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PubMed2002

Fully reversible procedure for silver staining improves densitometry of complex mixtures of biopolymers resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Due to its high sensitivity, silver staining is a widely popular method for the revelation of biopolymers separated by both native and denaturing electrophoresis. A step-by-step method for the destaining and restaining of overdeveloped/overloaded silver-stained bands is described that is applicable to both proteins and nucleic acids. The procedure significantly improves densitometric analysis of gels that have been silver stained with either commercial kits or solutions made in-house. The method permits reproducible densitometry of silver-stained gels and allows quantification of both main and minor components in complex mixture of molecules resolved on the same gel slab. All steps may be interrupted and are readily reversible, allowing for facile densitometric analyses and photographic recording under optimized conditions. Furthermore, common artifacts such as differential staining of the two gel surfaces, localized uneven yellow-ochre background, and the presence of fold marks and fingerprints can be easily removed.

Artifacts↗
PubMed2005

Robust enrichment of phosphorylated species in complex mixtures by sequential protein and peptide metal-affinity chromatography and analysis by tandem mass spectrometry.

Reversible protein phosphorylation mediated by kinases, phosphatases, and regulatory molecules is an essential mechanism of signal transduction in living cells. Although phosphorylation is the most intensively studied of the several hundred known posttranslational modifications on proteins, until recently the rate of identification of phosphorylation sites has remained low. The use of tandem mass spectrometry has greatly accelerated the identification of phosphorylation sites, although progress was limited by difficulties in phosphoresidue enrichment techniques. We have improved upon existing immobilized metal-affinity chromatography (IMAC) techniques for capturing phosphopeptides, to selectively purify phosphoproteins from complex mixtures. Combinations of phosphoprotein and phosphopeptide enrichment were more effective than current single phosphopeptide purification approaches. We have also implemented iterative mass spectrometry-based scanning techniques to improve detection of phosphorylated peptides in these enriched samples. Here, we provide detailed instructions for implementing and validating these methods together with analysis by tandem mass spectrometry for the study of phosphorylation at the mammalian synapse. This strategy should be widely applicable to the characterization of protein phosphorylation in diverse tissues, organelles, and in cell culture.

Animals↗
PubMed2005

The human brain mannose 6-phosphate glycoproteome: a complex mixture composed of multiple isoforms of many soluble lysosomal proteins.

The lysosome is a membrane delimited cytoplasmic organelle that contains at least 50 hydrolytic enzymes and associated cofactors. The biomedical importance of these enzymes is highlighted by the many lysosomal storage disorders that are associated with mutations in genes encoding lysosomal proteins, and there is also evidence that lysosomal activities may be involved in more widespread human diseases. The aim of this study was to characterize the human brain lysosomal proteome with the goal of establishing a reference map to investigate human diseases of unknown etiology and to gain insights into the cellular function of the lysosome. Proteins containing mannose 6-phosphate (Man6-P), a carbohydrate modification used for targeting resident soluble lysosomal proteins to the lysosome, were affinity-purified using immobilized Man6-P receptor. Fractionation by two-dimensional electrophoresis resolved a complex mixture comprising approximately 800 spots. Constituent proteins in each spot were identified using a combination of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (both peptide mass fingerprinting and tandem mass spectrometry) [corrected] on in-gel tryptic digests and N-terminal sequencing. In a complementary analysis, we also analyzed a tryptic digest of the unfractionated mixture by liquid chromatography MS/MS. In total, 61 different proteins were identified. Seven were likely contaminants associated with true Man6-P glycoproteins. Forty-one were known lysosomal proteins of which 11 have not previously been reported to contain Man6-P. An additional nine proteins were either uncharacterized or proteins not previously reported to have lysosomal function. We found that the human brain Man6-P-containing lysosomal proteome is highly complex and contains more proteins with a much greater number of individual isoforms than found in previous studies of Man6-P glycoproteomes.

Brain↗
PubMed1990

Quantification of monoclonal antibodies in complex mixtures by protein G high-performance liquid affinity chromatography.

High-performance liquid affinity chromatography (HPLAC) utilizing Protein G as a ligand has been evaluated for rapid quantification of monoclonal antibodies (MAbs) in various solutions. The results obtained by HPLAC agreed to within 10% of a standard enzyme-linked immunospecific assay (ELISA). A standard curve was prepared by injection of known amounts of a purified murine IgG1 with the elution peak area analyzed by computer integration software. Accuracy of quantification was independent of the injection volume, solution compositions, or mouse IgG subclass. A method is described for using Protein G HPLAC to determine murine IgG levels in various complex mixtures within 15 min, compared to the ELISA which required 5 h.

Animals↗
PubMed2001

Gangliosides from human granulocytes: a nano-ESI QTOF mass spectrometry fucosylation study of low abundance species in complex mixtures.

Nano-ESI QTOF MS was used for sensitive mapping and sequencing of single molecular species in complex ganglioside mixtures obtained from human granulocytes, where the fucosylated carbohydrate chains of granulocyte gangliosides carry sLex and VIM-2 epitopes postulated to interact with E-selectin of the blood vessel wall in the early phase of the inflammation process. Functionally relevant components are expressed only at a low level, but using the negative ion detection it is possible to trace and identify such species, which were not detectable even by TLC. Advantage of the low-energy CID fragmentation for low abundance components of the complex ganglioside mixtures in the negative ion mode is to produce clear-cut series of fragment ions for sequencing. Fucosylation analysis carried out for each molecular species by MS/MS permits the clear distinction between sLex and VIM-2 epitope. VIM-2 epitope was expressed in all species with a longer sugar core, while in the short oligosaccharide chain with a sLex only, using biological material at a mid-femtomol level detection.

Animals↗
PubMed1975

Sterols of scallop. I. Application of hydrophobic sephadex derivatives to the resolution of a complex mixture of marine sterols.

Column chromatography on a hydroxyalkoxypropyl derivative of Sephadex LH-20 and on Anasil B has been applied to the resolution of complex marine sterol mixture in combination with argentation thin-layer chromatography and gas chromatography. This approach permits isolation in quantity of individual sterols from a complex mixture and separation of sterol mixtures that were not resolved without the modified Sephadex step. Seventeen sterols were detected in the scallop Placopecten magellanicus. 24-Methyl-cholesterol, 24-ethyl-cholesterol, 24-methyl-22-dehydrocholesterol and 24-ethyl-22-dehydrocholesterol, i.e. sterols whose configuration at C-24 had not been definitively established, were isolated in sufficient quantities for further study by nuclear magnetic resonance spectroscopy.

Animals↗
PubMed2006

Measurement of un-ionized ammonia in complex mixtures.

The toxicity of un-ionized ammonia, NH3 (aq), in anaerobic digestion of high-nitrogen wastes has been researched extensively. Previous estimates of NH3 (aq) concentration have relied on a simple speciation approach, based only on the acid dissociation constant and the sample pH and total ammonia concentration. The distinction between concentration and chemical activity has generally not been made, despite the potential for resulting errors in the calculation of NH3 (aq) concentration, and the greater applicability of activity to toxicity work. The currently accepted approach for estimating NH3 (aq) concentration is based on assumptions that are not valid in digested animal manure or other concentrated wastes. This work presents an approach for directly measuring NH3 (aq) activity in complex mixtures using gaseous/aqueous equilibrium across microporous tubing. Application of this approach to anaerobic digester samples confirms that the currently accepted approach is not accurate; it overestimated NH3 (aq) activity in unaltered samples by 45-200%. Previous work on the toxicity of ammonia to methanogenesis has probably overestimated the tolerance of consortia to NH3 (aq), due to overestimation of concentrations. The method introduced here is expected to be useful in a range of research on ammonia toxicity and volatilization.

Ammonia↗
PubMed1999

A simple two-dimensional high performance liquid chromatography/high performance capillary electrophoresis set-up for the separation of complex mixtures.

A two-dimensional high performance liquid chromatography/capillary electrophoresis (HPLC/CE) instrumental set-up was assembled from commercially available equipment. Fractions of the effluent from the HPLC system are collected into microtiter plates with a microfraction collector. The fractions are then dried under vacuum at room temperature, reconstituted, and analyzed by capillary zone electrophoresis (CZE). This method allows the collection of samples by time, drops, or external signal (peaks). Any size or type of HPLC or CE column can be used with no limitation on the amount of sample injected into the HPLC. Any CE detection, laser-induced fluorescence (LIF), mass spectrometry (MS), ultraviolet (UV) or other, can be used. This set-up is practical, simple, robust and allows the separation of complex mixtures. Preliminary results show the utility of this system for the analysis of protein digest.

Chromatography, High Pressure Liquid↗
PubMed2004

Trace labeling of proteins with stable isotopes to identify fragments in complex mixtures.

We describe a novel nonradioactive protein-labeling technique that permits mass spectrometric identification of fragments of labeled proteins. Proteins are labeled by modulating their content of carbon-13 and labeled fragments identified from the distinctive isotope pattern observed on MALDI-TOF mass spectrometry. We show that carbon-13 enrichment to just 2.3% of total carbon (about twice the natural abundance of 1.1%) is sufficient for all fragments to be distinguishable from fragments of natural carbon-13-content proteins. Distinguishing labeled fragments is easily accomplished by visual inspection of spectra, but importantly, we show that labeled fragments can also be identified by computer analysis of spectra using novel parameters we have derived. The technique is demonstrated for identification of fragments of carbon-13-enriched glutathione transferase within a complex mixture of unlabeled peptides by visual and computer analysis of MALDI-TOF mass spectra, but it could be developed to mass spectrometrically identify and characterize fragments of labeled proteins recovered from biological systems.

Automation↗
PubMedDate not supplied

Quantitative profiling of proteins in complex mixtures using liquid chromatography and mass spectrometry.

The objective of this study was to determine if liquid chromatography mass spectrometry (LC/MS) data of tryptic digests of proteins can be used for quantitation. In theory, the peak area of peptides should correlate to their concentration; hence, the peak areas of peptides from one protein should correlate to the concentration of that particular protein. To evaluate this hypothesis, different amounts of tryptic digests of myoglobin were analyzed by LC/MS in a wide range between 10 fmol and 100 pmol. The results show that the peak areas from liquid chromatography mass spectrometry correlate linearly to the concentration of the protein (r2 = 0.991). The method was further evaluated by adding two different concentrations of horse myoglobin to human serum. The results confirm that the quantitation method can also be used for quantitative profiling of proteins in complex mixtures such as human sera. Expected and calculated protein ratios differ by no more than 16%. We describe a new method combining protein identification with accurate profiling of individual proteins. This approach should provide a widely applicable means to compare global protein expression in biological samples.

Amino Acid Sequence↗
PubMed2003

Determination of potentially bioaccumulating complex mixtures of organochlorine compounds in wastewater: a review.

Organic chlorine compounds can be persistent environmental contaminants and may be accumulated through the food chain to the aquatic organisms, to fish and humans, depending basically on their hydrophobic properties. Consequently, there is an interest to measure these organic compounds from both the scientific and regulatory communities. The analytical essays have been improved for measuring specific organic chlorine compounds that present the most toxicological potential (polychlorinated biphenyls [PCBs], certain pesticides and dioxins), although they are tedious and time-consuming procedures. The existing tests to measure adsorbable organic halogens (AOX) or extractable organic halogens (EOX) do not distinguish the more hydrophobic organic chlorine matter. The intention of this paper is to make a review of the existing methods to measure the potentially bioaccumulating organochlorine compounds (OCs) from wastewater and propose a methodology to a standardisation procedure for complex mixtures of OCs in wastewater, such as pulp mill effluents. A new method has been proposed for determining the most hydrophobic part of the extractable organic halogens (EOX(fob)), the lowest reported value is 0.6 microg/l, expressed as chloride, and the relative standard deviation at 20 microg/l is 7% on laboratory samples and 30% on real effluents. This new procedure could be a valuable tool to complement environmental risk assessment studies of wastewater discharges.

Animals↗
PubMed1979

Bioassay of complex mixtures derived from fossil fuels.

The conversion or processing of shale, coal, or petroleum involves elevated temperatures and altered pressures, and under these conditions polynuclear aromatic hydrocarbons are likely to form. Certain compounds of this type exhibit carcinogenic activity for a variety of organ sites in experimental animals and epidemiological evidence strongly implicates their role as carcinogens in man. It is then not unexpected that many liquid fractions derived from shale and coal are carcinogenic when subjected to bioassay. Benzo(a)pyrene, [B(a)P], is frequently considered to be an indicator substance. It is clear that when a small quantity of B(a)P is present in a fraction, the fraction will exhibit carcinogenic activity in a bioassay (mouse skin). However, it does not follow that the lack of detectable B(a)P insures that the fraction will be noncarcinogenic. Several fractions have been analyzed for their content of B(a)P and then subjected to bioassay. A method for testing complex mixtures for their carcinogenic potential is described. The carcinogenic potency of these fractions are compared to petroleum fractions.

Animals↗
PubMed1990

Cytogenetic markers in detecting the effects of exposure to complex mixtures of chemicals.

A significant proportion of the mutations responsible for both inherited and acquired human disease are a consequence of chromosome structural changes and of losses or gains of whole chromosomes. Some of these changes are readily detectable by the cytogeneticist and their frequencies are increased following exposure of individuals to mutagens. Techniques to visualize human sperm chromosomes and to detect micronuclei in spermatids now allow the cytogeneticist to detect induced chromosomal mutations in human germ cells. Rapid advances in our understanding of the molecular organization of the human genome, in the application of recombinant DNA probes and in situ hybridization with isotopic and non-isotopic probes, now enable chromosome organelles and specific chromosomal regions to be uniquely identified. These approaches are being applied to analyse chromosomal changes in somatic cells and, coupled with the development of machines to rapidly analyse chromosomes and nuclei, provide a powerful means to utilize cytogenetic markers to detect the effects of exposure of people to complex mixtures of chemical mutagens.

Animals↗
PubMed2005

Identification of a multixenobiotic resistance mechanism in primary cultured epidermal cells from Oncorhynchus mykiss and the effects of environmental complex mixtures on its activity.

Multixenobiotic resistance (MXR) is a mechanism analogous to the mammalian multidrug resistance (MDR) phenotype, whereby, simultaneous resistance is conferred against the intracellular accumulation of structurally and functionally diverse, natural, endogenous and environmental toxicants. Expression of P-glycoproteins (P-gp), ATP-dependent transporters encoded for by the mdr1 gene that have been implicated in this xenobiotic efflux mechanism, have previously been detected in normal teleost tissues involved in a secretory, absorption or a barrier function. The presence of these proteins in the epidermis of fish species has not to our knowledge previously been investigated. In the present study, primary cultures of epidermis from the rainbow trout Oncorhynchus mykiss were employed to investigate whether an MXR mechanism is functional in the epidermis of fish. The efflux of the fluorescent mdr1 substrate rhodamine 123 from the cells was significantly inhibited by verapamil, a compound known to interfere with P-gp mediated transport. The cultured epidermal cells were also observed to accumulate this fluorescent dye in a verapamil sensitive manner, thus indicating the presence of an mdr1-like mechanism. Immunocytochemical analysis, using a monoclonal antibody (JSB1) directed against a conserved cytoplasmic P-gp epitope, also demonstrated the presence of P-gp-like proteins. Sediment elutriate extracts were employed as models of environmental complex mixtures to evaluate the potential of the epidermal cultures to discriminate between samples of varying contaminant burden using MXR activity as an endpoint. The induction of P-gp expression was found to be in accordance with the level of contamination detected in the sediments from which the elutriates were extracted. The findings of the functional study also demonstrated that environmental pollutants, which interfere with P-gp function, could be identified using this model.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1997

O6-methylguanine-DNA methyltransferase activity in human buccal mucosal tissue and cell cultures. Complex mixtures related to habitual use of tobacco and betel quid inhibit the activity in vitro.

Extracts prepared from tissue specimens of normal, non-tumourous human buccal mucosa, and cultured buccal epithelial cells and fibroblasts, exhibited O6-methylguanine-DNA methyltransferase (MGMT) activity by catalysing the repair of the premutagenic O6-methylguanine lesion in isolated DNA with rates of 0.2 to 0.3 pmol/mg protein. An SV40 T antigen-immortalized buccal epithelial cell line termed SVpgC2a and a buccal squamous carcinoma line termed SqCC/Y1, both of which lack normal tumour suppressor gene p53 function, exhibited about 50 and 10% of the MGMT activity of normal cells, respectively. The normal, experimentally transformed and tumourous buccal cell types showed MGMT mRNA levels which correlated with their respective levels of MGMT activity. Exposure of buccal cell cultures to various organic or water-based extracts of products related to the use of tobacco and betel quid, decreased both cell survival (measured by reduction of tetrazolium dye) and MGMT activity (measured subsequently to the exposures in cellular extracts). Organic extracts of bidi smoke condensate and betel leaf showed higher potency than those of tobacco and snuff. An aqueous snuff extract also decreased both parameters, whereas an aqueous areca nut extract was without effect. The well-established sulph-hydryl-reactive agent Hg2+, a corrosion product of dental amalgam, served as a positive control and decreased MGMT activity following treatment of cells within a range of 1-10 microM. Taken together, significant MGMT activities were demonstrated in buccal tissue specimens and in the major buccal mucosal cell types in vitro. Lower than normal MGMT activity in two transformed buccal epithelial cell lines correlated with decreased MGMT mRNA and lack of functional p53. Finally, in vitro experiments suggested the potential inhibition of buccal mucosal MGMT activity by complex mixtures present in the saliva of tobacco and betel nut chewers.

Areca↗
PubMed1996

Measurement of DNA adducts in humans after complex mixture exposure.

In contrast to acute or chronic dosing experiments with a single chemical in animals, man is exposed to thousands of chemicals during a lifetime. Each of these may act alone, additively, synergistically or antagonistically in terms of biological effects, but most current risk assessment procedures fail to recognize such interactions. In carcinogenesis, a mutational process that is thought to occur through DNA damage by endogenous and/or exogenous agents, a wide variety of host factors is involved in disease outcome. These include absorption of chemicals, their distribution, metabolism and excretion. In addition, once metabolic activation has occurred, there is an array of protective mechanisms that cells have evolved to maintain DNA integrity, such as DNA repair, genetic redundancy and programmed cell death. One approach to risk assessment is to regard all DNA-damaging events as potentially leading to cancer and to measure DNA damage as the biologically relevant endpoint. The main method, if not the only method, presently available to assay a wide range of DNA adducts is 32P-postlabelling. This method has high sensitivity (limit of detection > 1 adduct per 10(10) nucleotides) and is capable of visualizing many different DNA adducts in a single analysis. Postlabelling is best suited for detecting hydrophobic adducts--low molecular weight adducts usually need a preliminary separation procedure prior to being postlabelled. This chromatographic procedure has been used to study DNA samples from human tissues of cigarette smokers, occupationally exposed groups and individuals living in polluted environments. Correlations have been found between the severity of exposure and the level of DNA adducts detected for human samples. However, most studies are single-time point studies, whereas for risk assessment purposes it may be better to use more quantitative and representative measures of long-term exposure, for example the number of adducts formed per annum. This article reviews methods of DNA adduct measurement, with particular reference to the 32P-postlabelling technique, which has been used to determine DNA adduct levels in populations exposed to complex mixtures.

DNA Adducts↗
PubMed2005

Link between spatial structure of microbial communities and degradation of a complex mixture of volatile organic compounds in peat biofilters.

AIMS: To investigate the relationships between the operation of the volatile organic compound (VOC) removal biofilter and the structure of microbial communities, and to study the impact on degradation activities and the structuring of microbial communities of biofilter malfunctions related to the qualitative composition of the polluted air. METHODS AND RESULTS: A microbiological study and a measurement of biodegradation activities were simultaneously carried out on two identical peat-packed columns, seeded with two different inocula, treating polluted air containing 11 VOCs. For both reactors, the spatial structure of the microbial communities was investigated by means of single-strand conformation polymorphism (SSCP) analysis. For both reactors, stratification of degradation activities in function of depth was observed. Oxygenated compounds were removed at the top of the column and aromatics at the bottom. Comparison of SSCP patterns clearly showed a shift in community structure in function of depth inside both biofilters. This distribution of biodegradation activities correlates with the spatialization of microbial density and diversity. Although the operating conditions of both reactors were identical and the biodegradation activities similar, the composition of microflora differed for biofilters A and B. Subdivision of biofilter B into two independent parts supplied with polluted air containing the complex VOC mixture showed that the microflora having colonized the bottom of biofilter B retained their potential for degrading oxygenated compounds. CONCLUSIONS: This work highlights the spatialization of biodegradation functions in a biofilter treating a complex mixture of VOCs. This distribution of biodegradation activities correlates with the spatialization of microbial density and diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: This vertical structure of microbial communities must be taken into consideration when dealing with the malfunctioning of bioreactors. These results are also useful information about changes in microbial communities following natural or anthropogenic alterations in different ecosystems (soils and sediments) where structuring of microbial communities according to depth has been observed.

Air Microbiology↗
PubMed2004

What contributes to the combined effect of a complex mixture?

The effect of a mixture of 10 compounds, which have previously been identified in an effect-directed analysis as potentially relevant for a specific contaminated riverine sediment (Brack et al. Arch. Environ. Contam. Toxicol. 1999, 37, 164), were investigated for the underlying joint effect. Components identified in an organic sediment extract included several PAHs (benzo[ghi]fluoranthene, benz[a]anthracene, fluoranthene, pyrene, 2-phenylnaphthalene, anthracene, and phenanthrene) plus prometryn, N-phenyl-2-naphthylamine, and parathion-methyl. Experiments were performed using a one-generation algal bioassay with the unicellular green algae Scenedesmus vacuolatus as well as chlorophyll fluorescence quenching analysis to describe the effects of the components and mixtures thereof. Analysis of the mixture effects based on concentration-response modeling of the effect data reveals that indeed effect contributions of several components can be expected although the mixture ratio is not equitoxic and the individual components vary greatly with respect to biological effect. Comparing predicted and observed mixture effects, the combined effect may not be attributed to a joint narcotic effect of the mixture components. Evidently, some of the components act specifically and dissimilar and may therefore be best described in their combined effect by response addition while for others a similar mode of action seems plausible. Chlorophyll fluorescence quenching analysis supports to discriminate between prometryn, N-phenyl-2-naphthylamine, and PAHs. A joint model for calculating the combined effect using concentration addition for the suspected unspecifically acting components in algae (PAHs and parathion-methyl) and subsequently response addition for this group and the other components clearly improves the description of the observed combined effect. Allocation of effect contributions to specific components using toxic units or effect contributions lead to different judgments. The observed combined effect of a 3-compound mixture of prometryn, N-phenyl-2-naphthylamine, and benzo[ghi]fluoranthene is indistinguishable from the effects of the original 10-compound mixture, demonstrating the need in site-specific assessment of complex contamination to account for the mode of action of contaminants. Implications forthe confirmation step in effect-directed analysis of substances causing effects in complex contaminated samples are discussed.

2-Naphthylamine↗