Metabolomics Core

Overview of Services

 

Biolog Phenotype Microarrays - A Breakthrough Technology

 

Phenotype MicroArrays (PMs) is an invitro assay that measures the kinetics of uptake/utilization of metabolites by actively growing cells in culture. PM Services accepts mycoplasma free-mammalian cells for testing, and uses Biolog PM Technology to deliver data in a fee for core service. These extensive analyses include up to ~2000 tests. Tests include utilization of a wide variety of C, N, P, and S sources (~800 tests), pH growth range (~100 tests), ionic and non ionic osmotic sensitivity (~100 tests), and sensitivity to chemical agents that disrupt various biological pathways (~1,000 tests). PM analysis has been successfully implemented to understand metabolite-dependence of normal and cancer cell lines as well as genetically engineered cells.

 

The Biolog portion of the Core will serve the utilization of a wide variety of C, N, P, and S sources using Biolog Phenotype Microarray (PM) MiroPlate (Biolog, Inc., Hayward, CA). PM technology is a high-throughput technology for testing the metabolic status of a cell line. The assay is performed in a 96-well format where each well contains a different metabolite. Utilization of the specific metabolite within the cell is detected colorimetrically by a tetrazolium-based redox dye. Reduction of this dye results in formation of specific color. The assay was performed as per manufactur’s instruction. Briefly, cells are counted, pelleted and resuspended in culture medium (M1-medium). 50 µl of the cell suspension containing 2x104 cells/well will then be transferred in to specific nutrient plates and incubated at 37°C in a humidified atmosphere with 5% CO2 for 24 hours. The next day 10 µl of redox dye (dye A) will be added into each of the well and plates will be assessed for metabolic activity by measuring the absorbance at 450 nm, automatically every 15 min for 1-6h using OmniLog instrument.

 

Metabolic Measurements Using Targeted Mass Spectrometry (Metabolomics)

 

The mass spectrometry-based metabolomics portion of the core will allow targeted measurement of candidate metabolites likely to be key to detection or disease progression. This will use protocols and similar MS instrumentation using a validated pipeline.

 

The laboratory currently measures over 300 metabolites using the targeted multiple reaction monitoring approach (MRM). MRM provides the lowest limits of detection with the highest confidence in metabolite identification. The laboratory has the capability for additional method development and has developed robust methods for handling cell line, flash frozen-tissue and plasma, specimens that will be routinely encountered in a core setting. The minimum amount of sample required for each of these matrices, to measure at least 10 metabolites is given below:

• Cell lines: 2 to 5 million
• Tissue: 10 to 25 milligrams
• Plasma: 100 to 500 ul

 

Procedurally, the investigator will be asked to provide the core with the biological specimen. The core personnel will perform extraction of metabolites from biological samples using a combination of aqueous and organic solvents in succession and carry out the mass spectral analysis and associated data pre‐processing. At the end of this pre-processing step, the investigator will be provided an excel file containing raw data for the metabolites measured in the biospecimen. Upon request, the core will also provide statistical help to analyze this data by performing, the various pre‐processing steps namely scaling, imputation and normalization determine class-specific metabolite levels. The latter will carried out by a biostatistician associated with the core on a fee-for service basis. Also whenever possible, upon request the core will also develop new methods to quantify additional metabolites. Because of the chemical diversity of metabolites, new method conditions will need to be determined on a case‐by‐case basis and would involve a determination of ionization conditions, single reaction monitoring (SRM) fragmentation conditions and mass transitions, and chromatographic behavior. For absolute quantification of specific metabolites, an additional fee for procurement of isotopic-labeled standards will be included.
The entire mass spectrometry-based metabolite-assessment will be performed on an Agilent 6490 triple quadrupole (QQQ) mass spectrometer based on instrument availability and the time line will be determined by the core director.

 

Getting Started

 

• Internal Users:  Click the link in the upper right corner to login or register for an iLab account using valid credentials.

 

• External Users:  Click the appropriate link in the upper right corner to login with your approved iLab credentials or to sign up for an iLab account.

 

• Visit the Customer Help Manual for further guidance using iLab's Core Facility Services.

 

 

Location and Hours of Operation

 

 

Contacts

Name	Role	Phone	Email	Location
George Costanza	Core Manager	123 456 7890	costanza@demo.edu	Vandelay industries
Cosmo Kramer	Director for Mass Spectrometry	123 456 7890	kramer@demo.edu	Kenny Rogers Chicken
Jerry Seinfeld	Biostatistics	123 456 7890	jerry@demo.edu	Del Boca Vista

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