[Show abstract][Hide abstract] ABSTRACT: Access to a wider range of quantitative protein assays would significantly impact the number and use of tissue markers in guiding disease treatment. Quantitative mass spectrometry-based peptide and protein assays, such as immuno-SRM assays, have seen tremendous growth in recent years in application to protein quantification in biological fluids such as plasma or urine. Here, we extend the capability of the technique by demonstrating the application of a multiplexed immuno-SRM assay for quantification of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) levels in cell line lysates and human surgical specimens. The performance of the assay was characterized using peptide response curves, with linear ranges covering approximately four orders of magnitude and limits of detection in the low fmol/mg lysate range. Reproducibility was acceptable with median coefficients of variation of approximately 10%. We applied the assay to measurements of ER and HER2 in well-characterized cell line lysates with good discernment based on ER/HER2 status. Finally, the proteins were measured in surgically resected breast cancers, and the results showed good correlation with ER/HER2 status determined by clinical assays. This is the first implementation of the peptide-based immuno-SRM assay technology in cell lysates and human surgical specimens.
[Show abstract][Hide abstract] ABSTRACT: High-throughput technologies can now identify hundreds of candidate protein biomarkers for any disease with relative ease. However, because there are no assays for the majority of proteins and de novo immunoassay development is prohibitively expensive, few candidate biomarkers are tested in clinical studies. We tested whether the analytical performance of a biomarker identification pipeline based on targeted mass spectrometry would be sufficient for data-dependent prioritization of candidate biomarkers, de novo development of assays and multiplexed biomarker verification. We used a data-dependent triage process to prioritize a subset of putative plasma biomarkers from >1,000 candidates previously identified using a mouse model of breast cancer. Eighty-eight novel quantitative assays based on selected reaction monitoring mass spectrometry were developed, multiplexed and evaluated in 80 plasma samples. Thirty-six proteins were verified as being elevated in the plasma of tumor-bearing animals. The analytical performance of this pipeline suggests that it should support the use of an analogous approach with human samples.
[Show abstract][Hide abstract] ABSTRACT: Wild stocks of Pacific salmon in the Northwestern United States have declined in recent years, and the major factors contributing to these losses include water pollution and loss of habitat. In salmon, sublethal chemical exposures may impact critical behaviors (such as homing, feeding, predator-avoidance) that are important for species survival. Therefore, understanding the potential for these species to biotransform organic compounds within sensitive target tissues such as liver, gills and olfactory region can help estimate or predict their susceptibility to pollutants. In this study, we used real-time quantitative polymerase chain reaction (Q-PCR), Western blotting, and catalytic assays to characterize the expression of Phase I biotransformation enzymes in coho salmon (Oncorhynchus kisutch), a sensitive species in the Pacific Northwest. Gene expression analysis using Q-PCR assays developed for coho genes revealed the presence of the predominant cytochrome P450 mRNAs (CYP1A, CYP2K1, CYP2M1, CYP3A27) in the olfactory rosettes and provided quantitative mRNA expression levels in coho liver and gills. Q-PCR analysis revealed relatively high expression of the major CYP isoforms in the liver and olfactory rosettes, which was generally confirmed by Western blotting. Extrahepatic CYP expression was generally higher in the olfactory rosettes as compared to the gills. Catalytic studies demonstrated functional CYP1A-dependent ethoxyresorufin-O-deethylase, CYP2-dependent pentoxyresorufin-O-dealkylase, CYP2K1-dependent testosterone 16beta-hydroxylase, and CYP3A27-dependent testosterone 6beta-hydroxylase activities in liver, but not at detectable levels in gills. In contrast, flavin-containing monooxygenase (FMO)-dependent thiourea S-oxidase activity was readily observed in the gills and was substantially higher than that observed in liver. Collectively, the results of this study suggest that the olfactory rosettes are important sites of extrahepatic biotransformation in coho salmon, and that tissue specific-differences in Phase I metabolism may lead to contrasting tissue-specific biotransformation capabilities in this species.
Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 02/2008; 147(1):78-84. · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The glutathione S-transferases (GSTs) are a family of phase II detoxification enzymes which protect against chemical injury. In contrast to mammals, GST expression in fish has not been extensively characterized, especially in the context of detoxifying waterborne pollutants. In the Northwestern United States, coho salmon (Oncorhynchus kisutch) are an important species of Pacific salmon with complex life histories that can include exposure to a variety of compounds including GST substrates. In the present study we characterized the expression of coho hepatic GST to better understand the ability of coho to detoxify chemicals of environmental relevance. Western blotting of coho hepatic GST revealed the presence of multiple GST-like proteins of approximately 24-26kDa. Reverse phase HPLC subunit analysis of GSH affinity-purified hepatic GST demonstrated six major and at least two minor potential GST isoforms which were characterized by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI MS-MS) and Fourier transform-ion cyclotron resonance (FT-ICR) MS analyses. The major hepatic coho GST isoforms consisted of a pi and a rho-class GST, whereas GSTs representing the alpha and mu classes constituted minor isoforms. Catalytic studies demonstrated that coho cytosolic GSTs were active towards the prototypical GST substrate 1-chloro-2,4-dinitrobenzene, as well as towards ethacrynic acid and nitrobutyl chloride. However, there was no observable cytosolic GST activity towards the pesticides methyl parathion or atrazine, or products of oxidative stress, such as cumene hydroperoxide and 4-hydroxynonenal. Interestingly, coho hepatic cytosolic fractions had a limited ability to bind bilirubin, reflecting a potential role in the sequestering of metabolic by-products. In summary, coho salmon exhibit a complex hepatic GST isoform expression profile consisting of several GST classes, but may have a limited a capacity to conjugate substrates of toxicological significance such as pesticides and endogenous compounds associated with cellular oxidative stress.
[Show abstract][Hide abstract] ABSTRACT: When present in the vadose zone, potentially toxic nitrate and perchlorate anions can be persistent sources of groundwater contamination. Gaseous electron donor injection technology (GEDIT), an anaerobic variation of petroleum hydrocarbon bioventing, involves injecting electron donor gases, such as hydrogen or ethyl acetate, into the vadose zone, to stimulate biodegradation of nitrate and perchlorate. Laboratory microcosm studies demonstrated that hydrogen and ethanol promoted nitrate and perchlorate reduction in vadose zone soil and that moisture content was an important factor. Column studies demonstrated that transport of particular electron donors varied significantly; ethyl acetate and butyraldehyde were transported more rapidly than butyl acetate and ethanol. Nitrate removal in the column studies, up to 100%, was best promoted by ethyl acetate. Up to 39% perchlorate removal was achieved with ethanol and was limited by insufficient incubation time. The results demonstrate that GEDIT is a promising remediation technology warranting further validation.
Water Environment Research 01/2007; 78(13):2436-46. · 1.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This report describes the demonstration and validation of a novel analytical technology: a bioavailable ferric iron (BAFeIII) assay. Demonstration and validation of the BAFeIII assay was conducted at four Department of Defense (DoD) installations.
[Show abstract][Hide abstract] ABSTRACT: Monitored natural attenuation (MNA) is a cost-effective remediation approach that is applicable to many sites and has been embraced by the U.S. Department of Defense (DOD). Monitored natural attenuation can be used to mitigate petroleum hydrocarbon, chlorinated hydrocarbon, and metal-contaminated sites as an alternative to groundwater pump and treat methods. Determination of MNA's technical applicability for a given site is based on sampling and analysis, data evaluation and modeling, and long-term monitoring. Parameters that are evaluated include concentrations of contaminants, electron acceptors, and electron donors. These concentrations in combination with hydrogeologic, soil, and microbial characteristics are used to assess the fate and transport of contaminants and the potential for natural attenuation. This report describes the demonstration of a novel analytical technology: a dissolved hydrogen (DH) analyzer. The report describes demonstration of the DH analyzer at three Department of Defense (DoD) sites as well as supplemental development of the DH analyzer.
[Show abstract][Hide abstract] ABSTRACT: A number of environmental chemicals have been shown to cause olfactory and neurobehavioral impairment in Pacific salmon. However, little is known about the ability of the salmon olfactory system to mediate the biotransformation of waterborne chemicals. In the current study, we analyzed the expression profiles of chemical biotransformation genes in olfactory rosettes of coho salmon (Oncorhynchus kisutch). Quantitative PCR (Q-PCR) assays were developed for the expression of coho cytochrome P450 (CYP) 1A, 2K1, 2M1, 3A27, and glutathione S-transferase (GST) pi, rho, and microsomal GST isoforms. In addition, olfactory mRNA expression was compared to gene expression in coho gills and liver. All of the genes analyzed were readily detected in olfactory tissues, with the relative levels of gene expression being CYP3A27>2K1>2M1>1A. Western blotting studies using salmonid class-specific CYP antibodies were consistent with the results obtained by Q-PCR. GST pi was the most abundant olfactory GST, but we also observed significant amounts of rho and microsomal GST isoforms. In some cases, CYP and GST isoform expression in the rosettes exceeded that in liver. Microarray analysis of coho olfactory tissues using a 16,000 element cDNA platform provided a more comprehensive profile of global biotransformation (phase I, phase II) and oxidative defense gene expression. The results of our study indicate a substantial biotransformation capacity of the olfactory system of coho salmon that may contribute to the detoxification of waterborne chemicals, and potentially limit chemical interactions with sensitive neuronal targets. Supported by NIH grants ES04696 and ES07033.