The following are metabolomic flux policies and guidelines for operation and sample preparation.
All projects require submission of a request for project (RFP) application through iLAB and approval for relevance and scientific merit. Before initiating the project and submitting samples, a meeting is required with the principle investigator requesting the service and core directors and biostatisticians for design of the project. At that time, investigators will receive additional information on sample preparation and scheduling of the project. At the completion of the project another exit meeting with the principle investigator is required to receive the report and explanation of the tier 1 data analysis. Data analysis will not be released without this meeting.
All users of the Core are required to sign up on iLAB for requesting services and provide account sources for core charges. Sample submission and preparation for analysis by the core must be accompanied by a completed sample submission form. Baylor investigators may complete and submit the form online (iLAB) or by email (MetabolomicsCore@bcm.edu) electronic copy and below to core director.
Nagireddy Putluri (email: Putluri@bcm.edu)
Vivekananda Shetty (email: email@example.com)
Feng Li (email: Feng.Li@bcm.edu)
Rules and Requirements for Collection and Submission of Samples
Guidelines for All Samples
- Notify us of any preservatives or additives that have been applied during the collection process.
- Use permanent markers (e.g. Sharpie) to directly label tubes since labels may fall off when frozen.
- Send an electronic document containing sample ID information—preferably in advance of sending the samples. Send the completed xl file to core directors.
- All Samples should be frozen and on dry ice. Tubes should be clearly labeled with sample identifiers and include a hard copy of the list of samples. Please include all available sample information when you submit the samples, excluding any patient identifiers.
Cell Line Samples
- Prepare the 13C labeled growth medium with the recommended concentrations. Nutrients labeled with 13C must be purchased from Cambridge Isotope Laboratories. Cells are grown in regular medium until 80% confluence, followed by overnight starvation in metabolite free medium and then addition of tracers L[U-13C5]glutamine, [1-13C]glutamine, or [5-13C]glutamine supplemented with medium containing [-]glutamine with 10% dialyzed FBS and 1% PS, or addition of D[U-13C6]glucose supplemented with media [-]glucose, 10% dialyzed FBS, and 1% PS. Culture medium is collected, cells are washed with PBS, and equal numbers of cells from each treatment are snap-frozen with liquid nitrogen. Cells are scraped into a 0.5-ml mixture of 1:1 water/methanol. (Note: Depends on the medium the concentration of L[U-13C5]glutamine or [U-13C6]glucose will change).
- Provide 500 ul of cell lysates to the core
- A minimum of 3-5 million cells/pellet is required. We recommend 4-5 biological replicates for each experimental group.
Investigator requires to submit the test samples (at least 3 samples) to check the labelling efficiency at different time point. For this there will additional change and need to discuss with core directors.
Additional details on cell cultures and culture conditions are required. This is critical as it can effect detection of metabolites. This information must be provided on sample submission form.
- For permanent cell lines provide cell line name. For primary cell cultures whether it is human patient derived.
- Provide detailed and accurate information on culture conditions and treatments.
- Culture medium, serum and other supplements.
- Provide information on any drug treatment of cell cultures: Drug name, dosage, and chemical structure . Drugs can potentially interfere with our analysis.
- Knockdown treatment of cells. (si or shRNA). Documentation of knockdown efficiency is required (protein data).
Sample Submission and Q/C Process
500 ul of above mentioned lysate will use further processing and mass spectrometry analysis performed. If sample amounts did not meet minimum criteria (ie number of cells) or quality (variation in cell numbers between samples) the core reserves the right to reject samples for further analysis. The core will consider analysis of rejected samples in cases when they are irreplaceable or too costly to replace. Under these circumstances analysis can only proceed with the understanding and acknowledgment by the Principle Investigator of the risk of failure to obtain quality results and an agreement to pay core charges regardless of the results.
A determination of optimal time points is required for each project. Preliminary samples for a pilot MS flux analysis is required in advance of analysis of the entire sample set to determine Q/C and to fine-tune parameters. If the pilot analysis fails to detect metabolites accurately the core will not proceed with the entire set of experimental sample. After pilot samples have passed Q/C the core will proceed with analysis of the experimental samples.
All samples of an approved project must be collected before submission to the core and for analysis. The core will not process partial samples from an experimental protocol. Samples will only be processed all together. After receiving samples that pass Q/C Core personnel will perform all procedures including extractions of samples for different class of metabolites, column chromatography for each class of metabolites, mass spectrometry analysis, Q/C and normalization of mass spectra and tier 1 analysis of data.
To test mechanisms that are responsible for altered regulation of steady-state levels of metabolites, in the cell, measurements of metabolic flux through pathways is required.
By providing stable-isotope labeled metabolic substrates (such as isotopic labeled glucose, glutamine, or lactate) to living cells, isotopomer patterns of key metabolites can be precisely measured using mass spectrometry. These analyses can provide valuable information on both pathway activities and metabolite pool sizes.
Since all metabolites are either reactants or products in metabolic pathways, changes in their levels due to either altered production or altered disposal are determined by the kinetic rates of key steps within those pathways. Nutrients or metabolic precursors labeled with stable isotopes can be used to feed cells in vitro or tissues in vivo in different mouse models, followed by measurement of the relative uptake and absorption of the isotopes and Metabolic flux analysis (MFA) to trace the usage and labeled products synthesized. This enables the measurement pre-cursor conversion to other biochemical compounds or end-products, or their utilization to synthesize larger biomolecules such as peptides and proteins.
The amount of label transferred from a precursor to its products is measured by mass spectrometry.
(Note: Services available currently use cell lines only and are confined to the targeted analysis of pathways as indicated below)
A. Glucose Metabolic Flux
Enrichment of [13C] glucose and other tracers in conjunction with glycolysis and pentose pathways will be performed. For comprehensive analysis of glucose turnover, [13C] glucose tracer is analyzed by LC/MS for mass isotopomer distribution.
Note: We cannot separate the isomeric compounds (G1P and F1P)
B. Citric Acid Cycle (TCA cycle) Metabolic Flux
The TCA flux is the key process in many metabolic pathways. To provide information on TCA cycle, quantitative analysis of lactic acid, pyruvic acid, citrate, cis-aconitate, isocitrate, ketoglutarate, succinate, fumarate, malate and oxaloacetate is determined by LCMS. To test possible mechanisms responsible for altered regulation, flux analysis is performed within the cycle via the isotopomer approach with 13C tracers.
C. Glutamine Flux
Glutamine is an alternative source of carbon for de novo fatty acid synthesis in some cancer cells. The pathway for fatty acid synthesis from glutamine may follow either of two distinct pathways after it enters the citric acid cycle. The glutaminolysis pathway follows the citric acid cycle, whereas the reductive carboxylation pathway travels in reverse of the citric acid cycle from α-ketoglutarate to citrate . To quantify fluxes in these pathways, cells are incubated with [U-13C]glutamine or [5-13C]glutamine [1-13C]glutamine and analyzed by the mass isotopomer distribution of key metabolites using models that fit the isotopomer distribution.
D. Fatty acid Metabolism
Acetyl-CoA is an important anabolic precursor for lipid biosynthesis. In the conventional view of mammalian metabolism, acetyl-CoA is primarily derived by the oxidation of glucose-derived pyruvate in mitochondria. When cells are grown under conditions of hypoxia or with defective mitochondria, a major fraction of acetyl-CoA is produced by an alternate route, via reductive carboxylation of glutamine-derived α-ketoglutarate (catalyzed by reverse flux through isocitrate dehydrogenase, IDH). A quantitative flux model using [13C] glutamine or [13C] glucose can be used to show oxidative and reductive flux respectively in hypoxia and in cells with defective mitochondria.
Currently core can measure only palmitic acid, oleic acid, and steric acid.
Investigator vs Core Responsibilities
Core: Targeted metabolic flux services will include consultation for protocol design based on the specific questions and hypotheses of the investigators (including hypotheses derived from metabolomic analyses), preparation of cell extracts, appropriate MS analysis of samples (GC/QQQ, LC/QQQ and LC-QTOF) , primary data analysis and assistance with interpretation of data.
Investigator: is responsible for providing and preparing cell cultures, purchasing isotopic tracers and labeling cell cultures. This must be done in consultation with the Core using protocols supplied by the core after an initial meeting with core directors to design the experiments.
Metabolomics Flux Primary (Tier 1) Data Analysis
Metabolomics flux primary (Tier 1) data analysis provided as a package with the MS analysis.
The statistical analyses will be carried out by the Dr. Cristian Coarfa associated with Alkek Center for Molecular Discovery, at Baylor College of Medicine. He has been working with Metabolomics core over the past two years and has been developing algorithms for analyses of metabolomics datasets. Accordingly, total percentage incorporation of C13 into is calculated with respect to each metabolite for all experimental time points and biological replicates respectively and are normalized for natural abundance in each isotope. Statistical significance is obtained using two sided t-test [1-5].
Metabolomics Flux Tier 2 Data Analysis:
Higher level data analysis, graphical representation and comparative analysis can be provided upon request as a separate negotiation with Dr. Coarfa.
- Bhowmik, S.K., et al., Application of 13C isotope labeling using liquid chromatography mass spectrometry (LC-MS) to determining phosphate-containing metabolic incorporation. J Mass Spectrom, 2013. 48(12): p. 1270-5.
- Dasgupta, S., et al., Coactivator SRC-2-dependent metabolic reprogramming mediates prostate cancer survival and metastasis. J Clin Invest, 2015. 125(3): p. 1174-88.
- Kang, Y.K., et al., CAPER is vital for energy and redox homeostasis by integrating glucose-induced mitochondrial functions via ERR-alpha-Gabpa and stress-induced adaptive responses via NF-kappaB-cMYC. PLoS Genet, 2015. 11(4): p. e1005116.
- Terunuma, A., et al., MYC-driven accumulation of 2-hydroxyglutarate is associated with breast cancer prognosis. J Clin Invest, 2014. 124(1): p. 398-412.
- Zaslavsky, A.B., et al., Platelet-Synthesized Testosterone in Men with Prostate Cancer Induces Androgen Receptor Signaling. Neoplasia, 2015. 17(6): p. 490-6.