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PubMed2003

Proteome analysis of intact proteins in complex mixtures.

Analysis of intact protein mixtures by electrospray ionization mass spectrometry requires the resolution of a complex, overlapping set of multiply charged envelopes. To ascertain the ability of a moderate resolution mass spectrometer to resolve such mixtures, we have analyzed the soluble proteins of adult chick skeletal muscle. This is a highly specialized tissue showing a marked bias in expression of glycolytic enzymes in the soluble fraction. SDS-PAGE-resolved proteins were first identified by a combination of matrix-assisted laser desorption ionization time-of-flight (TOF) and electrospray ionization tandem mass spectrometry. Then the mixture of intact proteins was introduced into the electrospray source of a Q-TOF mass spectrometer either by direct infusion or via a C4 desalting trap. In both instances, the complex pattern of peaks could be resolved into true masses, and these masses could in many instances be reconciled with the masses predicted from the known protein sequences when qualified by expected co- and post-translational modifications. These included loss of the N-terminal initiator methionine residue and N-terminal acetylation. The ability to resolve such a complex mixture of proteins with a routine instrument is of considerable value in analyses of protein expression and in the confirmation of post-translational changes in mature proteins.

Amino Acid Sequence↗
PubMed1993

Utility of controlled human exposure studies for assessing the health effects of complex mixtures and indoor air pollutants.

The study of health effects induced by exposure to mixtures of pollutants is a complex task. The purpose of this paper is to identify areas of research in which the conduct of human controlled exposure (clinical) studies may contribute to better understanding health effects of exposure to indoor air and other mixtures. The strengths and weaknesses of clinical studies in general are reviewed, as well as examples from the literature of approaches that have been used. Human chamber studies play an important role alongside epidemiologic and animal toxicologic studies in such research. Human chamber studies are limited with regard to assessing chronic effects, rare effects, or effects from long-duration exposures but are powerful in assessing acute, reversible effects from short-duration exposures in humans. The areas in which human chamber studies are most likely to contribute include identification of effects or markers of effects for exposure to a given pollutant or mix of pollutants; direct dose-response assessment of effects for individual compounds and mixtures of set composition; identification of individual compounds responsible for the effects of a mixture; study of the joint effects of a binary mixture; development of markers of acute exposure for particular compounds; development of outcome measurements to be used in the field; and identification, characterization, and testing of sensitive subpopulations.

Air Pollutants↗
PubMed2003

Phosphoprotein isotope-coded solid-phase tag approach for enrichment and quantitative analysis of phosphopeptides from complex mixtures.

Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated beta-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography-tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line.

Amino Acid Sequence↗
PubMedDate not supplied

Simple luminescence method for estimation of benzo[a]pyrene in a complex mixture of polycyclic aromatic hydrocarbons without a pre-separation procedure.

Benzo[a]pyrene causes cancer at cellular level and is widely present in the environment. Conventional spectroscopic methods for analysis of this compound need a pre-separation procedure due to severe spectral overlap from other polycyclic aromatic hydrocarbons. We report a simple method that avoids spectral overlap of benzo[a]pyrene from other impurities or polycyclic aromatic hydrocarbons (PAHs), thus it can easily identify benzo[a]pyrene in a complex PAH mixture. The method could easily identify benzo[a]pyrene in an 18-component PAH mixture. Calibration plots in methanol solution and in micellar media show a good linearity (R > 0.9997) in the benzo[a]pyrene concentration range generally found in the environment. The method gives a detection limit of 1.52 x 10(-9) mol/L in CTAB micellar medium and 2.55 x 10(-9) mol/L in methanol solution. The proposed method is selective, sensitive and fast. The fluorescence response of benzo[a]pyrene is found to be a potential candidate to sense the critical micellar concentration (CMC) of CTAB micelles.

Algorithms↗
PubMedDate not supplied

Protein identification in complex mixtures.

This paper investigates the prospects of successful mass spectrometric protein identification based on mass data from proteolytic digests of complex protein mixtures. Sets of proteolytic peptide masses representing various numbers of digested proteins in a mixture were generated in silico. In each set, different proteins were selected from a protein sequence collection and for each protein the sequence coverage was randomly selected within a particular regime (15-30% or 30-60%). We demonstrate that the Probity algorithm, which is characterized by an optimal tolerance for random interference, employed in an iterative procedure can correctly identify >95% of proteins at a desired significance level in mixtures composed of hundreds of yeast proteins under realistic mass spectrometric experimental constraints. By using a model of the distribution of protein abundance, we demonstrate that the very high efficiency of identification of protein mixtures that can be achieved by appropriate choices of informatics procedures is hampered by limitations of the mass spectrometric dynamic range. The results stress the desire to choose carefully experimental protocols for comprehensive proteome analysis, focusing on truly critical issues such as the dynamic range, which potentially limits the possibilities of identifying low abundance proteins.

Fungal Proteins↗
PubMed2000

Evaluation of cytotoxicity, dioxin-like activity and estrogenicity of complex environmental mixtures.

Complex organic extracts from soils and sediments collected in an industrial region of the Czech Republic were tested with in vitro recombinant cell lines for their potential dioxin-like and estrogenic activity and cytotoxicity. For dioxin-like toxicity tested on H4IIE-luc cells complete dose-responses were obtained with all extracts. The MCF-7-luc cell line used for determination of estrogen receptor-mediated activity was sensitive to the cytotoxic effects of the soil and sediment extracts. Dose dependent cytotoxicity was observed with most samples. Significant estrogenic activity was found in all samples. The toxic or estrogenic equivalents based on the analytically determined concentrations of organic pollutants with known dioxin-like or estrogenic potency were calculated and compared to the bioassay-derived estimates. Fractionation along with mass-balance calculation enabled identification of the most active fraction and classes of compounds. Polycyclic aromatic hydrocarbons were identified as the group of compounds responsible for most of the TCDD-like activity as well as for important portion of estrogenic activity.

Czech Republic↗
PubMedDate not supplied

Results of the IPCS collaborative study on complex mixtures.

The International Programme on Chemical Safety (IPCS) sponsored a collaborative study to examine the intra- and inter-laboratory variation associated with the preparation and bioassay of complex chemical mixtures. The mixtures selected were National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs). 20 laboratories worldwide participated in the collaborative trial. The participating laboratories extracted the organic portion of two particulate samples--an air-particulate sample and a diesel-particulate sample--and bioassayed the extracts. The laboratories simultaneously bioassayed a NIST-prepared extract of coal tar and two control compounds (benzo[a]pyrene, and 1-nitropyrene). The bioassay method used was the Salmonella/mammalian microsome plate-incorporation test using strains TA98 and TA100. Study design also allowed for a comparison of sonication and Soxhlet extraction techniques. The mean extractable masses for the air particles and diesel particles were approximately 5% and 17.5%, respectively. The particulate samples were mutagenic in both strains with and without activation in all 20 laboratories. For TA100 the with and without activation slope values for the air particulate were 162 and 137 revertants per mg particles, respectively. For TA98 the respective diesel slope values were 268 and 269. The mutagenicity slope values for the diesel particles ranged from 3090 (TA98, +S9) to 6697 (TA100, +S9) revertants per mg particles. The coal tar solution was negative for both strains when exogenous activation was not used but was mutagenic in both strains with exogenous activation. The benzo[a]pyrene and 1-nitropyrene were used as positive controls and gave results consistent with the literature. This paper provides a complete summary of the data collected during the collaborative study. Companion papers provide further analysis and interpretation of the results.

Air Pollutants↗
PubMed2002

Sensitivity of epigean and hypogean freshwater macroinvertebrates to complex mixtures. Part I: Laboratory experiments.

The impact of waste storage on aquatic systems is a regulatory requirement in Europe, but it is nowadays only considered to a limited extent. The complexity of mixtures, which contain many inorganic and organic compounds, requires the use of combining chemical measurements with ecotoxicological observations. This research employed an integrated laboratory and outdoor mesocosms approach to assess the effects of mixtures on freshwater macroinvertebrates. The effects of percolates coming from water having percolated through maturated secondary smelting slags, on freshwater macroinvertebrates (molluscs, crustaceans and insect larvae) were investigated under laboratory conditions using a continuous flow-through testing apparatus. Lethality (LC50 96 and 240 h) was chosen as the endpoint. The results indicate that the difference in sensitivity of macroinvertebrates is correlated with their ability to regulate or neutralize contaminants in the mixture during short-term exposure. Moreover, differences in sensitivity were dependent on duration of exposure. Because of the variability of toxicity among mixtures coming from the same waste, bioassays are required to determine the toxicities of these mixtures.

Animals↗