds respectively. Optimal reaction conditions were established for each group. The reactions were carried out in polar aprotic solvents. Alkyl ester alkyl ether derivatives were obtained quantitatively and rapidly in one step with n-alkyl (C1-C4) halides in the presence of sodium hydroxide. Methyl ester methyl ether derivatives have the highest volatility, but propyl ester propyl ether derivatives improved the separation of complex mixtures. The carbonyl group sometimes induced side-products, so the carbonyl group was converted into methoxime. Methoximation was achieved quantitatively by using methoxylamine in the presence of hydrochloric acid or sodium hydroxide, followed by alkylation in same reaction medium. Methoximation gave syn- and anti-isomers, which were separated chromatographically, decreasing the resolution for complex samples.

Alkylation↗
PubMed2003

Pre-mRNA splicing: awash in a sea of proteins.

What's in a spliceosome? More than we ever imagined, according to recent reports employing proteomics techniques to analyze this multi-megadalton machine. As of 1999, around 100 splicing factors were identified (Burge et al., 1999); however, that number has now nearly doubled due primarily to improved purification of spliceosomes coupled with advances in mass spectrometry analyses of complex mixtures. Gratifyingly, most of the previously identified splicing factors were found in the recent mass spec studies. Nonetheless, the number of new proteins emerging with no prior connection to splicing was surprising. Without functional validation, it would be premature to label these proteins as bona fide splicing factors. Yet many were identified multiple times in complexes purified under diverse conditions or from different organisms. Another recurring theme regards the dynamic nature of spliceosomal complexes, which may be even more intricate than previously thought.

Animals↗
PubMed2003

Aggregation and fluorescence quenching of chlorophyll a of the light-harvesting complex II from spinach in vitro.

The salt-induced aggregation of the light-harvesting complex (LHC) II isolated from spinach and its correlation with fluorescence quenching of chlorophyll a is reported. Two transitions with distinctly different properties were observed. One transition related to salt-induced fluorescence quenching takes place at low salt concentration and is dependent both on temperature and detergent concentration. This transition seems to be related to a change in the lateral microorganization of LHCII. The second transition occurs at higher salt concentration and involves aggregation. It is independent of temperature and of detergent at sub-cmc concentrations. During the latter transition the small LHCII sheets (approximately 100 nm in diameter) are stacked to form larger aggregates of approximately 3 microm diameter. Based on the comparison between the physical properties of the transition and theoretical models, direct and specific binding of cations can practically be ruled out as driving force for the aggregation. It seems that in vitro aggregation of LHCII is caused by a complex mixture of different effects such as dielectric and electrostatic properties of the solution and surface charges.

Chlorophyll↗
PubMed1990

Ion mobility detection of polydimethylsilicone oligomers following supercritical fluid chromatographic separation.

A mixture of polydimethylsilicones (Dow Corning 200), average molecular weight 2000 a.m.u., was separated by simultaneous density and temperature-programmed supercritical fluid chromatography and detected by ion mobility detection. Ion mobility spectra were captured by Fourier transform ion mobility spectrometry. Using information from these spectra it was possible to selectively detect a single compound in the complex mixture. A detector temperature investigation demonstrated that, for the efficient transfer of high-molecular-weight compounds from the column to the detector, the interface to the detector must be heated. Using a 50 microns I.D. column, a Guthrie-type restrictor and a detection temperature of 250 degrees C, as many as 70 oligomers were separated and detected.

Chromatography, High Pressure Liquid↗
PubMed1998

The influence of odor type on the discrimination and identification of odorants in multicomponent odor mixtures.

Using a limited set of odorants, previous studies have indicated that the ability of humans to discriminate and identify the components of olfactory mixtures is limited to approximately four. However, the ability to generalize these results may have been limited by specific neural or cognitive interactions among the particular odorants used. In the present experiment, 41 subjects examined the influence of odor type (different individual odorants), from two very different odor sets, on the perception of the components of complex mixtures. One set contained odors that were selected by an expert panel to blend well in mixtures (good blenders), whereas the other contained odors that blended poorly in mixtures (poor blenders). The stimuli were common, dissimilar odorants of equivalent, moderate intensity, each of which was a single chemical. A computer-controlled air dilution olfactometer delivered a single odorant or a mixture containing up to eight odorants. Although the poor blenders were more easily discriminated, this superiority was displayed within a narrow range, and the ability of subjects to identify mixture components with either odor set was limited to approximately four. The results indicate that, whereas odor type can alter which odorants will be perceived in a mixture, the limited capacity to discriminate mixture components is independent of the type of odorants. These findings are discussed in terms of their implications for olfactory coding.

Adolescent↗