Core B: Research Support Core

The Research Support Core (RSC) of the University of Kentucky Superfund Research Center (UK SRC) provides vital access to expertise, research resources and state of the art instrumentation to researchers engaged in all aspects of the biomedical and environmental science research projects. The RSC enhances the productivity, quality and consistency of these projects by providing a formalized mechanism for access to expert investigators and professional staff and by enabling efficient use of expensive and sophisticated instrumentation that would otherwise be beyond the capabilities of individual laboratories to acquire and support. The RSC provides services in “Quantitative Biology” encompassing biostatistics support for experimental design and computational infrastructure for data management, analysis and sharing. Bioanalytical services provide technologies for quantitation and structural analysis of environmental toxicants, nutritional protectants and markers and mediators of oxidative stress and inflammation. The RSC is now also developing advanced mass spectrometry-based strategies for identification of biomarkers of PCB toxicity and nutritional protection from PCB exposure. 

Establishment of a coordinated effort in the form of the Research Support Core maximizes the research efficiency and cost effectiveness of our Superfund Center. This Core is divided into two major components directed by an expert in each area:

  • Bioanalytical Component - Directed by Dr. Andrew Morris
  • Quantitative Biology Component - Directed by Dr. Arnold Stromberg

The research support core is directed by Dr. Morris who works with Dr. Hennig (the Director of UK SRC) and the Administration Core, and the Project Leaders of all individual grants to ensure operation of the core serves the overall goals of the center.

Aims

  • Provide unified infrastructure for data management that can be used to share information between the component projects enabling integration of observations that will illuminate common pathways of PCB toxicity and nutritional protection.
  • Provide expertise in biostatistics that is vitally necessary for the design and interpretation of experiments conducted by all of the participating investigators and to continue to expand and refine these capabilities to encompass informatics approaches to management of large data sets necessary for application and implementation of gene expression profiling and mass spectrometry.
  • Provide access to state of the art mass spectrometry based methods for detection and quantitation of PCBs, nutritional protectants and other small molecules and mediators that are of shared importance to the participating investigators for calibration, validation and monitoring of PCB sensing and remediation technologies by the environmental science projects and for ensuring consistency and relevance to human exposure of PCB doses used in studies with cultured cell and animal models.
  • Leverage recent institutional investments in advanced mass spectrometry instrumentation and research infrastructure for the further development of the metabolomics capabilities of the bioanalytical component of the RSC.
  • Provide a conduit for synergistic interactions between the UK SRC and clinical and translational researchers at the University of Kentucky who are engaged in studies of nutritional protection in case controlled clinical studies of cardiovascular and metabolic disease.

The Research Support Core is directed by Dr. Andrew J. Morris, who also oversees the Bioanalytical Component. Dr. Morris is a Professor of Cardiovascular Medicine. Dr. Morris’s personal research program concerns studies of pathways of lipid metabolism involved in inflammation, cardiovascular and metabolic diseases. Professional staff of the Research Support Core include Dr. M. Abdul Mottaleb, Dr. Jianzhong Chen and Ms. Yu Li. Dr. Mottaleb is an environmental analytical chemist with extensive experience gained in academia and governmental research laboratories including employment with the Environmental Protection Agency. Drs. Morris and Mottaleb, working with Dr. Chen and Ms. Li, are responsible for all aspects of the analytical chemistry component of the Core. Dr. Arnold J. Stromberg directs the Quantitative Biology Component. Dr. Stromberg is the Chair of the Statistics Department and has published extensively in the area of computational analytical methods for large scale analysis of gene expression data.​

Instrumentation

​​Bioanalytical instrumentation is housed in the Biological Biomedical Sciences Research Building at the University of Kentucky and at the adjacent Lexington Veterans Affairs Medical Center. 

University of Kentucky:  Mass Spectrometry Laboratory

This instrumentation is contained in ~2000 square feet of laboratory space in the basement of the Biological Biomedical Sciences Research Building that includes separate rooms for most of the instrumentation and a common area for bench work including sample preparation. An adjoining room houses two 5HP powerex scroll air compressors with storage tanks that supply compressed air to Parker Balston nitrogen and tri gas generators that service the individual mass spectrometer systems and a small nitrogen generator that supplies gas to solvent evaporators.

  • AB Sciex 4000 Q-Trap triple quadrupole linear ion trap mass spectrometer. This instrument is configured for use with either an  ABSciex “Turbo V” electrospray and chemical ionization source or an ABSciex Flashquant vaccum MALDI ion source. It is interfaced with a Shimadzu prominence HPLC system comprising an autosampler, column oven, dual pumps, switching valves and a controller and is connected to a workstation computer running AB Sciex Analyst and Multiquant Software for instrument control and data analysis.
  • AB Sciex 4000 Q-Trap triple quadrupole linear ion trap mass spectrometer. This instrument is equipped with an ABSciex “Turbo V” electrospray and chemical ionization source and is interfaced with a Shimadzy Nexera UHPLC system comprising an autosampler, dual pumps, switching valves and a controller and is connected to a workstation computer running AB Sciex Analyst and Multiquant Software for instrument control and data analysis.
  • ABSciex 5600 Quadrupole time of flight mass spectrometer. This instrument incorporates an AB Sciex electrospray and chemical ionization source and is configured for operation with either a complete automated Shimadzu HPLC system, or with a Eksigent microflow HPLC system that is configured for use with either capillary HPLC columns or perform automated direct infusion. This microflow system operates with a low dispersion electrode insert that provides some of the sensitivity benefits of nano flow electrospray ionization with increased robustness in comparison to glass emitter electrodes. This instrument can also be configured with an Advion Nanomate robotic chip based nano electrospray ionization source for sample analysis by direct infusion.
  • Thermo Q-Exactive quadrupole orbitrap mass spectrometer. This is a quadrupole orbitrap mass spectrometer system that provides high sensitivity and high mass resolution for quantitative and qualitative analysis. The system is configured for use with a Thermo/Dionex U3000 UPLC chromatography system and is interfaced with a workstation computer running software for instrument control, data acquisition and analysis including Thermo Compound Discoverer and Lipid Search Software.
  • Sample preparation and biochemical analysis. The laboratory contains two Gilson Gradient HPLC systems with absorbance, fluorescence and evaporative light scattering detectors. Other equipment in the laboratory for sample preparation for GC or LC MS includes two N-Evap N2 evaporator systems, three Biotage Turbo Vap evaporators (one configured for 4 ml vials, the other for 96 well plates), two LabConco centrifugal evaporator/concentrators, a Genevac Rocket Solvent Evaporator System, a Thermo/Dionex Accelerated Solvent Extraction System, two Supelco Solid Phase extraction systems, a Matrix Scientific Well Mate multiwell plate dispenser, a Gilson 215 Robotic Liquid Handling System configured for multiwell plate and cartridge based Solid Phase Extraction with workstation computer running Gilson Trilution Software, two multi sample vortexers, heating blocks for lipid phosphate analysis and chemical derivatization and a Biotek Multimode absorbance/fluorescence reader, biotek powerwave absorbance plate reader.
  • Data analysis. In addition to the instrument control computers, the facility contains two workstation computers running instrument control software and software packages for data analysis that are available for off-line processing of data collected using the primary instrument control computers. Proprietary and open source software packages are available for compound identification, metabolomics and lipidomics. This includes Thermo Compound Discoverer and Lipid Search and ABSciex Peakview/Masterview and Lipidview. All of the computer systems in the laboratory have redundant hard drives and are networked for data transfer and backup.

Lexington Veterans Affairs Medical Center 

This instrumentation is contained in ~1000 square feet of laboratory space at the adjacent Lexington Veterans Affairs Medical Center. The space is assigned to Dr. Morris in his capacity as a VA funded research investigator. Use of this instrumentation for UK Superfund Center Research has been approved by the associate chief of staff for research and development.

  • ABSciex 6500 plus Q-Trap triple quadrupole linear ion trap mass spectrometer system. The instrument is configured with a two separate Shimadzu Nexera UPLC system including a rack changing autosampler and column oven and connected to a workstation computer running AB Sciex Analyst and Multiquant Software for instrument control and data analysis. One of these UHPLC systems is dedicated for measurements of trace levels of environmental chemicals in clinical samples. A Parker Balston TGN 30 integrated gas generator supplies nitrogen zero and dry air to the instrument. The instrument control and data acquisition computer can be connected to the University of Kentucky computer network for data transfer and storage including integration with the LIMS system.
  • Aglilent Intuvo 9000GC/Aglient 5977B MSD. This instrument is configured with an electron impact source, integrated autosampler and microfluidics cartridge and has a direct heat column oven for ultra fast gas chromatography. The system is interfaced with a workstation computer running Agilent Chemstation software for instrument control and data analysis.
  • Sample preparation. An adjacent laboratory assigned to Dr. Morris contains a 96 well format Biotage Turbo Vap and an organomation N-evap system that are supplied with gas from a small nitrogen generator and air compressor.

Established Analytical Methods to Support UK Superfund Researchers

Environmental Chemicals

 

Panel/Service

Analytes

Method

  A

Per and Poly Fluorinated Substances

 

Short-chain:  PFBS, GenX, 4.2 FTS, Long-chain, PFNA, PFOA, PFHxS, PFHpA, PFOS: Surrogates, 4:2 FTSL, PFOAL, PFNAL, PFOSL

 

HPLC MS/MS (triple quadrupole)

  B

Phthalates

 

Dimethyl phthalate (DMP), Diethyl phthalate (DEP) Diallyl phthalate (DAP) Dipropyl phthalate DPrP Diisopropyl phthalate DIPrP Dibutyl phthalate  Diisobutyl phthalate Bis(2-methoxyethyl) phthalate DMEP Dipentyl phthalate EPA DPP Diisopentyl phthalate DIPP Bis(2-ethoxyethyl) phthalate DEEP Benzyl butyl phthalate Diphenyl phthalate DPhP Dicyclohexyl phthalate DCHP Bis(4-methyl-2-pentyl) phthalate Dihexyl phthalate DHXP Di-n-heptyl phthalate DHP Bis(2-n-butoxyethyl) phthalate DBEP Bis(2-ethylhexyl) phthalate Di-n-octyl phthalate Diisononyl ortho-phthalate Diisodecyl ortho-phthalate

 

HPLC MS/MS (triple quadrupole)

  C

Environmental Phenols

 

4-t-Octylphenol, Benzophenone-3 (Oxybenzone), Bisphenol A (BPA), o-Phenylphenol, Parabens, Triclocarban, Triclosan

HPLC MS/MS (triple quadrupole)

  D

Polychlorinated Biphenyls

Selected congeners and mixtures

GC MS, GC ECD, GC MS/MS

  E

Polychlorinated Dibenzofurans

 

2,3,7,8-TCDF​; 1,2,3,7,8-PeCDF; 2,3,4,7,8-PeCDF; 1,2,3,4,7,8-HxCDF; 1,2,3,6,7,8-HxCDF; 2,3,4,6,7,8-HxCDF; 1,2,3,7,8,9-HxCDF; 1,2,3,4,6,7,8-HpCDF; 1,2,3,4,7,8,9-HpCDF; OCDF          

GC MS, GC MS/MS

  F

Polychorinated dibenzo-p-dioxins

 

2,3,7,8-TCDD; 1,2,3,7,8-PeCDD; 1,2,3,4,7,8-HxCDD; 1,2,3,6,7,8-HxCDD; 1,2,3,7,8,9-HxCDD​; 1,2,3,4,6,7,8-HpCDD; OCDD

GC MS, GC MS/MS

 

Behavior/Lifestyle Associated Environmental Stressors

 

Panel/Service

Analytes

Method

  A

Smoking/e-cigarette use

Nicotine, Cotinine, trans 3 OH cotinine and glucuronides

Serum, plasma, urine, tissues  LC MS/MS (triple quadrupole)

  B

Tobacco smoking

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN)4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and their glucuronides

Serum, plasma, urine, tissues  LC MS/MS (triple quadrupole)

  C

Common opiates

codeine, morphine, hydrocodone, hydromorphone, oxycodone, oxymorphone

Serum, plasma, urine, tissues  LC MS/MS (triple quadrupole)

 

Metabolomics/Lipidomics

 

Panel/Service

Analytes

Method

  A

Lipidomics

~1200 individual lipid species with quantitation using lipid class specific standards.  Plasma, serum, other biofluids or tissues

Validated for plasma/serum, tissues.  Reverse phase UHPLC, positive and negative mode HRMS using Orbitrap or Q-Tof Platforms.  Data analysis using Lipidsearch or Lipidview software validation using targeted measurements with triple quadrupole instrument

  B

Metabolomics

~ 400 widely targeted polar small molecule metabolites.  Plasma, serum, other biofliuds or tissues

Validated for plasma/serum, tissues.  HILIC UHPLC positive and negative mode HRMS using Orbitrap or Q-TOF platforms, data analysis using compound discoverer validation using targeted measurements with triple quadrupole instrument

  C

Bioinformatics support

Analysis and visualization of lipidomics/metabolomics data.  Formatting and submission to online repositories.  Integrated/multiomics pathway analysis

Proprietary and open source software- could be done collaboratively with Superfund Data Management Core

 

Biomedical Project Services

 

Panel/service

Analytes

Method (validated for plasma/serum and tissues)

  A

Methylamines

Choline, Betaine, TMAO, Creatinine

LC MS/MS (triple quad)

  B

Acyl CoA

Short-long chain (C2-24)

LC MS/MS (triple quad)

  C

Acyl Carnitines

Short-long chain (C2-24))

LC MS/MS (triple quad)

  D

Fatty Acids

C4->C30 branched, unsaturated

Methyl esters by GC MS, MS/MS  Methyl pyridinium derivatives by LC MS/MS (triple quad- targeted, Q-TOF, untargeted

  E

Sterols and Bile Acids

~20 species includes plant sterols and cholesterol metabolites (microbiome, peroxisomes)

GC MS, LC MS/MS (triple quad)

  F

Glucose homeostasis

Glucose/deuterated Glucose

Derivatization GC MS

  H

Lipogenesis, triglyceride synthesis using deuterated   water

Fatty acids, triglycerides, glycerol

Derivatization GC MS, LC MS/MS

  I

Glycerophospholipids (positive mode)

Phosphatidylcholines, phosphatidylethanolamines

LC MS/MS (triple quadrupole)

  J

Glycerophospholipids (negative mode)

Phosphatidylserines, phosphatidylinositols

LC MS/MS (triple quadrupole)

  K

Sphingophospholipids

Ceramides, sphingomyelins

LC MS/MS (triple quadrupole)

  L

Bioactive Lysophospholipids

Lysophosphatidic acids, sphingosinse 1 phosphate

LC MS/MS (triple quadrupole)

  M

Phosphoinositides

Phosphatidylinositol phosphates (PI, PIPs, PIP2, PIP3)

LC MS/MS (triple quadrupole) of methylated derivatives

  N

Isoprostanes

F2-isoprostane, PGF2 alpha

LC MS/MS (triple quadrupole)

  O

Eicosanoids

Bioactive arachidonic acid metabolites ~10

LC MS/MS (triple quadrupole)

  P

Glycolysis/pentose phosphate/TCA cycle

fructose 1,6,bisphosphate, phosphoenolpyruvate etc

LC MS/MS (triple quadrupole)

  Q

Redox/Bioenergetics

ATP, ADP, AMP, NAD, NADH, NADP, NADPH, glutathione

LC MS/MS (triple quadrupole)

Trainees

Jianzhong Chen
(Postdoctoral Trainee)

Pan Deng
(Postdoctoral Trainee)

Greg Hawk
(Graduate Trainee)