@article {89, title = {A rapid and sensitive fluorometric method for determination of aldehyde oxidase activity.}, journal = {Toxicol Appl Pharmacol}, volume = {341}, year = {2018}, month = {2018 02 15}, pages = {30-37}, abstract = {

Previous research has characterized the important role of aldehyde oxidases (AOX) in biotransformation of N-heterocyclic therapeutic drugs and environmental contaminants in mammals. Research pertaining to AOX activity in non-mammalian vertebrates, however, is scarce, despite its biological role as a potentially important metabolic pathway for xenobiotics. One of the limiting factors of research on AOX is that available photometric methods are relatively insensitive, limited in throughput, and prone to cross-reactivity from other enzymes. Therefore, this study aimed to develop a novel and improved fluorometric AOX assay. This assay is based on the conversion of the exogenous aldehyde substrate 4-(dimethyl)amino cinnamaldehyde to its corresponding fluorescent acid by AOX, and was evaluated using partially purified hepatic cytosol from rat, human, and rainbow trout. Purification of native cytosol by heat treatment and ammonium sulfate precipitation resulted in increased specific activity of AOX. Michaelis-Menten kinetic parameters (Kand V) were comparable to values previously generated by photometric methods. Furthermore, effects of the inhibitor hydralazine on AOX activity revealed half maximal inhibitory concentrations comparable to those generated using conventional methods. Product identity was confirmed by liquid chromatography and mass spectrometry. In summary, this study successfully developed a rapid and sensitive assay for determination of AOX activity in across different vertebrate species that is 4- to 10-fold more sensitive compared to conventional absorbance-based methods. It can be applied in environmental, toxicological, and pharmacological studies relating to identification of AOX substrates, as well as the induction of AOX expression through drugs and environmental contaminants.

}, keywords = {Aldehyde Oxidase, Animals, Chromatography, Liquid, Dose-Response Relationship, Drug, Enzyme Activation, Enzyme Inhibitors, Fluorometry, Humans, Hydralazine, Mass Spectrometry, Oncorhynchus mykiss, Rats, Time Factors}, issn = {1096-0333}, doi = {10.1016/j.taap.2018.01.006}, author = {Apenova, Nancy and Peng, Hui and Hecker, Markus and Brinkmann, Markus} } @article {90, title = {Mass spectrometry methods to study protein-metabolite interactions.}, journal = {Expert Opin Drug Discov}, volume = {12}, year = {2017}, month = {2017 12}, pages = {1271-1280}, abstract = {

INTRODUCTION: To ​​​​understand and manipulate biochemical processes and signaling pathways, the knowledge of endogenous protein-metabolite interactions would be extremely helpful. Recent developments in precision mass spectrometry, high-throughput proteomics and sensitive metabolomic profiling are beginning to converge on a possible solution, heralding a new era of global metabolome-proteome {\textquoteright}interactome{\textquoteright} studies that promise to change biomedical research and drug discovery. Areas covered: Here, we review innovative mass spectrometry-based methods and recent pioneering studies aimed at elucidating the physical associations of small molecule ligands with cellular proteins. The technologies covered belong to two main categories: tag-based and tag-free methods. We emphasize the latter in this review, and outline promising experimental workflows and key data analysis considerations involved. Expert opinion: Recent ground-breaking advances in chemical-proteomics technology and allied computational methods now make the global detection of protein-ligand engagement an increasingly attractive research problem. Despite ongoing challenges, rapid progress in the field is expected these coming next few years, leading to a refreshed systems biology research paradigm and much needed new opportunities for improving sparse drug discovery pipelines.

}, keywords = {Biomedical Research, Drug Discovery, High-Throughput Screening Assays, Humans, Ligands, Mass Spectrometry, Metabolome, Metabolomics, Proteins, Proteome, Proteomics, Systems Biology}, issn = {1746-045X}, doi = {10.1080/17460441.2017.1378178}, author = {Guo, Hongbo and Peng, Hui and Emili, Andrew} } @article {107, title = {Detection, identification, and quantification of hydroxylated bis(2-ethylhexyl)-tetrabromophthalate isomers in house dust.}, journal = {Environ Sci Technol}, volume = {49}, year = {2015}, month = {2015 Mar 03}, pages = {2999-3006}, abstract = {

Ultra-High Resolution LC/mass spectrometry (LC-UHRMS; Thermo Fisher Q-Exactive) was used to identify two novel isomers of hydroxylated bis(2-ethylhexyl)-tetrabromophthalate (OH-TBPH) which were unexpectedly observed in a commercial standard of TBPH. By combining ultra-high resolution (UHR) mass spectra (MS(1)), mass errors to theoretical [TBPH-Br+O](-) were 2.1 and 1.0 ppm for the two isomers, UHR-MS(2) spectra and NMR analysis; the structures of the two compounds were identified as hydroxylated TBPH with a hydroxyl group on the aromatic ring. Relatively great proportions of the two isomers of OH-TBPH were detected in two technical products, Firemaster 550 (FM-550; 0.1\% and 6.2\%, respectively) and Firemaster BZ 54 (BZ-54; 0.1\% and 7.9\%), compared to a commercial standard (0.4\% and 0.9\%). To simultaneously analyze OH-TBPH isomers and TBPH in samples of dust, a method based on LC-UHRMS was developed to quantify the two compounds, using negative and positive ion modes, respectively. The instrumental limit of detection for TBPH was 0.01 μg/L, which was 200-300 times better than traditional methods (2.5 μg/L) based on gas chromatography-mass spectrometry. The analytical method combined with a Florisil cleanup was successfully applied to analyze TBPH and OH-TBPH in 23 indoor dust samples from Saskatoon, Saskatchewan, Canada. Two OH-TBPH isomers, OH-TBPH1 and OH-TBPH2, were detected in 52\% and 91\% of dust samples, respectively. Concentrations of OH-TBPH2 (0.35 {\textpm} 1.0 ng/g) were 10-fold greater than those of OH-TBPH1 (0.04 {\textpm} 0.88 ng/g) in dust, which was similar to profiles in FM-550 and BZ-54. TBPH was also detected in 100\% of dust samples with a mean concentration of 733 {\textpm} 0.87 ng/g. A significant (p \< 0.001) log-linear relationship was observed between TBPH and OH-TBPH isomers, further supporting the hypothesis of a common source of emission. Relatively small proportions of OH-TBPH isomers were detected in dust (0.01\% {\textpm} 0.67 OH-TBPH1 and 0.1\% {\textpm} 0.60 OH-TBPH2), which were significantly less than those in technical products (p \< 0.001). This result indicated different environmental behaviors of OH-TBPH and TBPH. Detection of isomers of OH-TBPH is important, since compounds with phenolic groups have often shown relatively greater toxicities than nonhydroxylated analogues. Further study is warranted to clarify the environmental behaviors and potential toxicities of OH-TBPH isomers.

}, keywords = {Chromatography, Liquid, Dust, Flame Retardants, Limit of Detection, Mass Spectrometry, Phthalic Acids}, issn = {1520-5851}, doi = {10.1021/es505743d}, author = {Peng, Hui and Saunders, David M V and Sun, Jianxian and Codling, Garry and Wiseman, Steve and Jones, Paul D and Giesy, John P} } @article {105, title = {Untargeted Identification of Organo-Bromine Compounds in Lake Sediments by Ultrahigh-Resolution Mass Spectrometry with the Data-Independent Precursor Isolation and Characteristic Fragment Method.}, journal = {Anal Chem}, volume = {87}, year = {2015}, month = {2015 Oct 20}, pages = {10237-46}, abstract = {

While previous studies have found that unknown natural and synthetic organo-bromine compounds (NSOBCs) contributed more than 99\% of the total organic bromine (Br) in the environment, there was no efficient method for untargeted screening to identify NSOBCs in environmental matrixes. A novel untargeted method for identifying NSOBCs, based on ultrahigh-resolution mass spectrometry (UHRMS) with the Q Exactive instrument was developed. This method included a data-independent precursor isolation and characteristic fragment (DIPIC-Frag) procedure to identify NSOBCs. A total of 180 successive 5-m/z-wide windows were used to isolate precursor ions. This resulted in a sufficient dynamic range and specificity to identify peaks of Br fragment ions for analysis. A total of 2520 peaks of NSOBC compounds containing Br were observed in sediments from Lake Michigan, United States. A new chemometric strategy which combined chromatographic profiles, isotopic peaks, precursor isolation window information, and intensities was used to identify precursor ions and chemical formulas for detecting NSOBCs. Precursor ions for 2163 of the 2520 NSOBCs peaks (86\%) were identified, and chemical formulas for 2071 NSOBCs peaks (82\%) were determined. After exclusion of isotopic peaks, 1593 unique NSOBCs were identified and chemical formulas derived for each. Most of the compounds identified had not been reported previously and had intensities which were 100- to 1000-fold greater than the congeners of polybrominated diphenyl ethers (PBDEs). In extracts of sediments, these compounds exhibited variations in intensities (\<10(3) to \~{}10(8)), m/z values (170.9438-997.5217), retention times on a C18 column (1.0-29.3 min), and the number of Br atoms (1-8). Generally, compounds with greater m/z values had longer retention times and greater numbers of Br atoms. Three compounds were used in a proof-of-concept experiment to demonstrate that structures of some of the screened NSOBCs could be further predicted by combining searching of database libraries and high-resolution MS(2) spectra.

}, keywords = {Databases, Chemical, Geologic Sediments, Hydrocarbons, Brominated, Lakes, Mass Spectrometry}, issn = {1520-6882}, doi = {10.1021/acs.analchem.5b01435}, author = {Peng, Hui and Chen, Chunli and Saunders, David M V and Sun, Jianxian and Tang, Song and Codling, Garry and Hecker, Markus and Wiseman, Steve and Jones, Paul D and Li, An and Rockne, Karl J and Giesy, John P} }