CNS-Met Metabolomics Core

Purpose

Director: Lance Johnson, Ph.D.  

Co - director: Samir Patel, Ph.D. 

The overarching goal of the CNS-Met Metabolomics Core is to provide CNS-MET COBRE-funded researchers access to cutting-edge metabolomics and mitochondrial bioenergetics techniques. The core will assist in experimental design, sample preparation, data acquisition, and analysis of brain metabolism data.

To assist fellow COBRE-affiliated researchers the core will:

  • 1.) Provide experimental design consultation and project-specific study planning. Proper experimental design and sample preparation will lay the foundation for a successful metabolomics experiment.
  • 2.) Provide technical expertise throughout all phases of metabolomics experiments and offer training on instrumentation, data analysis and interpretation.

The core will utilize established workflows which ensure proper experimental design, provide rapid and consistent preparation of biological samples (including sample fixation, metabolite extraction, and data acquisition), and robust data curation/analysis in addition to assisting in biological interpretation to facilitate the success of COBRE researchers.

Facilities & Other Resources

 

CNS-Met Metabolomics

The CNS-Met Metabolomics Core is housed in the Healthy Kentucky Research Building (HKRB), occupying >900 sq ft on the first floor in a state-of-the-art research facility.

The CNS-Met Metabolomics Core consists of a set of combined instruments to provide analysis and interpretation services for a wide range of metabolites.

Metabolite Panels

 
Central Carbon Metabolism Panel*: Based on the Fiehn metabolomics GCMS approach, this method profiles up to 100 polar metabolites from central carbon metabolism (metabolites involved in glycolysis, TCA cycle and pentose phosphate pathway) with high confidence. Our personnel have tailored this method for use in analyzing brain tissue; however, it is especially useful to profile biofluids, such as cerebrospinal fluid and plasma, which contain abundant volatile small metabolites as well as amino acids. In addition to polar samples, this system will be utilized to analyze biomass after polar extraction (i.e. the insoluble pellet) that contains protein, DNA, RNA and glycogen. 

*Compatible with 13C stable isotope tracers.

Fatty Acid Panel: This workflow detect fatty acids ranging from eight to 24 carbons in length, with enough resolution to distinguish between varying degrees of saturation. This method can reliably detect 20 different fatty acids in brain tissue using a simple, yet robust, methylation via acidification with methanolic hydrogen chloride yielding fatty acid methyl esters (FAMEs) which are distinguishable on our GCMS system. Using a biphasic extraction process, this method can be used in conjunction with polar metabolite panels (central carbon metabolism) without the need for additional biological samples.

 

Major Equipment:

  • Agilent 5977B single quadrupole GCMS
    This mass spectrometer is the most popular Agilent mass spectrometer on the market for analytics. It is routinely used for environmental, chemical, food, forensic, and pharmaceutical applications. The 5977B has a femtogram detection limit, is coupled to a 8890 GC ideal for separation of small molecules and organic compounds, and is equipped with a 150-sample auto-sampler that is designed for high sample numbers. It is a workhorse instrument built to operate 24-hours/day with minimal downtime while providing outstanding consistency and reliability.


     
  • Agilent 6545 quadrupole time of flight LC/CE-QTOF
    This high-end accurate-mass mass spectrometer is used for SIRM analysis, lipidomics and unknown-discovery metabolomics. The 6545 maintains femtogram sensitivity with a mass resolution of 40,000 FWHM. This detection range is perfectly suited for distinguishing between different isotopologues for entry-level SIRM experiments and initial identification of different lipid species. Coupled orthogonally to either a HPLC or CE, the 6545 can analyze metabolites ranging from 100 Da to 10,000 Da. This unit is currently used to assign over 50,000 polar mass features that correspond to over 1200 polar metabolites covering major central carbon metabolic pathways from glycolysis to nucleotide sugar biosynthesis. Further, we can define 13C isotopologues in many of these metabolites when enriched with stable tracer. 

 

Data Analytics

The core utilizes an array of software tools to assist in data curation, statistical analysis, and interpretation of metabolomics data. We currently use the following platforms:

  • Agilent MassHunter Qualitative & Quantitative Analysis
  • DExSI (Data Extraction for Stable Isotope metabolites)
  • Mass Profiler Professional
  • Metaboanalyst
  • Prism GraphPad

Mitochondrial Bioenergetics Core

 

The Mitochondrial Bioenergetics Core is housed in the Biomedical & Biological Science Research Building (BBSRB), occupying >900 sq ft on the fourth floor in a state-of-the-art research facility. 

The Mitochondrial Bioenergetics Core consists of a set of combined instruments to provide analysis and interpretation assistance for cellular bioenergetic and metabolic processes to the Junior PIs and Pilot Projects associated with COBRE grant.

Major Equipment

  • Seahorse XFe24 and XFe96 Flux Analyzers (Agilent Technologies)

These instruments provide an advanced and high-throughput real-time metabolic analysis of various types of biological materials. The core has the 24-well (xfe24) and 96-well (xfe96) units. These formats allow for detecting the changes in cellular bioenergetics including respiration rates, glycolysis, pH shifts and ATP production using combinations of different substrates, inhibitors, uncouplers, drugs, peptides etc. The center is housing an expertise, provide support through standard and inhouse developed protocols for carrying out functional mitochondrial analysis including primary and stable cell lines, isolated mitochondria, platelets and frozen tissue samples. 

  • BIOTEK Cytation 5 Multimode Reader

The BioTek Cytation-5 reader provides a versatile mode of analysis ranging from plate-based assays to high-throughput microscopic image analysis. Detection modes are available in UV-vis absorbance, fluorescence, and luminescence detection. Fluorescence microscopy to 60x magnification to do immunohistological analysis. Cytation 5 is linked to the Seahorse XFe96 for automated normalization of cell numbers after the routine metabolic analysis. The center provides expertize for Cytation-5 based mitohondrial analysis for membrane potential assays, ROS determination, Ca buffering assay and mitochondrial enzyme complex assays. Gen5 software is included with the system and is designed for uncomplicated data collection and processing for the most complex assays.

  • Oroboros O2K-oxygraph 

The Oroboros O2K-oxygraph provides state-of-the-art, high resolution respirometry coupled with high resolution fluorometry using dual Clark-type oxygen electrode micro chambers that are coupled with fluorescent fiber optic probes. This allows for simultaneous measurement of mitochondrial or cellular respiration, membrane potential, Ca2+ cycling and ROS production in either isolated mitochondria (reported 1-5 ug/sample), minced/ homogenized tissue, permeabilized tissue, whole cells etc. A principle benefit of the Oroboros is its precise calibration, sensitivity and high fidelity which provides ability to measure mitochondrial function in small tissue samples without having to isolate mitochondria directly. 

For Further Assistance

For assistance with Metabolite Panels, please contact COBRE Core Director Dr. Lance Johnson (Johnson.Lance@uky.edu) and COBRE Core Manager Dr. Holden Williams (Holden.Willams@uky.edu) for assistance with experimental design, sample preparation/processing, data analysis/interpretation and scheduling. 

For assistance with Mitochondrial Bioenergetics measures, please contact COBRE Core Co-Director Dr. Samir Patel (skpate2@uky.edu) or COBRE Bioenergetics Core Manager Dr. Hemendra Vekaria (hemendravekaria@uky.edu) for assistance with experimental design, sample preparation/processing, assays, data analysis/interpretation and access to equipment scheduler.