The Donald B. and Catherine C. Marron Cell Metabolism Core Laboratory provides technologies and expertise allowing investigators at Memorial Sloan Kettering to measure small molecules in biological samples and characterize the metabolism of cancer cells.
The chemical diversity of small molecules and lipids present in biological systems means that multiple analytical approaches are required to provide complete coverage and comprehensively assess metabolic changes. The Core Laboratory therefore provides researchers with access to a variety of complementary state-of-the-art technologies including gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) instrumentation for targeted and untargeted metabolomic studies. In addition, we provide access to non-mass spectrometry technologies, including enzymatic assays for nutrient analysis in cell culture media and instrumentation to measure cell viability and oxygen consumption.
The laboratory uses a YSI 7000 enzymatic analyzer to measure glucose, glutamine, and lactate levels in cell culture media to determine cellular nutrient consumption rates.
Hansatech and Instech oxygen consumption chambers are used to interrogate cellular and mitochondrial respiratory function. The laboratory also has a Seahorse XF96 Extracellular Flux Analyzer for measurement of oxygen consumption and extracellular acidification of cells grown in a 96-well format, and a hypoxia chamber for studying the growth of cells in low oxygen.
The ACEA xCELLigence MP platform is available to monitor cellular proliferation and viability in real-time and in a label-free format. The instrument records changes in impedance when cells are grown in 96-well plates with an electrode surface. A single 96-well plate instrument is also available for comparing the growth of cells under low oxygen conditions.
Sample Preparation and Metabolite Extraction
We provide access to a variety of instrumentation and protocols for sample disruption, homogenization, and metabolite extraction from cells, tissue, blood plasma, and urine. A Gerstel MPS robotic system provides off-line derivatization and microscale solid phase extraction (SPE) chemistries.
Gas Chromatography–Mass Spectrometry
Two Agilent GC-MS systems with 5975 mass selective detectors are used for detection of chemically derivatized organic acids, amino acids, fatty acid methyl esters, and short chain fatty acids. Methods are available for both relative and absolute quantitation.
Liquid Chromatography–Mass Spectrometry Targeted Analysis
Agilent 6230 time-of-flight (TOF) LC-MS instrument is equipped for basic relative quantitation of polar metabolites from cells, blood plasma, and tissue extracts. We also use a Thermo Vantage triple quadrupole LC-MS system with a MayLab column switcher to transfer dedicated targeted methods enabling higher sample throughput.
Liquid Chromatography–Mass Spectrometry Profiling
The Agilent 6230 TOF and Agilent 6550 Q-TOF are used for a variety of profiling applications including dedicated methods for polar metabolites, lipidomics, and bile acids. Both instruments are equipped with dual Agilent 1290 high-pressure pumps to allow sequential analysis of samples by both reverse phase (RP) and aqueous normal phase (ANP) chromatographic methods.
Liquid Chromatography–Mass Spectrometry Unknown Identification
Experiments performed using the 6230 TOF and 6550 Q-TOF accurate mass instruments can identify compounds of interest for which a formula can be assigned. To assign an identity to such compounds, the laboratory has a variety of workflows available utilizing both bioinformatics approaches and the MS/MS capabilities of the 6550 Q-TOF and Vantage Triple Quad.
Isotope Tracing and Flux studies
Studies using 13C and 15N stable isotope pathway tracing and flux analysis are available on a collaborative basis. In addition to Agilent MassHunter software the IsoCor and MAVEN software packages are available to help with the analysis of labeled metabolite extracts.
Analytical Pharmacology Services
We provide a variety of services for the targeted quantitation of small molecule pharmaceutics. These activities support research at the preclinical and phase I/II clinical trial stages of drug development. Assays can be preformed under good laboratory practice (GLP) conditions when required. Targeted methods for new compounds are developed for an AB SCIEX API 4000 LC-MS system or Shimadzu HPLC with fluorescence and UV detection.
The analytical pharmacology services available for the Core Laboratory include:
- Pharmacokinetic (PK) analysis
- Drug metabolism studies and metabolite identification
- Time to peak dose and total exposure calculations
Data Mining and Statistical Analysis
The laboratory maintains a variety of unit mass and accurate mass metabolite libraries and pathways tools based on the KEGG, WikiPathways and BioCyc repositories. Data filtering and statistical analysis is preformed using Agilent Mass Profiler Professional and GeneSpring GX packages. A suite of four data processing computers is available to core lab users and training on the various software packages is available on request.