Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low µg/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.Proteomic technologies based on mass spectrometry (MS) have emerged as preferred components of a strategy for discovery of diagnostic, prognostic and therapeutic protein biomarkers. Because of the stochastic sampling of proteomes in unbiased analyses and the associated high false-discovery rate, tens to hundreds of potential biomarkers are often reported in discovery studies. Those few that will ultimately show sufficient sensitivity and specificity for a given medical condition must thus be culled from lengthy lists of candidates -a particularly challenging aspect of the biomarker-development pipeline and currently its main limiting step. In this context, it is highly desirable to verify, by more targeted quantitative methods, the levels of candidate biomarkers in body fluids, cells, tissues or organs from healthy individuals and affected patients in large enough sample numbers to confirm statistically relevant differences 1, 2. Verification of novel biomarkers has relied primarily on the use of sensitive, specific, high-throughput immunoassays, whose development depends critically on the availability of suitable well-characterized antibodies. However, antibody reagents of sufficient specificity and sensitivity to assay novel protein biomarkers in plasma are generally not available. The high cost and long development time required to generate high-quality immunoassay reagents, as well as technical limitations in multiplexing immunoassays for panels of biomarkers, is strong motivation to develop more straightforward quantitative approaches exploiting the sensitivity and molecular specificity of mass spectrometry.Recently, multiple reaction monitoring (MRM) coupled with stable isotope dilution (SID)-MS for direct quantification of proteins in cell lysates as well as human plasma and serum has been shown to have considerable promise 3- RESULTS Study de...
Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants in consumer goods, such as plastics, electronics, textiles, and construction material. PBDEs have been found in human milk, fat, and blood samples. Rodent studies indicate that PBDEs may be detrimental to neurodevelopment, possibly by lowering thyroid hormone concentrations in blood. In the present study, we determined concentrations of PBDEs and thyroid hormones in human fetal and maternal serum. Patients presenting in labor to Indiana University and Wishard Memorial County hospitals in Indianapolis, who were older than 18 years, were recruited to participate. Twelve paired samples of maternal and cord blood were obtained and analyzed using gas chromatographic mass spectrometry; thyroid hormone concentrations were determined by radioimmunoassay. Six congeners of PBDE were measured in maternal and fetal serum samples. The concentrations of total PBDEs found in maternal sera ranged from 15 to 580 ng/g lipid, and the concentrations found in fetal samples ranged from 14 to 460 ng/g lipid. Individual fetal blood concentrations did not differ from the corresponding maternal concentrations, indicating that measurement of maternal PBDE blood levels is useful in predicting fetal exposure; similarly, other reports have shown a high correlation between PBDE in mother's milk and fetal exposure. In accord with reports on other biologic samples, the tetrabrominated PBDE congener BDE-47 accounted for 53-64% of total PBDEs in the serum. The concentrations of PBDEs found in maternal and fetal serum samples were 20-106-fold higher than the levels reported previously in a similar population of Swedish mothers and infants. In this small sample, there was no apparent correlation between serum PBDEs and thyroid hormone concentrations. Our study shows that human fetuses in the United States may be exposed to relatively high levels of PBDEs. Further investigation is required to determine if these levels are specific to central Indiana and to assess the toxic potential of these exposure levels. Key words: brominated diphenyl ethers, cord blood, human, pregnancy, serum.
Few studies have measured the flame retardants polybrominated diphenyl ethers (PBDEs) in the indoor environment. Here, we report measurements of PBDEs in house dust samples collected from the Washington, D.C. metropolitan area in the United States. Dust samples were analyzed for 22 individual PBDE congeners and our results found PBDEs present in every sample. Concentrations of total PBDEs ranged from 780 ng/g dry mass to 30 100 ng/g dry mass. The dominant congeners observed in the dust samples were congeners associated with the pentaBDE and decaBDE commercial mixtures. Ancillary data were collected on the homes and examined for any correlations with total PBDE concentrations. No correlations were observed with year of house construction, type of flooring (i.e., hardwood vs carpet) or the number of television sets or personal computers in the home. However, a significant inverse correlation (p < 0.05) was observed between the area of the home and the contribution of BDE 209 to the total PBDE concentration in dust. Using estimates of inadvertent dust ingestion (0.02-0.2 g/day) by young children (ages 1-4), we estimate ingestion of total PBDEs to range from 120 to 6000 ng/day. Clothes dryer lint was also sampled and analyzed for PBDEs from five of the homes and were present in all five samples ranging from 480 to 3080 ng/g dry mass. This study demonstrates that PBDEs are prevalent at relatively high concentrations within homes where people, and particularly young children, may be susceptible to exposure.
Air samples were analyzed from urban, rural, and remote sites near the Great Lakes to investigate the occurrence, concentrations, and spatial and temporal differences of polybrominated diphenyl ethers (PBDE) in air. The concentrations of PBDEs were compared to those of other organohalogen compounds such as PCBs and organochlorine pesticides. The samples were collected in 1997-1999 as part of the Integrated Atmospheric Deposition Network (IADN). To minimize the variability of the data, we selected only samples taken when the atmospheric temperature was 20 +/- 3 degrees C. PBDEs were found in all samples, indicating that these compounds are widely distributed and that they can be transported through the atmosphere to remote areas. The total concentrations of PBDEs were similar to some of the organochlorine pesticides such as sigmaDDT and ranged from 5 pg/m3 near Lake Superior to about 52 pg/m3 in Chicago. In fact, the spatial trend was well correlated to those of PCBs. Our results indicate a relatively constant level from mid-1997 to mid-1999. At 20 +/- 3 degrees C, about 80% of the tetrabromo homologues are in the gas phase and about 70% of the hexabromo homologues are associated with the particle phase. Thus, particle-to-gas partitioning in the atmosphere is an important process for these compounds.
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