Metabolomics Innovation
Resource (MIR)

Analytical Services

If you are new to working with the Metabolomics Core Facility, we strongly encourage you to read our sample preparation advice and recommendations. 

The MIR is equipped with four mass spectrometers: two GC/MS, UPLC/QQQ and UPLC/QTOF.  The facility staff also has access to the NMR spectrometers at the QANUC NMR facility and the MUHC-RI Platform for drug discovery.  This allows the MIR to provide a wide array of analytical platforms for metabolite analysis.  We offer both targeted metabolite and untargeted services.

In order to establish the best relationship possible with the investigators using the MIR, we ask you to review our “Terms of Use” document and provide us with a signed copy of the “Acknowledgment for conditions of use” document. 

When you are ready to start a project with us, please read our General Recommendations if you have not worked in the area of metabolomics or metabolite profiling previously.

1. Discussion with MIR Staff Member
2. Cell/Tissue/Biofluid extraction protocol will be provided
3. A pilot study on a few samples will be run for you free of charge to ensure
   1. ou are able to follow our extraction protocol for your specific project.
   2. Determine that the extraction protocol works for your specific project
4. Ensure that the MIR can measure the metabolites of interest discussed in part #1
   After a successful pilot study any minor changes or modifications to the protocols will be discussed.
5. A larger study will be designed, planned & scheduled.

It is imperative that we communicate well every step of the way for a successful metabolomics project!

Our services are always expanding. See our services below to learn more.

Targeted Analysis

Targeted analysis is performed to analyze or quantify specific metabolites. The instruments used for targeted analysis are NMR, GC/MS and LC-MS/MS. Targeted analysis depends on the following:

1. The ability to obtain authentic or synthetic standards
2. The ability to detect the standard (using any of the listed instruments)
3. The ability to efficiently extract the endogenous compound(s) from the sample(s) and derivatize if necessary (GC/MS).
4. The ability to have good chromatography (either GC or LC) of the endogenous compounds to cleanly separate them from other confounding metabolites.

We have attempted to group targeted metabolites into logical families. The families tend to be grouped either by metabolic pathway or chemical behavior or class. Note that there is overlap between groupings. When more than one analysis is requested, we do our best to try to analyze as much as possible from the given sample. 

If you cannot find the metabolites you are interested in targeting, please make an appointment to talk with us. Our group has the expertise to create new methodologies specific to your needs. Custom analysis charges will apply (staff & instrument time) unless otherwise directed by the MCF oversight committee.

Amino Acids and Derivatives

alanine, beta-alanine	glycine	phenylalanine
allo-isoleucine	histidine	phospho-serine
arginine	homocysteine	proline
asparagine	homocystine	pyroglutamate
aspartic acid	hydroxyproline	sarcosine
betaine	hypotaurine	serine
citrulline	isoleucine	taurine
cysteine	leucine	threonine
cystine	lysine	tryptophan
glutamic acid	methionine	tyrosine
glutamine	ornithine	valine
gamma-aminobutyric acid

The MCF offers both free and total amino acid analysis either by NMR, GC/MS or LC/MS depending on other desired analyses. The current library of amino acid and amino acid derivatives are listed

Citric Acid Cycle Intermediates

The citric acid cycle intermediates may be measured by LC/QQQ or GC/MS.

Note that oxaloacetate, pyruvate and a-ketoglutaric are not stable intermediates. For GC/MS analysis these intermediates are reduced using NaBD4 to their more stable alpha-hydroxy acids. 

The intermediates are quantified by stable isotope dilution (ref: http://link.springer.com/article/10.1007/s11306-013-0521-1#page-1). 

The following metabolites are quantified:

Lactic acid	Alpha-ketoglutaric acid
Pyruvic acid	Succinic acid
Citric acid	Fumaric acid
Isocitric acid	Oxaloacetic acid
Oxaloacetic acid*	Malic acid
2-hydroxyglutaric acid

Free or Total Fatty Acids

Fatty acids generally vary in length from 4 to 28 carbons with various levels of unsaturation.  Free fatty acids can be metabolized as a fuel (free fatty acids) or used as building blocks for cell walls, tri-glycerides and other lipids.  Saponification allows for full fatty acid analysis compared to the generally lower level of free fatty acids. 

The MCF provides GC/MS based untargeted analysis for fatty acids.  Our fatty acid library is listed below.  Other fatty acids of special interest can be added to our library assuming an authentic standard can be obtained. 

Currently we provide ratios of fatty acid to an internal standard (D27-myristic).  Absolute quantitation can be done by special request.

Current list of fatty acids in our library (more can be added!):

Montanic C28:0	Eicosapentaenoic C20:5 {{5,8,11,14,17}}	Palmitic C16:0
Cerotic C26:0	Eicosapentaenoic C20:5 {{5,8,11,14,17}} Stearic C18:0	Palmitoleic C16:1 {{9}}
Lignoceric C24:0	Oleic C18:1 {{9}}	Palmitelaidic C16:1 {{t9}}
Nervonic C24:1 {{15}}	Petroselinic C18:1 {{6}}	C15:0
Behenic C22:0	Vaccenic C18:1 {{11}}	Myristic C14:0
Erucic C22:1 {{13}}	Linoleic C18:2 {{9,12}}	Myristoleic C14:1 {{9}}
Arachidic C20:0	α-Linolenic C18:3 {{9,12,15}}	C13
Eicosenoic C20:1 {{11}}	γ-Linolenic C18:3 {{6,9,12}}	Lauric C12:0
Eicosatrienoic C20:3 {{8,11,14}}	Margaric C17:0	Capric C10:0
Arachidonic C20:4 {{5,8,11,14}}		Caprylic C8:0
β-Hydroxy Acids
Decanoic C10:0 3-OH	Undecanoic C11:0 3-OH	Lauric C12:0 3-OH
Myristic C14:0 3-OH	C15:0 3-OH	Palmitic C16:0 3-OH

Glycolytic Intermediates

Glycolytic intermediates are measured by LC/QQQ and quantified by external calibration curves. Due to structural and molecular weight similarity it is not possible to resolve some intermediates.

Note also that glycolytic intermediates are normally found at very low concentrations, and therefore may be difficult to measure.

The intermediates in our protocol are as follows:

Glucose	Glyderaldahyde 3-phosphate
Glucose 6-phosphate / Fructose 6-phosphate	Dihydroxyacetone phosphate
Fructose 1,6 bis-phosphate	Phosphoenolpyruvate
2-Phosphoglycerate	Pyruvate
3-Phosphoglycerate	Lactate

Nucleotide Analysis

The nucleotide analysis includes many mono, di and triphosphates, such as ATP, ADP and AMP for energy charge analysis.
This analysis also includes creatine and phosphocreatine since the ratio of these metabolites also contributes to the energy charge of the cell. Nucleotides are measured by LC/QQQ and quantified based on external standard calibration curves run at the same time as the samples of interest.

Our current library of nucleotides is listed below:

Adenosine monophosphate  (AMP)	Inosine 5’ monophosphate (IMP)	Flavin adenine dinucleotide (FAD)
Adenosine diphosphate (ADP)	Inosine 5’ diphosphate (IDP)	beta-Nicotinamide adenine dinucleotide (NAD+ )
Adenosine triphosphate (ATP)	Inosine 5’ triphosphate (ITP)	beta-Nicotinamide adenine dinucleotide reduced (NADH)
Adeosine 3,5, cyclic monophosphate (cAMP)	Cytidine 5’ monophosphate (CMP)	beta-Nicotinamide adenine dinucleotide phosphate (NADP+ )
Guanosine monophosphate (GMP)	Cytidine 5’ diphosphate (CDP)	beta-Nicotinamide adenine dinucleotide phosphate reduced (NADPH)
Guanosine diphosphate (GDP)	Cytidine 5’ triphosphate (CTP)	Uridine 5’-diphosphoglucose (UDP-glucose)
Guanosine triphosphate (GTP)	Thymidine 5’ monophosphate (TMP)	Uridine 5’- diphosphogalactose (UDP-galactose )
Uridine 5’ monophosphate (UMP)	Thymidine 5’ diphosphate (TDP)	Uridine 5’- diphosphoglucuronic acid (UDP-glucuronic acid )
Uridine 5’ diphosphate (UDP)	Thymidine 5’ triphosphate (TTP)	Uridine 5’ diphospho–N-acetylglucosamine (UDP-N-acetyl glucosamine)
Uridine 5’ triphosphate (UTP)

We can add additional nucleotides upon request assuming authentic standards are available.

Nucleoside Analysis

The nucleoside analysis includes the nitrogenous bases, ribonucleosides, and deoxyribonucleosides. Nucleosides are measured by LC/QQQ and quantified based on external standard calibration curves run at the same time as the samples of interest.
Our current library of nucleosides is listed below. We can add additional nucleotises upon request assuming authentic standards are available.

Adenine	Adenosine	2'-Deoxyadenosine
Guanine	Guanosine	2'-Deoxyguanosine
Cytosine	Cytidine	2'-Deoxycytidine
Thymine	5-Methyluridine	2'-Deoxythymidine
Uracil	Uridine	2'-Deoxyuridine

Water Soluble Oxidative Stress Indicators

Please note that some metabolites listed here are redundant to the Amino Acids analysis. We attempt to capture reduced and oxidized forms of important metabolites involved in reducing oxidative stress.

These partially cover folate metabolism and the methionine cycle.
Taurine and hypotaurine can be easily added upon request.

Reduced Glutathione	Cystathionine	Folate
Oxidized Glutathione	Homocysteine	Dehydrofolate
Ascorbic Acid	Methionine	Tetrahydrofolate
Dehydroascorbic acid	S-adenosylmethionine (SAM)
Cystine	S-adenosylhomocysteine (SAH)
Cysteine	gamma-glutamylcysteine

Pentose Phosphate Pathway

The pentose phosphate pathway intermediates are quantified by LC/QQQ using external calibration curves.  The following metabolites may be measured:

Glucose	Ribose
Glucose-6 phosphate	Ribulose
5-Phospho-D-Ribose 1 diphosphate	Glucosaminic Acid
6-Phosphogluconic acid	Ribose 5-phosphate
Gluconate	Ribulose 5-phosphate
Gluconolactone	2-Deoxy-ribose 5-phosphate
Sedoheptulose 7-phosphate	2-Deoxyribose

Untargeted Analysis

Due to the time commitment of untargeted analysis, the MCF currently only offers this service to GCRC members.  We hope to offer this service more broadly in the near future.
Untargeted analysis will be managed as a close collaboration between the MCF and the investigator.
Three instruments can be used for untargeted analysis: LC/QTOF, GC/MS and NMR.
A general workflow is described below. This workflow is flexible and will be modified to meet the needs of the project.

Samples

Samples are extracted according to the chemistry of the metabolites that the scientist is most interested in (e.g. polar metabolites vs fatty acids).

Data acquisition

• NMR: Samples are prepared and data are acquired 
• GC/MS: samples are derivatized (usually MOX/TMS or MOX/TBDMS) and GC/MS data collected
• LC/MS: an appropriate column and buffer conditions are chosen depending on the class of molecules of interest (e.g. organic acid vs nucleotide) and LC/QTOF data are acquired.

Data alignment or recursive analysis

• NMR data are fit using Chenomx software or other software as defined by the project.
• GC/MS data are deconvoluted by chromatography and retention time using AMDIS or MassHunter Qualitative software (Agilent).
• LC/QTOF data are processed for molecular features including all ions (plus their isotopes) and adducts. This may be repeated in a recursive workflow using MassHunter Qualitative software and Mass Profiler Professional (MPP) (Agilent).

Statistical analysis

• NMR assigned metabolites, bins, or modeled peaks are subjected to statistical analysis (PCA, PLSDA, etc) using MPP (Agilent) or MetaboAnalyst.  Metabolites or peaks causing the differentiation between groups are identified.
• GC/MS spectra are tentatively identified using Feihn, Bains (Steadman Metabolism laboratory, Duke University) or NIST11 databases. The identified and unidentified metabolites are subjected to data alignment across different samples and statistical analysis using MPP. Identified and unidentified metabolites that cause differentiation between groups are “tagged” for further identification. 
• LC/QTOF extracted metabolites or “features” are subjected to data alignment across different samples and statistical analysis using MPP. Differentiating “features” are “tagged” for identification.

Metabolite identification

• NMR : metabolites identified using 1D and 2D NMR techniques. Significant metabolites may be spiked into samples to confirm chemical shift, spectral patterns and a quantitative increase in the metabolite’s concentration. The HMDB , MMSD and BMRDB are used to help identify metabolites and their spectra.
• GC/MS based metabolites are identified and ideally new samples are split where one is spiked with the authentic metabolite standard(s) confirming an increase in the putatively identified metabolite and confirmation of mass spectrum and retention time.
• LC/MS: samples are re-run where the statistically significant differentiating metabolites are targeted for MS/MS analysis using different collision energies (Product ion scanning). These data are then used to identify the metabolites (tentative). The METLIN accurate mass and MS/MS database as well as the Human Metabolome Database are used to help the identification. If possible, samples are spiked with authentic metabolite standards to confirm an increase in the significant metabolite peak intensity as well as to confirm retention time, mass spectrum and fragmentation pattern.

Validation

Once metabolites have been identified, the pathways where they are up-regulated and down-regulated need to be elucidated. Using Pathway Analyst (Agilent), Cytoscape (NRNB), MetPA (University of Alberta) etc, can facilitate the identification of active pathways. This data can be compared to other «omics» data (transcription, protein levels etc). Further analyses such as pathway inhibitors, genetic manipulations and flux analysis are encouraged to confirm pathway use and directionality.