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Redox Metabolism Shared Resource Facility (RM SRF)

Director: D. Allan Butterfield, PhD

  • Mission Statement

    The mission of the Redox Metabolism Shared Resource Facility (RM SRF) is to provide expertise and services in redox metabolism, oxidative stress, mitochondrial function, proteomics, and metabolomics to basic and clinical investigators of the Markey Cancer Center (MCC) doing basic, pre-clinical, and clinical research.

  • Rationale

    Cancer cells demonstrate increased free radical biology and altered metabolism. Often these changes are manifested by elevated markers of oxidative stress, modified mitochondrial and cellular metabolism, and since proteins carry out metabolic processes, by differential protein expression and/or posttranslational modification. Among the latter are specific oxidative and epigenetic modifications. 

    To investigate these integrated aspects of redox metabolism, the RM SRF performs four major services:

    1. Analysis of markers of oxidative and nitrosative stress
    2. Seahorse FX-based analyses of mitochondrial function and glycolysis
    3. Proteommics or redox proteomics analyses of protein expression or oxidatively or covalently modified proteins
    4. Profiling and Stable Isotope Resolved Metabolomics (SIRM)
  • How the RM SRF Furthers Markey Science

    The role of free radicals and altered metabolism is increasingly apparent in various aspects of cancer biology and cancer therapy. Analysis of the roles of free radicals in cancer biology requires highly skilled ability to:

    • Measure the damage induced by free radicals in various tissues, cells, and fluids associated with different cancers.
    • Measure molecules responsible for free radical production, oxidant scavenging, and free radical damage.
    • Use proteomics to identify proteins in tissues, cells, and fluids that have differential levels or have been oxidatively modified.
    • Measure mitochondrial function, since mitochondria are a major source of free radicals in cells.
    • Provide metabolic information about altered metabolites, enzymes, and pathways in cancer and following cancer treatments.

    A fundamental cornerstone for the RM SRFs metabolomics component is stable-isotope labeling during the biological experiment, meaning experimental design must be integrated with analysis and informatics. Because of this, a simple fee-for-service model is unlikely to yield very useful results for cancer researchers, and metabolomics services are structured for extensive advising and collaboration.

    Importantly, and unique to this region of the United States, the RM SRF is one of only a few cancer center-resident resources in the world with the knowledge and reagents necessary to provide the redox and metabolism services needed by MCC members. Personnel in the RM SRF are highly knowledgeable about free radicals, oxidative stress, metabolism, sample handling and preparation, and the advantages and limitations of each assay employed, including bioenergetics, metabolomics, and proteomics. The Resource Center for Stable Isotope-Resolved Metabolomics (RC-SIRM), housed in the Center for Environmental and Systems Biochemistry (CESB), is one of the six metabolomics centers supported by the NIH Common Fund. These unique investments provide a distinct advantage to MCC researchers in sample use, analysis, and interpretation of results.

  • Prioritization of Services

    If multiple principal investigators (PIs) request simultaneous analyses, the RM SRF leadership determines priority based on the following criteria:

    • Level 1: Samples from MCC members with NCI funding or cancer-related federally funded peer reviewed studies
    • Level 2: Samples from MCC investigators preparing cancer-related, peer reviewed grant applications
    • Level 3: Samples from MCC investigators with a cancer-related project funded by MCC pilot studies or non-peer reviewed source
    • Level 4: Samples from MCC investigators without funding whose analysis would lead to preliminary data and the likelihood of a subsequent extramural proposal submission
    • Level 5: Samples from non-MCC investigators.
  • Acknowledging the RM SRF

    Investigators are required to acknowledge the Markey Cancer Center Redox Metabolism Shared Resource Facility (RM SRF) in any publications that result from the use of Redox Metabolism services or information received through the RM SRF. For your convenience, you are welcome to use the following statement. Please include the names of individuals from the RM SRF if they provided any intellectual input or additional effort.

    The research was supported by the Redox Metabolism Shared Resource Facility of the University of Kentucky Markey Cancer Center (P30CA177558).

  • Forms and Services

    Forms

    Before the RM SRF can begin work, researchers are required to complete the online RM SRF Sample Submission Form and the RM SRF Biosafety Questionnaire via iLab. Both forms may be accessed by visiting the iLab page.

    If you experience any problems trying to submit your service request to iLab, contact Mr. Michael Alstott for troubleshooting. 

    NOTE: RM SRF services cannot be initiated without a researcher account number and authorization within the iLab submission form.

    RM SRF Main Services (Fee-for-Service Basis) 

    Service 1: Analysis of markers of oxidative and nitrosative stress. Assays of total protein oxidation and lipid peroxidation ($20/per oxidative modification/per membrane used).

    • Indices of protein oxidation (protein carbonyls and 3-nitrotyrosine).
    • Index of lipid peroxidation (protein-bound 4-hydroxy-2-trans-nonenal, HNE).
    • Index of DNA or RNA oxidation (8-hydroxy-2 deoxy-guanosine or 8-hydroxy-2-guanosine, respectively).
    • Analyses of antioxidant enzyme activities and levels.
    • Analyses of reduced and oxidized glutathione (GSH/GSSG) and  NAD+/NADH, NADP+/NADPH.
    • Interpretation of results and suggestions for use of these indices in the four MCC research programs

    Service 2: Molecular biological manipulation of biological systems with which to investigate redox signals, including measurements of mitochondrial function. Seahorse 96 well analysis ($50/sample plate). Seahorse 8 well analysis ($25/sample plate). Molecular biological manipulation of redox signaling ($50/sample). Assay of redox enzymes ($15/sample).

    • Seahorse Biosciences instrumental analyses to monitor changes on oxygen consumption and pH in intact cells simultaneously using a microtiter plate platform (for example, to facilitate dose-response studies of chemotherapeutics). 
    • cDNA probes coding for primary antioxidant enzymes.
    • Stable and transient transfection of redox-related proteins (including those that regulate the redox status of cells, scavenge free radicals, and repair oxidative/nitrosative damage) into cells.

    Service 3: Proteomics identification of proteins. Proteomics or redox proteomics analyses of protein expression or oxidatively or covalently modified proteins. The MCC subsidizes proteomics services for MCC members at a rate of 40%. For instance, LC-MS/MS analysis of protein modifications using Orbitrap is $90 for MCC members instead of $150. To comply with the University policy that all users should be charged with the same rate, MCC provides a $60 subsidy for this analysis. The commitment from MCC will enable the investigators to utilize the state-of-the-art technology in their cancer research programs.

    • Proteomics identification of proteins with differential expression, deferential oxidative modification or differential covalent modification in systems of interest: protein separation, digestion, and ESI-MS/MS sequence analysis of tryptic peptides on an orbitrap MS instrument. 
    • Imaging software-mediated determination of proteins to be evaluated.
    • Spot excising and protein digestion.
    • Database interrogation to identify proteins.
    • Validation of identification by Western blotting or other means.
    • Functional analysis of oxidatively modified proteins (can also be performed for other post-translational modifications).
    • Analysis of protein-protein interactions involved in redox signaling (can also be applied to other signaling pathways).
    • Interpretation of results in terms of pathways and functions modulated by protein oxidation.

    Service 4: Metabolomics. Two broad categories of metabolomics services are performed: “profiling” and Stable Isotope Resolved Metabolomics (SIRM). Profiling refers to targeted or untargeted experimental designs to determine the amounts of features in analytical platforms (e.g. different types of MS or NMR) or the identities and amounts of compounds in samples. SIRM enables simultaneous quantitative analysis of many metabolic "pathways" and fluxes that are of especial relevance in the context of metabolic reprogramming that occurs in cancer, including energy production, anabolic pathways necessary for proliferation, and survival pathways including oxidative stress metabolism. 

    A detailed list of metabolomics services and rates can be found at http://bioinformatics.cesb.uky.edu/bin/view/RCSIRM/ServicesAndRates 

    Guide to selecting services

    At your initial consultation with Dr. Mitov, which will be arranged via iLab, we will conduct an assessment of your particular needs and which services are necessary.

    If Service 1 or Service 2 is needed, Dr. Mitov will direct you to the RM SRF facility in 227 HSRB, and either he or Mr. Alstott will work with you.

    If you are interested in Service 3 (proteomics), Dr. Mitov will direct you to Dr. Haining Zhu (haining@uky.edu; 859-323-3643).

    If you are interested in Service 4 (metabolomics), Dr. Mitov will direct you to Dr. Rick Higashi (rick.higashi@uky.edu). 

    Prior to contacting Dr. Higashi, investigators should begin preparing to discuss questions such as: what is the problem; what is the experimental system and why; what information is already available; and whether Seahorse analyses are appropriate to conduct. Once a researcher has answers to these questions, they should contact one of the four Center for Environmental and Systems Biochemistry (CESB) directors listed below to begin consultation. Like all components of the RM SRF, the metabolomics component operates on a charge-back basis. It is very common for data analysis to take 90% of metabolomics labor, so MCC researchers should be prepared to discuss time frame and cost structure with this consideration.

    Ancillary Services (Gratis)

    In addition, the RM SRF offers the following ancillary services:

    • Consulting with Dr. Mitov for assessment of services, design and interpretation of experiments involving oxidative stress and Seahorse XF analyses, directing investigators to proteomics or metabolomics components of RM SRF as needed.
    • Education of MCC investigators on ways to prevent artifactual results and, consequently, obtain reliable and precise data. Please schedule appointments with Dr. Mitov via iLab at https://ukmcc.ilabsolutions.com/service_center/show_external/3590/
    • Writing technical descriptions, result sections and discussion paragraphs for posters, papers and grants. Assistance to investigators on grant proposals and manuscripts by providing technical information or preliminary data.
    • Provision of templates for protocols of indices of oxidative stress, Seahorse technology, proteomics and metabolomics.
    • Pursuit of new applications of expression and redox proteomics to identify other protein post-translational modifications at the cancer interface (e.g., methylation, acetylation). 
    • Cross-validation studies of analyses conducted by RM SRF and FCCS SRF; additional methods involving tools of FCCS SRF for protein oxidation.
  • Technologies Used by the RM SRF

    Measures of oxidative/nitrosative stress
    The RM SRF offers highly sensitive (ng of protein) immunochemical analyses of total oxidative and/or nitrosative stressDr. Butterfield, has published extensively on validating this method for assessment of global oxidative stress. If desired by MCC investigators, redox-coupled reduced glutathione and oxidized glutathione are determined by fluorescence or HPLC methods, and NAD(P)H and NAD(P)+ are determined by HPLC with electrochemical detection. In the rare event that equivocal results are obtained, MS-based detection of protein carbonyls will be employed to validate the immunochemical methods or LC-MS will be used for analysis of small molecule markers of cellular redox state.  

    Mitochondrial biology
    Several indices of mitochondrial changes that occur in conditions of oxidative/nitrosative stress are carried out using state-of-the-art Seahorse technology. Dr. Mitov trained in the Seahorse Bioscience company headquarters and has many years’ experience in designing, performing experiments and data analysis. Mr. Alstott, MS has also been trained on use of the Seahorse instrument. The Warburg effect is a preponderance of energy production by glycolysis rather than through the mitochondrial electron transport chain in cancer cells. It can easily be determined on intact cells using the 96-well Seahorse instrument to determine cellular sequelae of oxidative/nitrosative stress. This platform allows dose-response studies to be carried out using mitochondrial biology as an endpoint. Determination of exo- and endogenous fatty acid utilization can be also investigated using the XF Analyzer. 

    Expression proteomics
    Identification of proteins with differential expression is important to relate gene and protein concordance in cancer cells and to help identify key transcription factors that may play a role in cancer. Expertise in expression proteomics also exists in the RM SRF. Learn more about our Proteomics Core Facility.

    Redox proteomics identification of oxidized proteins
    A key RM SRF service, redox proteomics, originated in the Butterfield laboratory. Principal redox proteomics approaches used include identification of proteins with excess protein carbonyls, 3-nitrotyrosine, or protein-bound HNE. Identification of proteins with excess nitrosylation of cysteine residues is also available. Once identified, such oxidatively modified proteins are placed into their respective molecular pathways to determine cellular sequelae of oxidatively dysfunctional proteins and provide new research of importance in cancer. Moreover, MS/MS sequence analyses of tryptic peptides used in redox proteomics permit identification of the amino acids on which the oxidative modification exists. 

    Proteomics to identify covalently modified proteins
    Acetylation and methylation are two principal covalent modifications to proteins with significant relevance to cancer. Proteomics identification of acetylated or methylated proteins is an important tool in the RM SRF.

    Metabolomics 
    “Metabolomics” is the technical means to analyze metabolism by identifying and quantifying a large fraction of all of the metabolites present in a cell and how they change in response to perturbations of relevant metabolic networks. “Metabolomics” is not synonymous with “metabolism” because it is completely possible to carry out metabolomics analyses without gaining any insight, discovery or understanding of metabolism.

    Metabolomics requires appropriate metabolome coverage, and this in turn requires very high-end analytical instrumentation. Together, mass spectrometry and NMR are the most appropriate technologies worldwide. Informatics is the third critical component of metabolomics.

    Metabolomics services fall into two broad categories, namely “profiling” and “stable isotope resolved metabolomics, or SIRM.” Profiling refers to targeted or untargeted experimental designs to determine the amounts of features in analytical platforms (e.g. different types of MS or NMR) or the identities and amounts of compounds in samples.

    SIRM refers to the tracing of individual atoms from stable isotope-enriched source molecules through biochemical transformations into a variety of intermediates and products, for the purpose of pathway analysis and flux measurement 1-9.

    In general, the choice of the metabolic source determines the biochemical network being probed. Listed below are a few of the thousands of possible probes:

    • [U-13C]-Glucose: survey 13C enters amino acids, nucleotides, lipids (glycolysis, PPP, CAC), hexosamine
    • 13C113C2-Glc: discrimination between oxidative and non-oxidative branches of the Pentose Phosphate Pathway; PC anaplerosis
    • 13C15N Gln: glutaminolysis, nucleotide biosynthesis, energy metabolism (CAC), FA biosynthesis
    • 13C FA: b-oxidation, FA biosynthesis
    • 13C Ser: serine metabolism; 1-C metabolism; lipid metabolism
    • 13C glycerol: lipid backbone biosynthesis

    The analytical platforms available include high resolution NMR, high-resolution (>400,000) high mass accuracy mass spectrometry (direct infusion or LC), GC-MS. These platforms provide information about the amounts and nature of metabolites and pathways and networks involved in central metabolism that can be probed using tracer experiments with combined isotopomer and isotopologue analysis, and multiple 13C/15N enriched precursors 1-9:  

    1.   Aerobic glycolysis: oxidation of glucose to pyruvate by NAD+

    2.   Lactic fermentation: reduction of pyruvate to lactate

    3.   Rate of glucose consumption normalized to measures of cell numbers or volumes

    4.   Fraction of glucose consumed converted to excreted lactate

    5.   Fractional enrichment of excreted lactate from 13C sources (e.g. glucose, Gln etc.)

    6.  Quantification of free NAD+, NADH

    7.   Pentose phosphate pathway: discrimination between oxidative (NADPH producing) and non-oxidative branches- ribose synthesis in ribonucleotide pool

    8.   Glycogenolysis

    9.   Serine/glycine pathways from 3PGA

    10. Gluconeogenesis from e.g. lactate or other glucogenic precursors

    11. Krebs cycle and anaplerosis (including via PC, and canonical and non canonical glutaminolysis)

    12. Amino acid oxidation other than glutaminolysis (e.g. serinolysis, branched chain amino acid oxidation into TCA, GDH etc.)

    13. Nucleotide synthesis- purine and pyrimidine pathways

    14. Fatty acid oxidation

    15. Lipid synthesis and turnover (cf both acyl chain and glycerol headgroups from DHAP)- analysis of isotopologue distribution in most major lipids classes, >500 species

    16. Hexosamine pathway

    17. GSH synthesis and oxidation- GSSH/GSSG/GR cycle

    18. Lipid peroxidation

    Generally speaking, there is no single marker per pathway in an embedded network, rather the combination of several isotopomers/isotopologues are needed for unambiguous assignments.

    The specific isotopomer distributions also enable (in some circumstances) discrimination between the same biochemical reactions occurring in different compartments.  

    The total number of named molecules including the lipids is >700. Including isotopologues and isotopomers it is several thousand. 

    State-of-the-art mass spectrometry and NMR spectrometers tailored for applications in SIRM are used.

    Useful Metabolomics Links

     

  • Faculty and Staff

    D. Allan Butterfield, PhD, Director of the Redox Metabolism Shared Resource Facility 

    Allan Butterfield
    dabcns@uky.edu; 859-323-1106  
    Dr. Butterfield is the UK Alumni Association Endowed Professor of Biological Chemistry. His expertise is in detection of free radicals, measures of oxidative stress, and proteomics identification of oxidatively modified proteins.


    Haining Zhu, PhD, Associate Director of the RM SRF for Proteomics and Director of the University of Kentucky Proteomics Facility 

    Haining Zhu
    haining@uky.edu; 859 323-3643  
    Prof. Zhu is primarily responsible for overseeing proteomics analyses of proteins of interest to MCC investigators.


    Richard Higashi, PhD, Associate Director of the RM SRF for Metabolomics and Director of the Resource Center for Stable Isotope-Resolved Metabolomics (RC-SIRM)

    Richard Higashi
    rick.higashi@uky.edu; 859-218-1027   
    Dr. Higashi, Professor of Toxicology and Cancer Biology, is an expert on mass spectrometry, identifying and quantitating metabolites of importance in cancer.


    Michael Alstott, MS, Senior Laboratory Technician

    Michael Alstott
    michael.alstott@uky.edu; 859-323-1106 
    Mr. Alstott assists in all aspects of the daily laboratory operation, including oxidative stress and Seahorse analyses. He also provides experimental administrative assistance to the Director and RM SRF service users.