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PubMed1991

Nanoscale packed-capillary liquid chromatography coupled with mass spectrometry using a coaxial continuous-flow fast atom bombardment interface.

Nanoscale packed-capillary liquid chromatography (LC) columns have been coupled with mass spectrometry (MS) using a coaxial continuous-flow fast atom bombardment interface. The combined system has been applied to the analysis of mixtures of peptides, including synthetic mixtures of bioactive peptides and tryptic digests of proteins. Nanoscale packed-capillary columns offer two principal advantages for LC/MS analysis--high chromatographic separation efficiencies and low mobile-phase flow rates. The high separation efficiencies facilitate the separation of complex mixtures, and the low mobile-phase flow rates reduce problems with coupling the LC effluent with the high-vacuum, high-voltage environment of sector MS ion sources. The columns used in this work were 50- or 75-micron i.d., 1-2 m long, packed with 10-micron C18 particles, using mobile-phase flow rates of 50-350 nL/min.

Animals↗
PubMed1999

Use of mass spectrometry To rapidly characterize the heterogeneity of bovine alpha-lactalbumin.

From a bovine whey protein fraction the nonglycosylated and glycosylated alpha-lactalbumin fractions were isolated by gel-permeation chromatography followed by reversed-phase high-performance liquid chromatography. Both fractions were studied by electrospray-ionization mass spectrometry (ESI-MS). For the nonglycosylated fraction, a mass of 14 178 Da was found, which was in accordance with the known amino acid sequence of the protein. The glycosylated fraction appeared to be a mixture of components with mass values ranging from ca. 15 840 to 16 690 Da. Given the published carbohydrate composition of the whole glyco-alpha-lactalbumin fraction, these masses could be matched to those of 14 differently glycosylated forms of alpha-lactalbumin. Further support for these forms was obtained from the results of a separate mass spectrometric analysis of the mixture of oligosaccharides released from the protein by treatment with peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase F. ESI-MS was found to be a powerful tool to gain insight into the composition of the complex mixture of glycoforms of alpha-lactalbumin without the need of further purification of these forms or of the oligosaccharides released thereof.

Animals↗
PubMed2005

Thyroid-hormone-disrupting chemicals: evidence for dose-dependent additivity or synergism.

Endocrine disruption from environmental contaminants has been linked to a broad spectrum of adverse outcomes. One concern about endocrine-disrupting xenobiotics is the potential for additive or synergistic (i.e., greater-than-additive) effects of mixtures. A short-term dosing model to examine the effects of environmental mixtures on thyroid homeostasis has been developed. Prototypic thyroid-disrupting chemicals (TDCs) such as dioxins, polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers have been shown to alter thyroid hormone homeostasis in this model primarily by up-regulating hepatic catabolism of thyroid hormones via at least two mechanisms. Our present effort tested the hypothesis that a mixture of TDCs will affect serum total thyroxine (T4) concentrations in a dose-additive manner. Young female Long-Evans rats were dosed via gavage with 18 different polyhalogenated aromatic hydrocarbons [2 dioxins, 4 dibenzofurans, and 12 PCBs, including dioxin-like and non-dioxin-like PCBs] for 4 consecutive days. Serum total T4 was measured via radioimmunoassay in samples collected 24 hr after the last dose. Extensive dose-response functions (based on seven to nine doses per chemical) were determined for individual chemicals. A mixture was custom synthesized with the ratio of chemicals based on environmental concentrations. Serial dilutions of this mixture ranged from approximately background levels to 100-fold greater than background human daily intakes. Six serial dilutions of the mixture were tested in the same 4-day assay. Doses of individual chemicals that were associated with a 30% TH decrease from control (ED30), as well as predicted mixture outcomes were calculated using a flexible single-chemical-required method applicable to chemicals with differing dose thresholds and maximum-effect asymptotes. The single-chemical data were modeled without and with the mixture data to determine, respectively, the expected mixture response (the additivity model) and the experimentally observed mixture response (the empirical model). A likelihood-ratio test revealed statistically significant departure from dose additivity. There was no deviation from additivity at the lowest doses of the mixture, but there was a greater-than-additive effect at the three highest mixtures doses. At high doses the additivity model underpredicted the empirical effects by 2- to 3-fold. These are the first results to suggest dose-dependent additivity and synergism in TDCs that may act via different mechanisms in a complex mixture. The results imply that cumulative risk approaches be considered when assessing the risk of exposure to chemical mixtures that contain TDCs.

Animals↗
PubMed2003

Identification of protein ligands in complex biological samples using intensity-fading MALDI-TOF mass spectrometry.

The easy detection of biomolecular interactions in complex mixtures using a minimum amount of material is of prime interest in molecular and cellular biology research. In this work, a mass spectrometry MALDI-TOF based approach, which we call intensity-fading (IF MALDI-TOFMS), and which was designed for just such a purpose, is reported. This methodology is based on the use of the MALDI ion intensities to detect quickly the formation of complexes between nonimmobilized biomolecules in which a protein is one of the partners (protein-protein, protein-peptide, protein-organic molecule, and protein-nucleic acid complexes). The complex is detected through the decrease (fading) of the molecular ion intensities of the partners as directly compared to the MALDI mass spectrum of the mixture (problem and control molecules) following the addition of the target molecule. The potential of the approach is examined in several examples of model interactions, mainly involving small nonprotein and protein inhibitors of proteases, at both the qualitative and semiquantitative levels. Using this method, different protein ligands of proteolytic enzymes in total extracts of invertebrate organisms have been identified in a simple way. The proposed procedure should be easily applied to the high-throughput screening of biomolecules, opening a new experimental strategy in functional proteomics.

Animals↗
PubMedDate not supplied

Synthesis of selenocysteine peptides and their oxidation to diselenide-bridged compounds.

Using the Fmoc/tBu protection scheme and the p-methoxybenzyl derivative of selenocysteine, the synthesis of related peptides in the selenol-protected form could be optimized by operating the coupling steps in the absence of auxiliary bases and by reducing the piperidine treatment to the minimum time required for quantitative Fmoc cleavage. Under these conditions, beta-elimination of the p-methoxybenzylselenol as the main side reaction of these syntheses, as well as epimerization of the protected selenocysteine, was largely suppressed. Conversion of the selenol- and thiol-protected bis-selenocysteine and selenocysteine, cysteine peptides into the related cyclic monomeric forms by iodine-mediated oxidation failed since a complex mixture of compounds was produced. Cleavage of the selenoether bond with mercuric acetate was found to proceed smoothly, but displacement of the heavy metal ions by treatment with excesses of thiols or hydrogen sulphide was unsuccessful since a stable Hg2+ diselenide complex was obtained. However, oxidation was achieved in good yields by the dimethylsulphoxide/trifluoroacetic acid procedure and the peptides were then used for determining the redox potential of the diselenide and selenide/sulphide bridge, respectively.

Organoselenium Compounds↗
PubMed2006

Hydrothermal reactions of pyruvic acid: synthesis, selection, and self-assembly of amphiphilic molecules.

Selection and self-assembly of organic compounds in aqueous phases must have been a primary process leading to emergent molecular complexity and ultimately to the origin of life. Facile reactions of pyruvic acid under hydrothermal conditions produce a complex mixture of larger organic molecules, some of which are amphiphiles that readily self-assemble into cell-sized vesicular structures. Chemical characterization of major components of this mixture reveals similarities to the suite of organic compounds present in the Murchison carbonaceous chondrite, some of whose molecules also self-assemble into membranous vesicles. Physical properties of the products are thus relevant to understanding the prebiotic emergence of molecular complexity. These results suggest that a robust family of prebiotic reaction pathways produces similar products over a range of geochemical and astrochemical environments.

Catalysis↗
PubMed2004

Characterization of mixtures of alkyl polyglycosides (Plantacare) by liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry.

PURPOSE: Industrial products of alkyl polyglycosides (APGs) are widely used as well tolerated surfactants in washing and cleaning agents. APGs belong to the class of nonionic surfactants and are mixtures of molecules consisting of a hydrocarbon chain with Y carbon atoms linked to X sugar residues. Physicochemical properties of APG products strongly depend on the molecular composition. Therefore, a detailed analytical investigation of technical-grade APGs is presented. METHODS: Complex mixtures of alkyl/alkenyl polyglycosides (APGs, Plantacare) and mixtures of APGs with other surfactants were analyzed by reversed-phase liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (LC/ESI-QTOF-MS) using methanol-water as mobile phase and gradient elution. RESULTS: Analytes were separated according to the chain length of the alkyl homologs. Under the soft ionization of the ESI technique, mainly [M+Na]+ ions were observed, which proved to be very stable. Additionally [M+H]+ ions were detected. The QTOF mass spectrometer allows to identify even high molecular mass components in the mixtures, and APGs up to eight sugar residues were found. From these data, the alkyl/alkenyl chain lengths and the degree of oligomerization of the sugar moiety for different technical-grade APGs were calculated. Using MS/MS experiments, additional structural and chemical information was obtained. CONCLUSIONS: The presented LC/ESI-QTOF-MS approach allows to analyze and characterize various APG products, such as Plantacare. The ability of this LC/ESI-QTOF-MS approach to analyze mixtures of APGs with other surfactants is demonstrated.

Chemistry, Pharmaceutical↗
PubMed2002

A mathematical algorithm to identify toxicity and prioritize pollutants in field sediments.

In order to evaluate sediment toxicity, a mathematical algorithm was developed to compute the toxicity of multiple component mixtures acting in an additive manner. A statistical approach was devised to determine the presence of potential interactive effects among mixture components. The algorithm uses three kinds of data to obtain an integrative approach to sediment toxicity assessment: Microtox toxicity data (EC50 values), sediment pollutant concentration measurements, and sequential extraction (SEQ) data to investigate metal partitioning. To simplify the analysis of complex mixtures using a prioritization scheme based on intrinsic toxicity and relative abundance, a toxicity index (TI) was employed as an indicator of adverse ecological impact. In general, the ranking of contaminants using the TI approach was found to be most efficient in reducing computational time, and concentrations using bioavailability data from SEQ was found to be the best theoretical predictor of the experimental mixture toxicity value. Only a few pollutants that were present at the greatest abundance were needed to provide a good approximation of the calculated EC50 found when all components were included. Not only does this substantially reduce the computational time needed to determine the EC50, it could in some cases dramatically reduce the pollutant monitoring effort required to track toxicity effectively. This approach would have substantial implications for both risk assessment and for remediation strategies, making them more efficient by focusing on the priority pollutants identified.

Algorithms↗
PubMed1990

A comparison of the results of sequential hydrazinolysis-nitrosation and alkali-mediated cleavage-nitrosation of the O-linked oligosaccharides of gastric mucus glycoproteins.

Analysis of the oligosaccharides released from pig gastric mucus glycopolypeptides by hydrazinolysis showed that degradation had occurred. Nitrosation of the products followed by reduction gave a mixture that had a low content of 2,5-anhydro-D-talitol, which implied destruction of much of the terminal reducing 2-amino-2-deoxy-D-galactose. Under the conditions of hydrazinolysis, cellobiose was largely unchanged but laminaribiose gave a complex mixture that probably contained glucose hydrazone (13C-n.m.r. data). In order to avoid degradation, the hydrazinolysis-nitrosation sequence should be applied to the reduced oligosaccharides released on cleavage with alkali.

Alkalies↗
PubMed1997

Cuticular extracts of five common mantids (Mantodea:Mantidae) of the eastern United States.

We undertook a preliminary investigation of the cuticular extracts of five common mantid species in the eastern United States: Tenodera sinensis (Saussure), T. angustipennis (Saussure) and Mantis religiosa (Linnaeus) introduced from the Old World and Stagmomantis carolina (Johannson) and Bruneria borealis (Scudder), which are New World species. The major components of these mixtures were normal alkanes, predominately hentriacontane, or in the case of the parthenogenic species B. borealis, tritriacontane. Tricontanal was detected in the extracts of all five species, and smaller amounts of other aldehydes and n-tricontanol were detected in some species. Complex mixtures of methyl and dimethylalkanes also were present in these extracts. The composition of the cuticular hydrocarbons of these mantids may be an adaptation for reduction of evaporative water loss in these insects that inhabit open fields.

Alkanes↗
PubMed2001

Mass spectrometry screening of combinatorial mixtures, correlation of measured and predicted electrospray ionization spectra.

Methodology was developed to afford rapid characterization of multicomponent mixtures of small organic molecules prepared by split-and-mix combinatorial synthesis. This methodology involved the use of liquid chromatography mass spectrometry (LC/MS) combined with correlation analysis of measured versus predicted electrospray ionization mass spectra. Low-resolution mass spectra of complex mixtures revealed predictable patterns that confirm library products, assisted in identifying chemical synthesis errors, and assessed overall library integrity. In general, equal signal intensities were observed for most combinatorial mixture components, indicating that differences in electrospray ionization efficiency was not a major limitation to this approach. High-throughput data processing programs and informatics tools were used to speed data analysis and to simplify the presentation of the library characterization results. This approach has been used to characterize combinatorial libraries that were synthesized for a variety of drug-discovery programs. Examples are shown for library formats of 1, 40, 66, 280, and 400 component(s)/well. The applicability of this approach to large combinatorial mixtures should allow direct characterization of massive combinatorial libraries.

Journal Article↗
PubMed2002

Chromatographic isolation of methionine-containing peptides for gel-free proteome analysis: identification of more than 800 Escherichia coli proteins.

A novel gel-free proteomic technology was used to identify more than 800 proteins from 50 million Escherichia coli K12 cells in a single analysis. A peptide mixture is first obtained from a total unfractionated cell lysate, and only the methionine-containing peptides are isolated and identified by mass spectrometry and database searching. The sorting procedure is based on the concept of diagonal chromatography but adapted for highly complex mixtures. Statistical analysis predicts that we have identified more than 40% of the expressed proteome, including soluble and membrane-bound proteins. Next to highly abundant proteins, we also detected low copy number components such as the E. coli lactose operon repressor, illustrating the high dynamic range. The method is about 100 times more sensitive than two-dimensional gel-based methods and is fully automated. The strongest point, however, is the flexibility in the peptide sorting chemistry, which may target the technique toward quantitative proteomics of virtually every class of peptides containing modifiable amino acids, such as phosphopeptides, amino-terminal peptides, etc., adding a new dimension to future proteome research.

Algorithms↗
PubMed2000

A multiple-purpose design approach to the evaluation of risks from mixtures of disinfection by-products.

Drinking water disinfection has effectively eliminated much of the morbidity and mortality associated with waterborne infectious diseases in the United States. Various disinfection processes, however, produce certain types and amounts of disinfection by-products (DBPs), including trihalomethanes (THM), haloacetic acids, haloacetonitriles, and bromate, among others. Human health risks from the ubiquitous exposure to complex mixtures of DBPs are of concern because existing epidemiologic and toxicologic studies suggest the existence of systemic or carcinogenic effects. Researchers from several organizations have developed a multiple-purpose design approach to this problem that combines efficient laboratory experimental designs with statistical models to provide data on critical research issues (e.g., estimation of human health risk from low-level DBP exposures, evaluation of additivity assumptions as useful for risk characterization, estimation of health risks from different drinking water treatment options). A series of THM experiments have been designed to study embryonic development, mortality and cancer in Japanese medaka (Oryzias latipes) and liver and kidney endpoints in female CD-1 mice. The studies are to provide dose-response data for specific mixtures of the 4 THMs, for the single chemicals, and for binary combinations. The dose-levels and mixing ratios for these experiments were selected to be useful for development and refinement of three different statistical methods: testing for departures from dose-additivity; development of an interactions-based hazard index; and use of proportional-response addition as a risk characterization method. Preliminary results suggest that dose-additivity is a reasonable risk assessment assumption for DBPs. The future of mixtures research will depend on such collaborative efforts that maximize the use of resources and focus on issues of high relevance to the risk assessment of human health.

Alanine Transaminase↗
PubMed1976

Structure of flavin adducts with acetylenic substrates. Chemistry of monoamine oxidase and lactate oxidase inhibition.

The photoreaction of flavoquinones (lumiflavin, riboflavin, FMN etc. and their 3-alkylated derivatives) with propargylamine-type acetylenic substrates, R4 -Calpha identical to Cbeta -CgammaHR3 -NR2R1, yields a mixture of two adducts,which result from covalent Calpha fixation of the Cgamma-deprotonated substrate to either position C(4a) or N(5) in the flavin nucleus. The N(5) adduct is a dihydroflavin-5-trimethine-cyanine with very intense (xi greater than 20000 M-1 cm-1) absorption maxima in the region 380-450 nm depending on the R1,R2. This absorption allows recognition of minute amounts of this species of flavocyanine even in complex mixtures. Flavocyanines can be reconverted to starting flavin by base. It spectral properties are identical with those obtained for the pargyline-flavin inhibitor complex from bovine kidney or pig liver monoamine oxidase.

Acetylene↗
PubMed2005

Characterization of lipids in complex samples using comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry.

Most lipids are a complex mixture of classes of compounds such as fatty acids, fatty alcohols, diols, sterols and hydroxy acids. In this study, the suitability of comprehensive two-dimensional gas chromatography coupled to a time-of-light mass spectrometer is studied for lipid characterization in complex samples. With lanolin, a refined wool wax, as test sample, it is demonstrated that combined methylation plus silylation is the preferred derivatization procedure to achieve (i) high-quality GC x GC separation and (ii) easily recognizable ordered structures in lipid analysis. Optimization of the GC x GC column combination, the influence of the temperature programme on the quality of the separation, and the potential and limitations of automated TOF-MS-based identification are discussed. The combined power of a 2D separation, ordered structures and MS detection is illustrated by the identification of several minor sample constituents.

Gas Chromatography-Mass Spectrometry↗
PubMed2005

The wide diversity and complexity of peptides bound to class II MHC molecules.

The identification and quantitation of peptides selected by class II MHC molecules during natural processing of proteins is of key importance in understanding the repertoire and distribution of T cells. The examination of peptides selected by class II MHC molecules has depended greatly on mass spectrometry, a powerful technique that identifies and sequences peptides in complex mixtures with great sensitivity and precision. Such analysis has resulted in the identification of several factors, including the repertoire of peptides selected by MHC molecules during natural processing of proteins, motifs important for selection of processed peptides, conformational isomers of peptide-MHC complexes, and post-translational changes to the peptides.

Amino Acid Sequence↗
PubMed2001

Characterization of an intense bitter-tasting 1H,4H-quinolizinium-7-olate by application of the taste dilution analysis, a novel bioassay for the screening and identification of taste-active compounds in foods.

Thermal treatment of aqueous solutions of xylose and primary amino acids led to rapid development of a bitter taste of the reaction mixture. To characterize the key compound causing this bitter taste, a novel bioassay, which is based on the determination of the taste threshold of reaction products in serial dilutions of HPLC fractions, was developed to select the most intense taste compounds in the complex mixture of Maillard reaction products. By application of this so-called taste dilution analysis (TDA) 21 fractions were obtained, among which 1 fraction was evaluated with by far the highest taste impact. Carefully planned LC-MS as well as 1D and 2D NMR experiments were, therefore, focused on the compound contributing the most to the intense bitter taste of the Maillard mixture and led to its unequivocal identification as the previously unknown 3-(2-furyl)-8-[(2-furyl)methyl]-4-hydroxymethyl-1-oxo-1H,4H-quinolizinium-7-olate. This novel compound, which we name quinizolate, exhibited an intense bitter taste at an extraordinarily low detection threshold of 0.00025 mmol/kg of water. As this novel taste compound was found to have 2000- and 28-fold lower threshold concentrations than the standard bitter compounds caffeine and quinine hydrochloride, respectively, quinizolate might be one of the most intense bitter compounds reported so far.

Caffeine↗
PubMed2001

Antagonistic interactions among nephrotoxic polycyclic aromatic hydrocarbons.

Although the liver and pulmonary toxicity of polycyclic aromatic hydrocarbons (PAHs) has been extensively characterized, limited data concerning the nephrotoxic potential of these chemicals are available. The present studies were conducted to define the kidney cell-specific toxic responses to anthracene (ANTH), benzo[a]pyrene (BaP), and chrysene (CHRY). Given that exposure to environmental chemicals from a specific source is rarely limited to a single compound, a second goal was to evaluate the nephrotoxic potential of binary and ternary mixtures of these chemicals. Cultured rat glomerular mesangial cells (rGMCs) and porcine cortico-tubular epithelial kidney cells (LLCPK-1) were challenged with hydrocarbon concentrations ranging from 0.03 to 30 microM for up to 24 h and were processed for measurements of mitochondrial membrane permeability, trypan blue dye exclusion, cytoplasmic enzyme leakage, and protein synthesis. BaP induced a threefold increase in mitochondrial fragility, a modest increase in cellular death, and 40% decrease in the rate of protein synthesis in rGMCs. Anthracene was also cytotoxic to rGMCs, inducing a twofold increase in mitochondrial fragility and a 40% decrease in the rate of protein synthesis, but no changes in cellular viability. Although CHRY was devoid of toxicity to rGMCs, a 40% decrease in the rate of protein synthesis was observed in LLCPK-1 cells treated with this hydrocarbon. BaP and ANTH were not overtly cytotoxic to LLCPK-1 cells at any of the concentrations tested. Binary and ternary mixtures of BaP with ANTH and CHRY in rGMCs, and mixtures of CHRY with ANTH and BaP in LLCPK-1 cells, yielded antagonistic interactions. Based on these data, it is concluded that PAHs exhibit chemical- and cell-specific nephrotoxicity, but that toxicological outcomes are influenced by the presence of multiple hydrocarbons in complex mixtures.

Amino Acids↗