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PubMed2001

Quantum yield of singlet oxygen production by monomeric and aggregated forms of hematoporphyrin derivative.

Hematoporphyrin derivative (HpD) is a complex mixture of monoporphyrinic compounds, including hematoporphyrin, and oligomers containing up to eight porphyrin units. In methanol a sensitizer concentration-independent quantum yield of 0.64 is measured for the HpD-induced production of singlet molecular oxygen O2 (1Delta(g)). This finding is consistent with the dye components remaining unassociated in this solvent. In water pH 7.4 HpD consists of a complex mixture of non-aggregated and self- and cross-aggregated monoporphyrinic and oligomeric species, and the quantum yield of O2 (1Delta(g)) formation decreases significantly with increasing HpD concentration due to the lower quantum yield of aggregates. These variations can be quantitatively described in terms of a monomer-dimer equilibrium, with quantum yields of 0.64 and 0.11, respectively, for monomers and dimers. The yields of unassociated species are identical in methanol and water.

Dimerization↗

PubMed2004

Aerobic biodegradation of two commercial naphthenic acids preparations.

Naphthenic acids (NAs) have a variety of commercial uses including as emulsifiers and wood preservatives. They have been identified as being the main component responsible for the acute toxicity in produced waters in the oil sands operations in northeastern Alberta, Canada. NAs comprise a complex mixture of alkyl-substituted acyclic and cycloaliphatic carboxylic acids, with the general chemical formula CnH(2n+Z)O2, where n indicates the carbon number and Z specifies hydrogen deficiency from ring formation. In this study, commercial preparations of NAs were shown to be degraded in aerobic cultures from oil sands process-affected waters. High-performance liquid chromatography and gas chromatography-mass spectrometry (GC-MS) were used to monitor the concentrations and composition of the NA mixtures during biodegradation. Within 10 days of incubation, the NAs concentrations dropped from about 100 to <10 mg/L. This was accompanied by the release of about 60% of carbon from the NAs as CO2 and the reduction of toxicity of the culture supernatant, as measured by the Microtox assay. GC-MS results demonstrated that biodegradation changes the composition of the complex mixture of these NAs and that the lower molecular weight acids (with n = 5-13) were degraded more readily than the high molecular weight acids.

Bacteria, Aerobic↗

PubMed1996

Performance and application potential of ion-molecule reaction mass spectrometry (IMR-MS) in the analysis of complex gas mixtures.

The mild ionisation technique of IMR mass spectrometry in combination with a tuneable cell for the charge transfer reaction allows to study mixtures of small molecules with low fragmentation and chemical selectivity. Temporal resolution down to 100 ms and quasisimultaneous multicomponent trend analyses can be combined in a flexible way responding to changing analytical requirements. The research instrument of ATOMIKA provides digital control over a range of parameters and ionisation sources. Its performance is characterised in several applications ranging from purity tests over the detection of small oxygenate organic molecules to more complex reaction systems of heterogeneous catalytic performance testing.

Journal Article↗

PubMed2000

The relative toxicity of compounds in mainstream cigarette smoke condensate.

Many different in vivo and in vitro tests are currently used to assess the toxicity of chemicals and complex mixtures such as cigarette smoke condensate. In vivo tests include assays in rodents to determine carcinogenicity, tumorigenicity and reproductive effects In vitro tests of mutagenicity are conducted with both bacterial and mammalian cell systems. A first step towards lowering the toxicity of cigarette smoke condensate is the identification of the relevant compound However, changing the concentration of a given smoke component may not linearly alter the biological activity of the complex mixture due to interactive effects. The "effective toxicity" of a chemical constituent is a function of the concentration, the metabolic fate, the potency in in vivo and in vitro assays, and the ability to reach the target tissues. The logarithm of the octanol-water partition coefficient (log P) is an important parameter since it affects metabolism, biological transport properties and intrinsic toxicity. Using concentration data from the International Agency for Cancer Research (IARC), biological activity data from the Registry of Toxic Effects of Chemical Substances (RTECS) database and measured and calculated log P values, we have rank ordered some of the important compounds in cigarette smoke condensate by their measured or potential toxicity. Condensates from different cigarette brands, tar categories and styles vary in their concentrations of these compounds. Chemicals of greater commercial or scientific interest may be toxicity tested more extensively, thereby increasing the probability of positive test results and highlighting the need for consideration of structure-activity relationships.

Acetaldehyde↗

PubMed2006

Peptide-based phosphoproteomics with immobilized metal ion chromatography.

The identification of protein phosphorylation sites has always been a challenging task, traditionally involving large amounts of radioactive phosphorus and high-performance liquid chromatography separation and Edman sequencing of phosphopeptides. The rapid development of mass spectrometric methods has advanced protein research significantly, and the identification of in vivo post-translational modifications of even rare proteins is now possible. Even with the new generation of machines, however, phosphopeptides do not lend themselves easily to mass spectrometric analysis. In complex mixtures of peptides, phosphopeptides are often difficult to detect because of suppression effects during ionization. This problem can be largely solved by affinity purification/enrichment of the phosphopeptides, and immobilized metal ion affinity chromatography (IMAC) on chelated Fe3+ or other metal ions has emerged as the simplest and most useful method. IMAC has been useful to identify several in vivo phosphorylation sites of individual proteins, but is easier to apply to complex mixtures. We describe a complete protocol as it has been used for Arabidopsis plasma membranes, and note where it can be adapted for soluble protein mixtures.

Arabidopsis↗

PubMed2004

Fully automated chip-based mass spectrometry for complex carbohydrate system analysis.

Carbohydrates represent a major class of biopolymers, which occur in nature either as oligosaccharides or glycoconjugates, in which the sugar moiety is linked to proteins or lipids. The significance of mass spectrometry for highly sensitive analysis of complex carbohydrates increased after the introduction of the electrospray ionization and matrix assisted laser desorption/ionization methods and the possibility of tandem MS for sequencing of single molecular species in complex mixtures. Rapid and sensitive characterization of carbohydrates in biological systems by automated nanoscale liquid delivery and chip-based electrospray interface techniques have not been developed so far. In this contribution, the implementation and optimization of a fully automated chip-based nanoelectrospray assembly (NanoMate system), operating in the negative ion mode, in combination with QTOF-tandem MS for mapping/sequencing and computer-assisted structure assignment for carbohydrate components in complex mixtures is presented.

Automation↗

PubMed2000

Capillary electrochromatography of cholesterol and its ester derivatives.

Separation of cholesterol and its ester derivatives using micellar electrokinetic chromatography is a challenge due to the extreme hydrophobicity of these compounds. In this work, an isocratic capillary electrochromatography (CEC) method has been developed to separate a complex mixture of cholesterol and its 12-ester derivatives. The proportions of mobile phase (tetrahydrofuran, acetonitrile, water), as well as the effects of acid modifiers, buffer concentrations, voltage, and temperature on the separation of cholesterol derivatives were investigated. Addition of a polymeric surfactant, poly(sodium N-undecanoyl-L-glycinate), to the mobile phase reduced migration time and improved resolution of the analytes. The CEC method developed allows baseline separation of a complex mixture of cholesterol and 12 ester derivatives in less than 40 min. Finally, the method is applied to the characterization of cholesterol, cholesterol linoleate, and cholesterol oleate extracted from atherosclerotic plaque deposits in the arterial walls of a human aorta.

Aged↗

PubMed2002

Top down characterization of larger proteins (45 kDa) by electron capture dissociation mass spectrometry.

The structural characterization of proteins expressed from the genome is a major problem in proteomics. The solution to this problem requires the separation of the protein of interest from a complex mixture, the identification of its DNA-predicted sequence, and the characterization of sequencing errors and posttranslational modifications. For this, the "top down" mass spectrometry (MS) approach, extended by the greatly increased protein fragmentation from electron capture dissociation (ECD), has been applied to characterize proteins involved in the biosynthesis of thiamin, Coenzyme A, and the hydroxylation of proline residues in proteins. With Fourier transform (FT) MS, electrospray ionization (ESI) of a complex mixture from an E. coli cell extract gave 102 accurate molecular weight values (2-30 kDa), but none corresponding to the predicted masses of the four desired enzymes for thiamin biosynthesis (GoxB, ThiS, ThiG, and ThiF). MS/MS of one ion species (representing approximately 1% of the mixture) identified it with the DNA-predicted sequence of ThiS, although the predicted and measured molecular weights were different. Further purification yielded a 2-component mixture whose ECD spectrum characterized both proteins simultaneously as ThiS and ThiG, showing an additional N-terminal Met on the 8 kDa ThiS and removal of an N-terminal Met and Ser from the 27 kDa ThiG. For a second system, the molecular weight of the 45 kDa phosphopantothenoylcysteine synthetase/decarboxylase (CoaBC), an enzyme involved in Coenzyme A biosynthesis, was 131 Da lower than that of the DNA prediction; the ECD spectrum showed that this is due to the removal of the N-terminal Met. For a third system, viral prolyl 4-hydroxylase (26 kDa), ECD showed that multiple molecular ions (+98, +178, etc.) are due to phosphate noncovalent adducts, and MS/MS pinpointed the overall mass discrepancy of 135 Da to removal of the initiation Met (131 Da) and to formation of disulfide bonds (2 x 2 Da) at C32-C49 and C143-C147, although 10 S-S positions were possible. In contrast, "bottom up" proteolysis characterization of the CoaBC and the P4H proteins was relatively unsuccessful. The addition of ECD substantially increases the capabilities of top down FTMS for the detailed structural characterization of large proteins.

Amino Acid Sequence↗

PubMed2005

18O-labeling quantitative proteomics using an ion trap mass spectrometer.

We describe a method for simultaneous identification and quantitation of proteins within complex mixtures. The method consists of 18O-labeling, a simple stable isotope-coding that requires merely enzymatic digestion in 18O-water, in combination with a capillary-liquid chromatography electrospray ion-trap mass spectrometer. In a separate experiment using the same sample and a spike test, we demonstrate that the difference ration was calculated accurately using the 18O-labeling method even if the protein was part of a complex mixture. Our data also suggest that the accuracy of the quantitation can be improved by averaging the difference ratios of several peptides. In comparing our method with the isotope-coded affinity tag (ICAT) method, we show that the 18O-labeling method has the advantages of better recovery and fewer isotope effects. Therefore, the 18O-labeling method is a powerful tool for large-scale proteomics applications.

Animals↗

PubMed2005

A colorimetric sensor array for organics in water.

Molecular recognition of organic compounds in aqueous solutions is inherently challenging due to the potential interference from the very high concentration of water. Here we present a simple colorimetric sensor array that probes a wide range of chemical properties. By printing hydrophobic dyes on a hydrophobic membrane, sensor arrays are easily prepared that provide substantial chemical selectivity for the identification and quantification of various organics (both single compounds and complex mixtures) dissolved in water. It is possible to differentiate easily even among closely related organic compounds. Upon immersion in aqueous solutions, digital imaging of the dye array before and after exposure to an analyte provide a color change profile that is a fingerprint for the organic components of the solution. Facile identification of a wide variety of aqueous organic solutions is possible over a concentration range of 0.1 M to 1 muM. Complex mixtures present no inherent difficulty; for example, a series of commercial soft drinks were easily distinguished using the colorimetric sensor array approach.

Colorimetry↗

PubMed2004

Composition of the black crusts from the Saint Denis Basilica, France, as revealed by gas chromatography-mass spectrometry.

The organic fraction of black crusts from Saint Denis Basilica, France, is composed of a complex mixture of aliphatic and aromatic compounds. These compounds were studied by two different analytical approaches: tetramethyl ammonium hydroxide (TMAH) thermochemolysis in combination with gas chromatography-mass spectrometry (GC-MS), and solvent extraction, fractionation by silica column, and identification of the fraction components by GC-MS. The first approach, feasible at the microscale level, is able to supply fairly general information on a wide range of compounds. Using the second approach, we were able to separate the complex mixture of compounds into four fractions, enabling a better identification of the extractable compounds. These compounds belong to different classes: aliphatic hydrocarbons (nalkanes, n-alkenes), aliphatic and aromatic carboxylic acids (n-fatty acids, alpha,omega-dicarboxylic acids, and benzenecarboxylic acids), polycyclic aromatic hydrocarbons (PAH), and molecular biomarkers (isoprenoid hydrocarbons, diterpenoids, and triterpenoids). With each approach, similar classes of compounds were identified, although TMAH thermochemolysis failed to identify compounds present at low concentrations in black crusts. The two proposed methodological approaches are complementary, particularly in the study of polar fractions.

Carboxylic Acids↗

PubMed1989

Phagocytosis in Dictyostelium discoideum is inhibited by antibodies directed primarily against common carbohydrate epitopes of a major cell-surface plasma membrane glycoprotein.

Using a water-soluble, reversible biotinylating reagent, we retrieved three surface-exposed proteins from a complex mixture of crude membrane proteins. The compound, sulfosuccinimidyl 2-(biotinamido)ethyl-1-3'-dithiopropionate (sulfo-NHS-SS-biotin), which has a cleavable disulfide bond, was used to label Dictyostelium discoideum amebae. Cells were lysed and a crude membrane preparation was isolated and solubilized with Triton X-100. Biotinylated molecules were bound to immobilized streptavidin and then eluted from the affinity matrix with dithiothreitol. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that out of the original complex mixture of detergent-solubilized membrane proteins, three major species at 130, 100, and 77 kDa were specifically bound and eluted with thiol reagents. These three proteins were glycoproteins (gp) since they bound concanavalin A. As demonstrated by one-dimensional peptide mapping, the retrieved gp130 and gp100 also were present in specialized plasma membrane subdomains called contact regions which are regions of cell-cell cohesion isolated from aggregated, developed amebae. This finding provides preliminary evidence that the two proteins may be involved in cell-cell interactions during both the vegetative and aggregation stages of the D. discoideum life cycle. The retrieved gp130 species has a relative mobility on SDS-gels similar to that of gp126, a surface-exposed glycoprotein. gp126 has been suggested to play roles both as a phagocytosis receptor and as a cohesion molecule (C.M. Chadwick, J.E. Ellison, and D.R. Garrod, (1984) Nature (London) 307, 646). To test if the retrieved gp130 was the same as gp126, a polyclonal antiserum was raised against gel-purified, endoglycosidase F-treated gp130. The immune serum recognized epitopes, apparently carbohydrates, present on many D. discoideum membrane proteins. Univalent IgG fragments from this antiserum inhibited phagocytosis, suggesting that anti-carbohydrate activity was responsible for the functional inhibition of phagocytosis.

Dictyostelium↗

PubMed2002

Oligosaccharide composition and pheromonal activity of male tergal gland secretions of the German cockroach, Blattella gennanica (L.).

In the sequential courtship behavior of the German cockroach, Blattella germanica (Dictyoptera: Blattellidae), females feed on the tergal gland secretions from the male tergites. The phagostimulative components in the male secretions were composed of a complex mixture of oligosaccharides and phospholipids. Besides seven oligosaccharides previously identified, two new trisaccharides, O-alpha-D-glucopyranosyl-(1 --> 6)-alpha-D-glucopyranosyl alpha-D-glucopyranoside and O-alpha-glucopyranosyl-(1 --> 4)-O-alpha-glucopyranosyl-(1 --> 1)-myo-inositol, were characterized as the sugar components of the secretions. The activity of the sugar components was compared in combination with the lipid fraction. Individual oligosaccharides exhibited the activity either alone or synergistically with the phospholipids. A complex mixture of these primary substances in the tergal gland secretions, thus, serves as a pheromonal phagostimulant in the precopulatory behavior, strongly eliciting feeding response in the female cockroach.

Animals↗

PubMed1994

Evaluation of chemopreventive agents against breast cancer and proposed strategies for future clinical intervention trials.

This review is based on literature data pertaining to the use of single agents or complex mixtures that have been shown to offer protection against mammary cancer in rodents. The species and strains of animals, dose, route, frequency and duration of carcinogen administration, as well as types, levels, route of administration and duration of chemopreventive treatments are described in detail. A brief description regarding the mechanism(s) responsible for the chemopreventive action of each class of agent is provided. The reader is encouraged to use additional sources for detailed information. The epidemiological observations that implicate certain agents or complex mixtures as playing a role in reducing human breast cancer are also reported and a comparison is made with results from assays in laboratory animals. Possible intermediate biomarkers that indicate risk for breast cancer are discussed. The purpose of this review is to provide the reader with a fairly concise knowledge of the subject. The information in this review is gathered together to stimulate studies toward appropriate strategies for future clinical chemoprevention trials.

Animals↗

PubMed2000

The quantitive chlorioallantoic membrane test using trypan blue stain to predict the eye irritancy of liquid scintillation cocktails.

The chorioallantoic membrane-trypan blue staining assay (CAM-TBS) is used to evaluate the potential ocular irritation caused by liquid scintillation cocktails constituted by complex mixtures, including surfactants and other potential irritants. The harmful effect of these substances is determined by the amount of trypan blue adsorbed by the CAM. In the membrane previously treated with the scintillation liquids this amount was concentration dependent only in certain cases, irrespective of the water solubility of the mixtures. In general, it showed a high correlation (r=0.986) with the scores obtained in the Draize eye irritation test. In the present study, only two cocktails presented values of trypan blue adsorption higher than those recorded for their eye irritation in vivo, irrespective of the solvent nature. Unlike the classical HET-CAM procedure, this assay is objective and allows the evaluation of opaque and coloured substances without interfering in the determination of irritancy. Despite these advantages, the method is not suitable for complex mixtures of products that induce ocular irritation in small quantities.

Allantois↗

PubMedDate not supplied

Applications of an automated apparatus for two-dimensional electrophoresis, Model TEP-1, for microsequence analyses of proteins.

Two-dimensional electrophoresis has become a rapid and powerful separation method for the preparation of samples for sequence analysis depending on the development of automated microsequencers accompanied by a blotting technique. We have constructed an automated apparatus for two-dimensional electrophoresis, Model TEP-1, which allows precise analytical runs under computer-control with a fully automated transfer from the first- to the second-dimensional gel. In the present paper we describe several applications of TEP-1 followed by blotting and sequencing to reveal the primary structure of proteins in a complex mixture. Several known and unknown proteins were analyzed. Enzymatic digests of proteins could be directly applied onto the TEP-1, in which a gel of lower molecular weight proteins was used as the second dimension. Subsequent blotting and sequencing provided internal sequence information. The use of TEP-1, followed by blotting, provides spots containing ca. 10-100 pmol protein. These figures permitted the identification of 10-40 amino acid residues from the N-terminus. The TEP-1 provides a simple and rapid separation of complex mixtures obtained from natural sources and chemical, biological or genetic products.

Amino Acid Sequence↗

PubMed2003

LC-NMR-MS in drug discovery.

Nuclear magnetic resonance spectroscopy (NMR) is arguably the most versatile analytical platform for complex mixture analysis. Specifically, interfacing liquid chromatography with parallel NMR and mass spectrometry (LC-NMR-MS) gives comprehensive structural data on metabolites of novel drugs in development. Applications in natural product, combinatorial chemistry and drug metabolism studies are reviewed. Microcoil probes and capillary separation methods have enormous potential. Recent innovations to improve NMR detection limits include CryoFlowProbes and on-line solid-phase extraction (LC-SPE-NMR). These state-of-the-art analytical platforms are widely applicable to identifying novel candidate drugs from diverse complex mixtures within a drug discovery strategy.

Chemistry, Pharmaceutical↗

PubMed1995

Single cells as biosensors for chemical separations.

A biosensor system based on the response of living cells was demonstrated that can detect specific components of a complex mixture fractionated by a microcolumn separation technique. This system uses ligand-receptor binding and signal-transduction pathways to biochemically amplify the presence of an analyte after electrophoretic separation. The transduced signal was measured by means of two approaches: (i) fluorescence determination of intracellular calcium concentrations in one or more rat PC-12 cells and (ii) measurement of transmembrane current in a Xenopus laevis oocyte microinjected with messenger RNA that encodes a specific receptor. This analysis system has the potential to identify biologically active ligands present in a complex mixture with exceptional sensitivity and selectivity.

Acetylcholine↗