Metabolomics

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.

Metabolomics analysis was performed at the Metabolomics Core Facility at the University of Utah which is supported by 1 S10 OD016232-01, 1 S10 OD021505-01 and 1 U54 DK110858-01.

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.

GC-MS Metabolomic Analysis

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
Sample Numbers

For quality results we recomend at least six biological replicates for each condition, for example six cell culture samples treated with drug X and six cell culture samples treated with DMSO.

Cell Culture

We recommend 6 million cells minimal per analytical analysis. For instance for each GC-MS replicate it will take one 10 cm plate worth of cells. This is also true for LC-MS. For all analysis we recommend 6 biological replicates for each condition.

Once cell culture has come to desired confluence remove media.  Wash plate with PBS buffer then remove as much PBS as possible. At this stage you have several options.

Option 1) 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 and send us the sample via FedEx on dry ice. This is the preferred method. We will add several internal standards to the extraction for data normalization.

Option 2) Extract the cells yourself. After PBS add 1 mL of 80% MeOH solution to the plate (-20C), scrape cells into a microfuge tube, vortex and incubate for 1 hour at -20C. Centrifuge for 5 minutes, 4C at highest speed. Remove supernatant to new tube. Dry supernatant in a speed vac. Send us the samples in a zip lock bag with small desiccant bags added.

Plasma/Serum Preparation

Collect serum or plasma per usual. The only caveat is do not use sodium citrate in the preparation of samples. This strongly affects the results. Once you have serum collected snap freeze 20-50 uL of it for metabolomics analysis and bring to the Core. This is the preferred method of sample submission.

If you wish to extract the metabolites from serum/plasma follow these instructions. To 50 uL of serum add 450 uL of solution 90% methanol (-20C) and vortex the mixture for 10 seconds followed by 1 hour incubation at -20C.  Centrifuge the mixture at 14,000 rpm for 5 min. @ 4˚C to precipitate the protein.  Transfer the supernatant containing the metabolites to a fresh tube and dry this en vacuo using a Speed-Vac.  Perform the extraction on ice or in an -20C type enzyme caddy.

Yeast Preparation

For a single analysis (one GC-MS or one LC-MS) 5 total OD’s of cells are needed. For example for cells harvested at a 1 OD, 5 milliliters of this culture will be needed. For metabolite profiling pellet 5 OD’s of cells in a 15 mL conical tube, remove supernatant and snap freeze. Drop these samples off at the Core. This is the preferred method.

If you wish to extract the cultures follow this procedure. Heat up a water bath to 80C. Make a 75% ethanol solution diluting with water. Heat this solution in the water bath carefully. Once hot add 5 mL of this solution to each sample in the 15 mL tube, mix well by vortex and then incubate in the hot water bath for 5 minutes. Post incubation pellet the cell debris, transfer the supernatant to a fresh tube and completly dry using a Speed-Vac.

Tissue Preparation

We prefer to prepare all tissue samples in the Core. We use a high speed bead mill for this. Tissue preperation is sample dependent, please contact the Director for a discussion on methods.

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

Ren Miao, Ph.D.
ren.miao@path.utah.edu

Alan Maschek Ph.D.
alan.maschek@pharm.utah.edu

Oversight Committee

Dennis Winge
Eric Schmidt
Carl Thummel