Mass Spectrometry Core

The metabolome consists of hundreds to thousands of small molecules (< 50 Da to > 2000 Da) with vastly different chemistries; no single method is suitable to assess it in its entirety. To account for this, the Mass Spectrometry Core offers a range of chromatographic and MS methods to best address each research question.

1. Targeted metabolomics experiments are used to identify compounds in a pre-selected metabolite class or pathway. Targeted metabolite panels have been developed to provide high-confidence compound identification through direct comparison to known chemical standards.
2. Untargeted metabolomics analysis identifies all features in an experimental run and uses library matching for compound identification.

A metabolic tracing experiment provides an understanding of the metabolic flux within a biological model. In these studies, the researcher introduces a heavy stable isotope, such as 13C, into the experimental system. Then, the mass spectrometer detects alterations in the isotope patterns to determine the fraction of each metabolite pool that contains the heavy atom(s). These analyses can be either targeted or untargeted.

The Mass Spectrometry Core can provide guidance for the experimental design and sample handling for tracing experiments. For each experiment, the researcher must provide unlabeled control samples to be used for correction for naturally occurring isotopes already present in the system.

The Core has developed a lipidomics platform using the Thermo Scientific Orbitrap ID-X LC/MS, which can routinely annotate 500+ lipid species including phospholipids, ceramides, triacylglycerides, cardiolipins and cholesterol esters. The lipidomics method is semi-targeted, relying on previously run standards to confidently identify features, while also detecting any other features present in the sample.

For increased confidence in lipid identification, the EquiSPLASH®, Avanti Polar Lipids standard mixture can be used. The mixture of 13 deuterated lipid internal standards will be added to all samples, quality controls and blanks at a final resuspended concentration of 25 ug/mL post-metabolite extraction. This control serves to validate the accuracy of acquired data and, if needed, help normalize data for any fluctuations in metabolite abundances that are not the result of biological differences between samples. Due to the cost of the standard mixture, an additional fee is required for this option.

The metabolomics services described above provide relative quantitation, allowing comparison of metabolite pool sizes across experimental groups within a metabolite. Absolute quantitation requires preparation and analysis of the target metabolite(s) at known concentrations (i.e., a dilution curve of the chemical standard). The response of each known concentration can be used to quantitate the response from biological samples through a regression analysis.

An additional control required for targeted quantitation experiments is an isotopically labeled internal standard added to both the experimental and quantitation samples. The internal standard is used to correct for matrix effects as well as expected instrument variability during MS analysis.

Targeted quantitation experiments require metabolite- and matrix-specific method development to ensure accurate quantitation. The MSC routinely develops such methods, but planning, time, and researcher-provided pilot samples are required. The researcher must also provide the neat chemical standard(s) and isotopically labeled internal standard(s) to be used. After the initial method development, the method can be used routinely. The quantitation samples and internal standard will be run with all subsequent experiments.

Primary use: Semi-untargeted metabolite and lipid profiling and stable isotope labeling

Capabilities

• High-resolution/accurate mass data acquisition
• Up to four chromatographic separations per sample to maximize metabolite coverage
• MS2 fragmentation for compound identification and library matching
• MSn fragmentation for structural elucidation

Specifications

• Up to 500K resolution (FWHM)
• < 2 ppm mass accuracy
• Electrospray ionization
• Dual Vanquish UHPLC
• Data dependent acquisition
• CID and HCD fragmentation

Primary use: Stable isotope labeling of central carbon metabolism

Capabilities

• High-resolution/accurate mass data acquisition
• Duplexed ion-paired chromatography for reproducible retention times and maximum throughput
• MS2 fragmentation for compound identification and library matching

Specifications

• 240K resolution (FWHM)
• < 2 ppm mass accuracy
• Electrospray ionization
• Data-dependent acquisition

Primary use: Targeted polar metabolite profiling and custom targeted method development

Capabilities

• Ion-paired negative mode profiling of ~250 polar metabolites, including amino acids, nucleotides, carboxylates and sugar phosphates
• dMRM mode limits transition monitoring only to retention windows of interest to maximize compounds detected per run
• Highly reproducible ion-paired chromatography
• High sensitivity and specificity of targeted compounds
• Second UHPLC system for targeted method development of compounds not amenable to ion-pairing.

Specifications

• Sub-femtogram detection limits (compound dependent)
• Electrospray ionization

Primary use: Targeted metabolite profiling of central carbon metabolism and custom targeted method development

Capabilities

• Profiling, quantitation and stable isotope labeling of most amino acids and TCA cycle intermediates
• In-source compound fragmentation for positive ID

Specifications

• Sub-femtogram detection limits (compound dependent)
• Electron-impact ionization

The rates of oxygen consumption (OCR; i.e., mitochondrial ATP production) and extracellular acidification (ECAR; proportional to glycolytic rate in most systems) in live cells are determined in a convenient 96-well format under a variety of stimulation conditions. These data are often most robust when combined with metabolomic approaches as described above. The Seahorse can give functional significance to metabolomics findings; for example, if glucose more strongly labels the TCA cycle than glutamine in a SIL study, then this can be confirmed by OCR in the presence of glucose or glutamine. Importantly, a lack of phenotypic differences by Seahorse does not preclude a metabolomic phenotype.

Primary use: Measure OCR and ECAR of live cells in a 96-well plate format. OCR and ECAR rates are key indicators of mitochondrial respiration and glycolysis; these measurements provide a systems-level view of cellular metabolic function in cultured cells and ex vivo samples

Capabilities

• Four-port injection system and automated mixing allows real-time detection of responses to substrates, inhibitors and other compounds. Temperature is maintained at 37^o
• A 96-well plate allows multiple conditions to be run at once. The system is optimized for compound screening and dose-response studies.
• As few as 5,000 cells per well may be analyzed at high sensitivity (depending on cell type)
• System is compatible with many different sample types

Specifications

• 96 assay wells
• 2 µL microchamber volume

Primary use: Bottom-up and middle-down proteomics applications

Capabilities

• High-resolution/accurate mass data acquisition
• MSn fragmentation
• Data-dependent acquisition and data-independent acquisition
• Real-time search for TMT quantification
• High-sensitivity nano-flow UPLC

Specifications

• Up to 500K resolution (FWHM)
• < 2ppm mass accuracy
• Electrospray ionization
• CID, HCD and ETD fragmentation
• High field asymmetric waveform ion mobility (FAIMS)
• High mass range