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PubMed2002

Sensory evaluation of character impact components in an apple model mixture.

Food aromas generally are complex mixtures of volatiles. In the present study, we investigated the joint effects of hexyl acetate, trans-2-hexenal and 1-hexanol on the multi-attribute perception of an apple aroma. The first two substances were identified earlier as positive contributors to the apple aroma (high character impact), whereas the third component was identified as an irrelevant or negative contributor (low character impact). Aroma quality was quantified using a set of eight graphic rating scales. All three components had significant effects on the aroma profiles. These effects consist mainly of an effect of each component on the attribute that described its individual character and an effect of all three components on ratings on the main character attribute 'apple'. As expected, the high impact components increased 'apple' ratings, whereas the low character impact component decreased 'apple' ratings. Furthermore, intensity ratings on the attribute that corresponded with the odour of the low impact component were suppressed by the presence of high impact components. These results indicate that the contributions of odorants to the mixture's aroma are not linear combinations of separate odour intensities, because sensory interactions were observed. In addition, humans detect components in complex mixtures more accurately than studies on identification performance have suggested. We conclude that for an adequate assessment of the effects of multiple mixture components on changes in aroma perception, it is sufficient to employ multiple response scales measuring intensities of attributes that are distinctive with respect to the expected qualitative changes. Results of this approach should be subjected to multivariate methods of statistical analysis.

Acetates↗
PubMed1989

Toxicity of complex waste mixtures: a comparison of observed and predicted lethality.

The ability to predict the biological effect of complex waste mixtures from chemical characterization data was examined by comparing observed mortality to that predicted by a mathematical additivity model with literature LD50 values for the chemicals identified in the mixtures. Male F344 rats were exposed by gavage to 1 of 10 samples of complex industrial waste. Seven of the 10 waste samples caused death within 24 h of administration at dosages ranging from 1 to 5 ml/kg body weight. Two of the 7 lethal waste samples produced 100% mortality at a dosage of 2.5 ml/kg; another 2 waste samples produced 100% mortality at 5 ml/kg. The partial chemical analysis, although providing more extensive information on chemical composition than might normally be available for most complex waste mixtures, was not sufficient to distinguish lethal from nonlethal waste samples or to indicate lethal potency. Possible explanations for the apparent inability to predict readily lethality from the chemical characterization data include the possible inappropriateness of an additivity model due to the presence of interactions, such as synergism or antagonism; the kinetics of chemical absorption, distribution, and elimination, which may be affected by administration of the chemical in a complex matrix; and the presence of unidentified chemicals in the mixture that may have contributed to the observed toxicity.

Animals↗
PubMed2005

Predicting skin permeability from complex chemical mixtures.

Occupational and environmental exposure to topical chemicals is usually in the form of complex chemical mixtures, yet risk assessment is based on experimentally derived data from individual chemical exposures from a single, usually aqueous vehicle, or from computed physiochemical properties. We present an approach using hybrid quantitative structure permeation relationships (QSPeR) models where absorption through porcine skin flow-through diffusion cells is well predicted using a QSPeR model describing the individual penetrants, coupled with a mixture factor (MF) that accounts for physicochemical properties of the vehicle/mixture components. The baseline equation is log k(p) = c + mMF + a sigma alpha2(H) + b sigma beta2(H) + s pi2(H) + rR2 + vV(x) where sigma alpha2(H) is the hydrogen-bond donor acidity, sigma beta2(H) is the hydrogen-bond acceptor basicity, pi2(H) is the dipolarity/polarizability, R2 represents the excess molar refractivity, and V(x) is the McGowan volume of the penetrants of interest; c, m, a, b, s, r, and v are strength coefficients coupling these descriptors to skin permeability (k(p)) of 12 penetrants (atrazine, chlorpyrifos, ethylparathion, fenthion, methylparathion, nonylphenol, rho-nitrophenol, pentachlorophenol, phenol, propazine, simazine, and triazine) in 24 mixtures. Mixtures consisted of full factorial combinations of vehicles (water, ethanol, propylene glycol) and additives (sodium lauryl sulfate, methyl nicotinate). An additional set of 4 penetrants (DEET, SDS, permethrin, ricinoleic acid) in different mixtures were included to assess applicability of this approach. This resulted in a dataset of 16 compounds administered in 344 treatment combinations. Across all exposures with no MF, R2 for absorption was 0.62. With the MF, correlations increased up to 0.78. Parameters correlated to the MF include refractive index, polarizability and log (1/Henry's Law Constant) of the mixture components. These factors should not be considered final as the focus of these studies was solely to determine if knowledge of the physical properties of a mixture would improve predicting skin permeability. Inclusion of multiple mixture factors should further improve predictability. The importance of these findings is that there is an approach whereby the effects of a mixture on dermal absorption of a penetrant of interest can be quantitated in a standard QSPeR model if physicochemical properties of the mixture are also incorporated.

Algorithms↗
PubMedDate not supplied

Alterations in synaptic transmission and plasticity in hippocampus by a complex PCB mixture, Aroclor 1254.

Developmental exposure to polychlorinated biphenyls (PCBs) has been associated with a variety of neurological effects including cognitive dysfunction. The present study assessed the effects of acute in vitro exposure to a complex mixture of highly chlorinated PCBs, Aroclor 1254 (A1254), on synaptic transmission in the hippocampus of the rat. Increases in population spike (PS) amplitude were observed in field potentials recorded from the pyramidal cell layer of CA1 in response to 1.0-3 microg/ml of A1254. PS amplitude was increased by approximately 20% 5-10 min following the beginning of exposure to 3 microg/ml A1254, with some recovery towards baseline amplitudes occurring by 30 min, despite continuous perfusion. Longer exposures revealed that a maintained 10% increase in PS amplitude persisted beyond 90 min of exposure to 3 microg/ml A1254. A second recording electrode in the stratum radiatum revealed modest increases in EPSP slope (approximately 10%) that were transient. EPSP slope changes appeared within 5 min of exposure to A1254 (3 microg/ml), peaked at 10 min, and declined to baseline levels by 30 min, despite continued perfusion with A1254. Monitoring over a protracted period revealed relatively stable EPSP slope amplitudes following the return to baseline levels. Long-term potentiation (LTP) is a model of synaptic plasticity believed to encompass the physiological substrates of memory. Neither magnitude or persistence of PS potentiation recorded from the stratum pyramidale was affected by 3 microg/ml A1254. LTP of the EPSP slope recorded in the stratum radiatum was also induced to a comparable degree in control and A1254-treated slices. However, the augmentation in the dendritic response was not maintained in the treated slices over the 60-min posttrain recording period. Acute effects of PCBs on calcium homeostasis, protein kinase C translocation, dopaminergic function, and hormonal action may contribute to the pattern of effects seen in synaptic transmission and plasticity in the hippocampus.

Animals↗
PubMed2006

A suite of algorithms for the comprehensive analysis of complex protein mixtures using high-resolution LC-MS.

MOTIVATION: Comparing two or more complex protein mixtures using liquid chromatography mass spectrometry (LC-MS) requires multiple analysis steps to locate and quantitate natural peptides within a single experiment and to align and normalize findings across multiple experiments. RESULTS: We describe msInspect, an open-source application comprising algorithms and visualization tools for the analysis of multiple LC-MS experimental measurements. The platform integrates novel algorithms for detecting signatures of natural peptides within a single LC-MS measurement and combines multiple experimental measurements into a peptide array, which may then be mined using analysis tools traditionally applied to genomic array analysis. The platform supports quantitation by both label-free and isotopic labeling approaches. The software implementation has been designed so that many key components may be easily replaced, making it useful as a workbench for integrating other novel algorithms developed by a growing research community. AVAILABILITY: The msInspect software is distributed freely under an Apache 2.0 license. The software as well as a Zip file with all peptide feature files and scripts needed to generate the tables and figures in this article are available at http://proteomics.fhcrc.org/.

Algorithms↗
PubMed1989

Effects of complex waste mixtures on hepatic monooxygenase activities in brown bullheads (Ictalurus nebulosus).

Hepatic MFO components (cytochrome P-450, cytochrome b(5), and ethoxyresorufin O-deethylase, EROD) were measured in brown bullheads (Ictalurus nebulosus) inhabiting a creek receiving a complex mixture of organics and trace metals. The activities of these same enzymes were also measured in bullheads from an uncontaminated reference site to assess the relative ability of MFO parameters to serve as a biomarker of aquatic pollution. Bullheads analyzed from the polluted site had lower hepatic microsomal P-450 (p < 0.01) concentrations and similar EROD activities per mg protein as compared to bullheads from the reference site. However, analysis of enzyme turnover ratios revealed greater EROD activity per mg cytochrome P-450 (p < 0.05) in the fish from the polluted site. No difference in cytochrome b(5) activities were observed between the two groups. As compared to the reference site, bullheads collected from the polluted creek had an increased occurrence of lip and lower jaw lesions and liver damage, including elevated liver/body weight ratios. Accordingly, the monooxygenase activities measured in this study were not reliable indicators of chemical pollution or contaminant stress in bullheads in the polluted creek. Further research is needed concerning contaminant interactions, particularly among organic pollutants and metals and their effects on monooxygenase activities.

Journal Article↗
PubMed1991

Use of the spiral Salmonella assay to detect the mutagenicity of complex environmental mixtures.

The success demonstrated by the spiral Salmonella assay in a recent study of 20 pure prompted us to examine the effectiveness of this automated bacterial mutagenicity assay for testing complex environmental mixtures. Three sets of combustion emissions were selected for evaluation: automotive diesel exhaust, woodsmoke, and a coal combustion emission. Each sample was tested in the Salmonella mutagenicity assay according to standard protocol (plate incorporation) and spiral assay techniques. In the spiral assay, a specialized plating instrument dispenses the bacteria, test agent, and S9 mix in a spiral pattern onto a minimal agar plate supplemented with histidine and biotin. The components of the assay are administered in such a way that a uniform density of bacteria is exposed to a concentration gradient of the test agent on a single plate. When results are analyzed, a dose-response curve comprised of 13 data points is generated. A comparison of results from the two assays demonstrated the following: 1) Diesel exhaust was generally the most mutagenically potent sample in both assays, followed closely by the coal combustion emission. The woodsmoke sample was only weakly mutagenic in the standard assay but demonstrated higher mutagenic activity in the spiral assay. 2) Samples were more mutagenic on rev/microgram basis in the spiral assay, especially when metabolic activation was added. This disparity presumably was due to differences in the relative amounts of S9 administered across the dose range. 3) The spiral assay required 1/20 the sample mass of the standard assay to test equivalent doses; in addition, for some samples, 50 times more sample mass was required by the standard assay to generate a comparable dose response. 4) Dichloromethane extracts of the complex mixtures could be tested for mutagenicity in the spiral assay, thereby precluding solvent exchange (to dimethylsulfoxide) required by the standard assay for sample/bioassay compatibility.

Biotransformation↗
PubMed2000

Mathematical algorithm for qualitative and semiquantitative analysis of petroleum hydrocarbons in solid wastes using on-line gas chromatography.

Non-degradated mineral-oils like gasoline, solvent naphtha, diesel fuel, fuel and lubricating oils provide a characteristic fingerprint gas chromatogram. This visual classification, e.g. in solid wastes, is complicated due to the simultaneous presence of several mineral-oils. Therefore, a mathematical algorithm for the separation of gas chromatographic fingerprint of "single mixtures" of aliphatic hydrocarbons is developed. The technique is essential for analysis of time-overlapping "single mixtures" of petroleum hydrocarbons (so-called "complex mixtures") and it relies on the concentration-varying hydrocarbons during evaporation. It is possible to separate the data from the gas chromatogram of a "complex mixture" of hydrocarbons into the chromatograms of the pure "single mixtures" and to give their respective concentrations. A synthetic "complex mixture" of kerosene, diesel fuel and lubricating oil is used to illustrate the method.

Algorithms↗
PubMed2002

De novo peptide sequencing and quantitative profiling of complex protein mixtures using mass-coded abundance tagging.

Proteomic studies require efficient, robust, and practical methods of characterizing proteins present in biological samples. Here we describe an integrated strategy for systematic proteome analysis based on differential guanidination of C-terminal lysine residues on tryptic peptides followed by capillary liquid chromatography-electrospray tandem mass spectrometry. The approach, termed mass-coded abundance tagging (MCAT), facilitates the automated, large-scale, and comprehensive de novo determination of peptide sequence and relative quantitation of proteins in biological samples in a single analysis. MCAT offers marked advantages as compared with previously described methods and is simple, economic, and effective when applied to complex proteomic mixtures. MCAT is used to identify proteins, including polymorphic variants, from complex mixtures and measure variation in protein levels from diverse cell types.

Algorithms↗
PubMed1993

Simultaneous electroelution of whole SDS-polyacrylamide gels for the direct cellular analysis of complex protein mixtures.

A novel procedure which allow the rapid screening of complex protein mixtures in cellular assays is described. A device has been developed which allows a convenient, simultaneous electroelution of separated proteins from whole SDS polyacrylamide gels into narrow chambers each containing single or a few protein bands. We have optimized the conditions of the procedure and have obtained an efficient removal of SDS, leading to non-toxic protein fractions in a physiological buffer suited for direct testing in cell cultures. The responses generated by stimulating lymphocytes with the purified products have been compared to the native protein and a corresponding preparation of protein transferred to nitrocellulose particles. The method was used to investigate murine T cell responses to secreted mycobacterial antigens during infection with M. tuberculosis. A immunodominant secreted protein fraction was purified in a semipreparative scale by the procedure and used to immunize mice. The specificity of and lymphokine production by T cells generated in these animals were investigated. The device developed has various applications and provides a tool for the possible identification of new T cell antigens of importance for protective immunity.

Animals↗
PubMed2001

Solid-state NMR characterization of the mineral searlesite and its detection in complex synthesis mixtures.

The mineral searlesite (NaBSi(2)O(5)(OH)(2)) was synthesized and characterized by (1)H, (11)B, (23)Na, and (29)Si magic-angle spinning (MAS) NMR spectroscopy. From these spectra, the (11)B and (23)Na quadrupole coupling parameters and isotropic chemical shifts and the (29)Si chemical shift anisotropies have been precisely determined. These parameters are all consistent with the local environments obtained from the crystal structure for searlesite from X-ray diffraction, and they demonstrate that the synthetic sample has a high degree of both short- and long-range order. Furthermore, these anisotropic parameters are found to provide a unique fingerprinting of searlesite in complex mixtures where the presence of this mineral is not anticipated. This is demonstrated for product mixtures formed in attempts to incorporate boron in the structures of the layer silicates magadiite and kenyaite. These mixtures have been investigated by (11)B, (23)Na, and (29)Si MAS NMR which clearly reveal that the samples are mixtures of searlesite and magadiite/kenyaite and that searlesite production consumes all of the boron in these synthesis mixtures. However, the (29)Si MAS NMR spectra of these mixtures indicate that the presence of boron in the reaction mixtures nevertheless has an important influence on the quality of the magadiite and kenyaite layer silicates produced.

Journal Article↗
PubMed1997

Ceramide profiling of complex lipid mixtures by electrospray ionization mass spectrometry.

Ceramides and sphingoid bases are important intracellular second messengers that play a role in the regulation of cell growth, differentiation, and programmed cell death. Until now, quantitative and qualitative analysis of ceramide second messengers has been limited by a lack of analytical methods capable of detecting endogenous levels of, and differentiating between, individual ceramide species. Here we report the use of electrospray ionization tandem mass spectrometry for the qualitative and quantitative analysis of ceramides. Collision-induced fragmentation resulted in characteristic product ions for the sphingosine and dihydrosphingosine (sphinganine) head groups at m/z 264 and 282 and m/z 266 and 284, respectively, regardless of the length of the fatty acyl chains, with spectra being reproducible at concentrations as low as 25 nM (25 fmol/microliter). These reporter ions were used to detect both sphingosine- and sphinganine-based ceramides in complex mixtures using precursor ion scan analysis. We demonstrated the application of this method for profiling the composition of ceramides in a commercial preparation of bovine brain ceramides and, following minimal chromatographic separation, a lipid extract of cultured T cells. Furthermore, we easily detected relative differences in individual ceramide species levels by comparing the profiles of three related lymphocyte cell lines, Jurkat, U937, and WEHI 231. Finally, by the addition of a nonnaturally occurring internal standard, we show that the technique can be used to measure quantitative changes in ceramide levels in such biologically derived lipid preparations.

Animals↗
PubMed2005

Evaluating preparative isoelectric focusing of complex peptide mixtures for tandem mass spectrometry-based proteomics: a case study in profiling chromatin-enriched subcellular fractions in Saccharomyces cerevisiae.

We have evaluated the use of free-flow electrophoresis, an emerging separation method for preparative isoelectric focusing of complex peptide mixtures, as a tool for high-throughput tandem mass spectrometry-based proteomic analysis. In this study, we investigated the ability of free-flow electrophoresis to resolve and fractionate complex peptide mixtures and also the effectiveness of using peptide isoelectric point in conjunction with peptide match probability scoring in sequence database searching. As a model system for this study, we analyzed a chromatin-enriched fraction from the yeast Saccharomyces cerevisiae. This mixture was fractionated using preparative isoelectric focusing by free-flow electrophoresis, followed by online capillary liquid chromatography electrospray tandem mass spectrometry and sequence database searching. Our results demonstrate that (1) FFE effectively resolves and fractionates complex peptide mixtures on the basis of peptide isoelectric point and (2) the introduction of peptide pI is effective in minimizing both false positive and false negative sequence matches in sequence database searching of tandem mass spectrometry data.

Amino Acid Sequence↗
PubMed1996

Microheterogeneity of human filaggrin: analysis of a complex peptide mixture using mass spectrometry.

Filaggrin is the product of posttranslational processing of the large, epidermal protein profilaggrin, which consists of 10 or more tandem filaggrin domains plus an amino and a carboxyl domain. According to fragmentary cDNA sequences, the filaggrin domains in the human protein vary at 40% of the amino acid positions; hence, mature filaggrin is a population of homologous but heterogeneous proteins, even within one individual. Available gene sequences give only a limited picture of the heterogeneity of human filaggrin protein because no complete human profilaggrin gene has been sequenced. Questions about the extent of heterogeneity of filaggrin within and between individuals have not been answered, nor have questions concerning the limited proteolytic cleavage of human profilaggrin that generates filaggrin in vivo. In order to address these questions and to provide an analysis of the primary structure of human filaggrins, we employed various methods of mass spectrometry. The intact protein and a tryptic digest of the mixture of human filaggrins were examined by matrix-assisted laser desorption time-of-flight mass spectrometry. Tryptic digests of human filaggrin from single individuals were also separated and analyzed by liquid chromatography/mass spectrometry (LC/MS) (using electrospray mass spectrometry), and specific peptides were identified by tandem mass spectrometry (MS/MS). A robust data analysis program, Sherpa, was developed to facilitate the interpretation of both LC/MS and MS/MS. These experiments show that human filaggrin includes heterogeneity not yet seen in cDNA sequences, but that much structure is highly conserved. Interestingly, we found that the heterogeneity is conserved among individuals. An approximation of the regions linking filaggrins in human profilaggrin is developed. These investigations provide a unique test of the limits of tryptic mapping of complex mixtures using mass spectrometry.

Amino Acid Sequence↗
PubMed2005

Nanoflow liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry: sample preparation, data analysis, and application to the analysis of complex peptide mixtures.

We report the development of a robust interface for off-line coupling of nano liquid chromatography (LC) to matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) and its application to the analysis of proteolytic digests of proteins, both isolated and in mixtures. The interface makes use of prestructured MALDI sample supports to concentrate the effluent to a small sample plate area and localize the MALDI sample to a predefined array, thereby enriching the analyte molecules and facilitating automated MALDI-MS analysis. Parameters that influence the preparation of MALDI samples from the LC effluent were evaluated with regard to detection sensitivity, spectra quality, and reproducibility of the method. A procedure for data processing is described. The presented nano LC MALDI-MS system allowed the detection of several peptides from a tryptic digest of bovine serum albumin, at analyzed amounts corresponding to one femtomole of the digested protein. For the identification of native proteins isolated from mouse brain by two-dimensional gel electrophoresis, nano LC MALDI-MS increased the number of detected peptides, thereby allowing identification of proteins that could not be identified by direct MALDI-MS analysis. The ability to identify proteins in complex mixtures was evaluated for the analysis of Escherichia coli 50S ribosomal subunit. Out of the 33 expected proteins, 30 were identified by MALDI tandem time of flight fragment ion fingerprinting.

Animals↗
PubMed1999

Genotoxicity of complex PAH mixtures recovered from contaminated lake sediments as assessed by three different methods.

Although human exposure generally occurs to mixtures of chemicals, limited toxicological information is available to characterize the potential interactions of the components of environmental mixtures. This study was conducted to compare the genotoxicity of chemically characterized polycyclic aromatic hydrocarbon (PAH) mixtures using in vitro and in vivo techniques. A total of three extracts (E1-E3) were selected from sediment samples collected from a lake adjacent to an abandoned coal gasification site. Sediments were collected on a grid moving downstream and away from the most likely source of PAH contamination, with E1 collected closest to the shore, E2 at an intermediate distance, and E3 furthest from the shore. The sediment samples were extracted in methylene chloride and methanol, dried, and redissolved in an appropriate solvent for evaluation in a battery of genotoxicity assays. Samples were evaluated for their ability to produce point mutations in bacteria and DNA adducts in vitro without metabolic activation or in vivo. Samples were also analyzed using GC/MS. Sample E1 had both the highest concentration of benzo(a)pyrene (BP) (46.5 ppm) and carcinogenic PAHs and, using 32P-postlabeling, induced the highest adduct levels overall in vitro and in vivo. Sample E2, which had a BP concentration of 14 ppm, induced the greatest number of revertants in the bacterial mutagenicity assay. Sample E3, which had the lowest level of carcinogenic PAHs and BP, induced the lowest adduct levels. However, E3 was capable of inducing a positive genotoxic response in bacteria (with S9), although the slope of the response at lower doses was less than that of E2. The in vivo data showed that the major adduct formed by E1 and E2 was a BP adduct. This information could not have been obtained with the Salmonella or in vitro postlabeling tests. Among internal organs, the extracts of all three samples induced the greatest adduct levels in the lung, similarly to previous complex PAH mixtures studied. These data demonstrate the limitations of predicting genotoxic or carcinogenic potential based on chemical analysis or a single biological test. The results suggest that mixture interactions, cytotoxicity and metabolism are likely to have an influence on the potential of a complex mixture of chemicals to produce a carcinogenic effect. In addition, the concentration of genotoxic PAHs and both in vitro and in vivo DNA adduct formations were decreased with increasing distance from the shoreline.

Animals↗
PubMed1992

Purification of complex protein mixtures by ion-exchange displacement chromatography using spacer displacers.

The anion-exchange separation of complex protein mixtures by displacement chromatography using spacer displacers driven by high-affinity final displacers is demonstrated. Guinea pig serum was separated on a medium-resolution adsorbent using a single heterogeneous mixture of carboxymethyldextran displacers to space the protein components. Mouse liver cytosol was separated on a low-resolution adsorbent using six carboxymethyldextran spacer displacers of increasing column affinity. The demonstration of the purification of alkaline phosphatase from E. coli periplasm by displacement chromatography on a high-performance liquid chromatography column is reviewed. The benefits of spacer displacers for separating minor components from complex biological protein mixtures is discussed. A simplified method for preparing carboxymethyldextran displacers is presented.

Adsorption↗
PubMed2000

Separation and detection of intact noncovalent protein complexes from mixtures by on-line capillary isoelectric focusing-mass spectrometry.

Separation and mass spectrometric analysis of intact noncovalent protein-protein complexes from mixtures is described. Protein complexes were separated using isoelectric focusing in a capillary under native conditions. During the mobilization, molecular masses of the intact complexes were measured on-line (as they emerged from the capillary) using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. An FTICR "in-trap" ion cleanup procedure was necessary for some complexes to reduce high levels of adduction and to obtain accurate molecular mass measurements. Optimization of the conditions for analysis of different intact complexes is discussed. We have shown that either the intact noncovalent complexes or their constituent protein subunits can be detected by variation of sheath liquid (i.e., NH4OAc vs HOAc) added at the electrospray-mass spectrometer interface. Thus, two successive experiments permit a fast and efficient characterization of intact complex stoichiometry, the individual complex subunits and the possible presence of metal or other adducted species.

Animals↗