Metabolomics

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The Metabolomics Core provides metabolite profiling analysis to investigators at the University of Utah, the Huntsman Cancer Institute, and outside academic researchers. The Core was originally established 11 years ago as part of the Center of Excellence in Molecular Hematology (NIH 1P30DK072437) and originally provided GC-MS based metabolomics analysis to the hematology community. Since its founding the Core has expanded its capabilities to include LC-MS metabolomics, lipidomics and flux analysis. In short the Core is capable of fully and comprehensively profiling the metabolome.

 

To maximize the number of metabolites observed the Core is equipped with three chemical analysis platforms, GC-MS, LC-MS and NMR. Please refer to the Services section of the website for further details on the capabilities of each instrument. In addition, protocols for many of our assays are downloadable from this website in PDF form. Core staff will provide additional assay protocols and consultation upon request.

The Metabolomics Core offers 3 types of services; 1) GC-MS based non-targeted metabolomics, 2) LC-MS based non-targeted analysis, and 3) targeted assays. Metabolites observed are dependent upon the concentration found in the sample but a general outline of metabolites are found below.

 

GC-MS

GC-MS provides an excellent snapshot of central metabolism. It has proven to be highly reliable for the analysis of the metabolites found below.

  1. Amino acids: all but arginine. Cysteine analysis is unreliable due to its free thiol covalently linked to cysteine in proteins.

 

  1. TCA cycle and related metabolites: lactate, pyruvate, citrate, aconitate, isocitrate, succinate, fumarate, and malate.

 

  1. Other organic acids: glyoxylatate, ascorbate, 2-isopropylmalate, 2-ketoadipate, 3-methyl-2-oxopentanoate, 4-aminobutyrate, 3-hydroxypyruvate, 4-methyl-2-oxovalerate, cinnamate, citraconate, citramalate, glycerate, mevalonate, and pimelate.

 

  1. Free fatty acids and sterols: myristate, palmitate, oleate, linoleate, stearate, arachidonate and others.

 

  1. Glycolytic intermediates: glucose6P, fructose6P, glyceraldehyde3P, dihydroxyacetone phosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, and phosphoenolpyruvate.

 

  1. Carbohydrates: glucose, galactose, mannose, sorbitol, mannitol, xylose, fructose and ribose.

 

  1. Purine and pyrimidine bases. Adenine, guanine, thymine, cytosine, uracil, orotate, dihydroxyorotate, inosine as well as adenosine, guanosine, uridine and cytidine.

 

LC-MS

LC-MS provides an excellent platform for the analysis of redox metabolites, nucleotides and lipids.

  1. Redox metabolites: oxidized and reduced glutathione, NADP+, NADPH, NAD+, NADH.
  2. Coenzymes: acetyl-, malonyl-, succinyl-CoA.
  3. Nucleotides: AMP, ADP, ATP, GMP, GDP, GTP and many others.
  4. Lipidomics: Non-targeted lipidomics over 1000 individual lipid species, targeted assays for acyl-carnitines and ceramides routinely performed in the core.

 

Targeted Assays

The Core has over 600 purchased pure metabolite standards and routinely develops targeted quantitative assays for researchers. These are custom assays, please inquire with the Core for details on feasibility and pricing. Examples of past analysis include:

  1. Hepcidin quantification
  2. Acyl-carnitine quantification
  3. Creatine and phosphocreatine quantification

 

LC-MS Lipidomic Analysis

The Metabolomics Core offers three types of lipidomics services, 1) LC-MS based non-targeted lipidomics, 2) LC-MS based targeted lipidomics, and 3) GC-MS based fatty acid methyl ester (FAMES) analysis for any biological matrix.

 

LC-MS based non-targeted lipidomics

We employ the Agilent 6530 Accurate Mass Q-TOF dual ESI mass spectrometer operating in positive and negative ionization modes coupled with ultra-high performance liquid chromatography (UHPLC) to assay a broad range of lipid classes from any biological matrix. Most often, we examine the lipid differences found between biological samples after perturbation of a given system. While there is no single analytical platform that can fully characterize the highly diverse lipidome, our sample extraction and preparation strategy coupled with reversed-phase HPLC provides a robust tool to assess the lipid content of any biological system. Lipid class coverage includes PC, PE, PG, PI, PS, DAG, TAG, SM, CE, EE, CER, CL, and all related lyso and plasmalogen species. We typically identify >1000 unique lipid species from our untargeted lipidomics platform.

 

LC-MS based targeted lipidomics

When researchers wish to quantitate specific lipids in a targeted fashion, we employ the Agilent 6490 Triple Quadrupole mass spectrometer and the SCIEX QTRAP 6500 System coupled with UHPLC from any biological matrix. This type of project is often limited to 200 specific lipids from a subset of lipid classes and new assays require method development. We routinely run targeted assays for acylcarnitines and sphingolipids.

 

GC-MS based fatty acid methyl ester (FAMES) analysis

The metabolomics core utilizes an HP6890 GC-MS interfaced with a flame ionization detector (FID) to analyze FAMEs. Free fatty acids are esterified using acid-catalysis on a variety of biological matrixes.

 

Biological sample preparation

Sample collection is a vital part of lipid analysis. With the highly sensitive analysis methods available, only a very small amount of source material is needed. Two points should be kept in mind, 1) the small quantity of sample taken should be representative of the entire source material, and 2) just enough material, but not too much, should be sampled for all of the planned analysis. What is enough material? There is no clear answer for this as all source material will vary. Generally, we recommend a minimum of 5 mg of wet tissue, a million of cells, 20 µL of plasma, or 100 µg of protein from a membrane fraction. Too much material would be approximately 20-fold more than the minimum. We also require the researcher to record those values when preparing their samples. With few exceptions, all samples should be flash-frozen, stored under nitrogen if possible and shipped on dry ice. Researchers are free to label their samples as they see fit, although we do recommend a simple labeling scheme that refers back to detailed sample information. If possible, researchers should generate a blank (negative control) sample in their set.

 

Software and data analysis

LC-MS and tandem MS (MS/MS) data is analyzed using a suite of software packages including Agilent Mass Hunter Qual, Mass Hunter Quant and Profinder. Lipid annotation is provided by our core utilizing existing databases (LipidMaps, METLIN and Lipid Match). Data analysis is conducted using MetaboAnalyst (PCA, volcano plots, heatmaps).

 

Publications

Simcox, Judith et al. Global Analysis of Plasma Lipids Identifies Liver-Derived Acylcarnitines as a Fuel Source for Brown Fat Thermogenesis, Cell Metabolism, Volume 26, Issue 3, 509 – 522.e6

 

 

Email Dr. Alan Maschek for information.

Pricing

CIHD Internal Member Rates CIHD External Member Rates CIHD Internal Non-Member Rates CIHD External Non-Member Rates Commercial Rates
GC-MS Metabolomics
$25 $37.75 $45 $67.95 $90
LS-MS Metabolomics
$30 $45.30 $50 $75.50 $100
LS-MS Lipidomics $35 $52.85 $55 $83.05 $110
  1. These prices exclude a per hour charge for data analysis which ranges between $60-$100 per hour. Total time and cost will be quoted prior to start of project.
  2. Priority is given to CIHD members and University of Utah Investigators, followed by outside NIH funded investigators.

Sample replicates

For quality results, we recommend at least six biological replicates for each experimental condition.

 

Cell Culture

We recommend 6 million cells minimal for a complete metabolomic analysis, some assays require less (e.g., 1 million for lipidomics). Once cell culture has come to desired confluence, remove media, wash plate with PBS buffer and then remove as much PBS as possible. At this stage, you have several options.

Option 1 – Preferred) If you can remove the cells from the plate by trypsin or other method, transfer the cell culture to a microfuge tube, pellet by centrifugation then remove as much supernatant as possible. Snap freeze on dry ice, flush with nitrogen gas and send us the sample via FedEx on dry ice. We will add several internal standards to the extraction for data normalization.

Option 2) Extract the cells yourself. After PBS, add 1 mL of cold 80% MeOH solution to the plate, scrape cells into a microfuge tube, vortex and incubate for 1 hour at -20 °C. Centrifuge at max speed for 5 minutes at 4 °C. Remove supernatant, transfer to new tube and dry supernatant in a speedvac or under gentle nitrogen stream. Send us the samples in freezer box with small desiccant bags added.

 

Plasma/Serum Preparation

Collect serum or plasma per usual, but do not use sodium citrate in the preparation of samples as this strongly affects the results. Once you have serum collected snap freeze 20-50 µL of it for metabolomics analysis and deliver to the Core. This is the preferred method of sample submission. If you wish to extract the metabolites from serum/plasma, please follow these instructions. To 50 µL of serum, add 450 µL of cold 90% methanol and vortex for 10 seconds followed by 1 hour incubation at -20 °C.  Centrifuge the mixture at 14,000 rpm for 5 min at 4˚C to precipitate the protein, transfer to new tube and dry supernatant in a speedvac or under gentle nitrogen stream. Perform the extraction on ice or in an -20 °C type enzyme caddy.

 

Yeast Preparation

For a single GC-MS or LC-MS analysis, five total ODs of cells are required. For example, cells harvested at a 1 OD, 5 mL of this culture will be needed. For metabolite profiling pellet 5 OD’s of cells in a 15 mL conical tube, remove supernatant, snap freeze and deliver samples to the Core. This is the preferred method. If you wish to extract the cultures, please follow this procedure. Prepare and 75% EtOH solution and warm in a water bath to 80 °C. Once hot, add 5 mL of this solution to each sample in a 15 mL tube, vortex vigorously and incubate in the hot water bath for 5 minutes. Pellet the cell via centrifuge, transfer to new tube and dry supernatant in a speedvac or under gentle nitrogen stream.

 

Tissue Preparation

Analysis of tissue can be highly variable, for complete metabolic analysis we recommend a minimum of 20 mg of wet tissue. Tissues samples should be representative of the entire tissue, snap frozen and stored under nitrogen if possible. Researchers are required to record the masses of the tissue when preparing their samples. We prefer to extract all tissue samples in the Core laboratory using a high-speed bead mill for this. Tissue preparation is sample dependent; please contact the Director for a discussion on methods.

James Cox

15 N Medical Drive East

Bldg 565 Room A306

Salt Lake City UT 84112

801-587-7779

Hours of Operation

Monday-Friday 9am-5pm

Location

Emma Eccles Jones Medical Science Building (EEJ) Room A306
15 N Medical Dr East
Salt Lake City, UT 84112

Access is through the North or South stairwell as well as the service elevator.

Staff


James Cox, Ph.D. , Director
Phone: 801-587-7779
Fax: 801-585-6362
jcox@cores.utah.edu


Alan Mashchek, Ph.D.
alan.maschek@pharm.utah.edu


Krishna Parsawar, Ph.D.

krishna@genetics.utah.edu

Leon Catrow, Ph.D.
leon.catrow@utah.edu

Sandra Osburn, Ph.D.
sandra.osburn@cores.utah.edu

Tyler Van Ry, Ph.D.
tylerv@cores.utah.edu

Oversight Committee

Jared Rutter
Dennis Winge
Eric Schmidt
Carl Thummel

Metabolomics analysis was performed at the Metabolomics Core Facility at the University of Utah. Mass spectrometry equipment was obtained through NCRR Shared Instrumentation Grant 1S10OD016232-01, 1S10OD018210-01A1 and 1S10OD021505-01.

The Metabolomics Core processes every sample using two distinct but overlapping procedures, a targeted analysis and a non-targeted analysis. The targeted analysis is used to search every chromatogram for known metabolites in which the Core has purchased a standard  for and is highly confident in its identitiy. We use instrument specific software to report the area under the curve for each of these known metabolites and transfer this data to an Excel spreadsheet. We then perform a non-targeted analysis in which data mining software just detects chromatographic peaks that are altered in two different conditions. This is normally done by Priniciple Components Analysis (PCA) and Partial Least Squares-Discriminate Analysis (PLS-DA). Many times this detects known compounds but in the case where an unknown compound is found to be altered we add this to the Excel spreadsheet using a unique identifier. It is identified by its chromatographic retention time (rt) and a charecteristic mass (m/z) in the form of unrt_m/z. For example if an unkown was found at retention time of 10.78 minutes and had a mass of 347.0111 it would be labeled as un10.78_347.

The Core then uses the data generated in these two procedures for further basic analysis using a number of statistical packages including metaboanalyst.ca. The Core highly recomends users to visit this website and to further examine their data. The Core will provide users with a final report outling the experimental procedures performed and basic statistics analysis. Attached with this report is all the raw data so each user can further examine the results. Metabolomics related resources are listed in the Online Resorces tab.

Agilent 7200 GC-QTOF with EI and CI capabilities

This new, state of the art gas chromatograph-mass spectrometer is a high resolution/high mass accuracy quadrupole time-of-flight instrument. It is fitted with a CTC MPS auto sampler that automates sample preparation for high throughput sample analysis. This workhorse instrument is primary used for the analysis of the low molecular weight metabolome and for flux analysis.

Agilent 6550

An Agilent 6550 Quadrupole-Time-of-Flight (QToF) mass spectrometer coupled to an Agilent 1290 Ultra Pressure Liquid Chromatograph (UPLC) is used as our primary instrument for higher molecular weight metabolite analysis and for the profiling of metabolites not amenable to gas chromatography-mass spectrometry. Hydrophilic Liquid Chromatography (HILIC) is employed for metabolite separation prior to analysis. This instrument was purchased through NIH S10OD016232-01

Agilent 6530

An Agilent 6530 UPLC-QToF mass spectrometer is used as our primary instrument for non-targeted lipidomics analysis. Ultra pressure reversed phase liquid chromatography is employed for lipid separation prior to analysis. This instrument was purchased through a University of Utah Request for Instrumentation grant.

Agilent 6490

An Agilent 6490 Triple Quadrupole (QQQ) mass spectrometer coupled to an Agilent 1290 Ultra Pressure Liquid Chromatograph (UPLC) is used as our primary instrument for the targeted quantification of metabolites and drug molecules. Ultra-pressure reversed phase liquid chromatography is employed for rapid chromatographic fractionation prior to analysis. This instrument was purchased through a RIF grant.

Waters GCT Premier

A Waters GCT Premier is used for profiling the low molecular weight metabolome and is especially proficient at low molecular weight acids such as lactate, pyruvate, amino acids, TCA cycle intermediates, free fatty acids, and phosphorylated sugars. This is a Gas Chromatograph Time-of-Flight Mass Spectrometer (GC-ToF-MS) and was purchased through NIDDK grant P30DK072437.