The Metabolomics Core has 11 mass spectrometers divided into three categories: isotope ratio, single quadrupole and tandem.
Isotope ratio mass spectrometers (IR/MS)
Thermo DeltaV Advantage
Thermo DeltaV Advantage
This instrument has a dedicated gas chromatograph and online combustion system — the latter consisting of sequential oxidation and reduction furnaces plus water trap — and is used for [13C] measurements of amino acids, fatty acids and glucose, and [15N] in amino acids.
Typical analyses include the measurement of low enrichment levels of [ring-13C6]phenylalanine in muscle extracts such as mitochondrial and sarcoplasmic fractions, and [U-13C]palmitate in plasma and muscle extracts.
Thermo DeltaV Advantage.
The core has two of these new IR/MS systems, each accommodating different types of inlets. The first has a dual-inlet system plus H/device for the analysis of hydrogen deuteride (HD) in water, urine and plasma samples associated with total body water and doubly labeled water studies. The H/device utilizes a chromium furnace held at 825 degrees Celsius for the reduction of water to deuterium.
The second instrument has two inlets. The first is a gas bench inlet, which has a 96-port carrousel plus Leap autosampler and gas chromatograph (GC) for analyzing 13CO2 in expired air and 18O in water (by equilibration with CO2). The other inlet is a gas chromatograph and online combustion unit, which is a newer version of the inlet attached to the DeltaPlus.
This equipment permits the combustion of compounds that have been separated by gas chromatography, such as amino acids to CO2 and N2. This enables the analysis of low enrichment levels of [13C]-labeled compounds, such as [13C6]-phenylalanine (which is converted to 13CO2), to be performed. For [15N]-labeled amino acids, online reduction (NO to N2) allows the measurement of isotopic enrichments of nitrogen.
Single quadrupole gas chromatography mass spectrometers
Agilent Technologies 5973N MSD
Agilent Technologies 5975C MSD
Agilent Technologies 5973N MSD.
This is a bench-top quadrupole gas chromatograph/mass spectrometer (GC/MS) fitted with both electron impact (EI) and chemical ionization (CI) ion sources. It is used mainly for the measurement of enrichment levels of [1-13C]glucose and [6,6-2H2]glucose from plasma, and [13C2]oxalate levels in urine.
Agilent Technologies 5975C MSD.
This GC/MS instrument is a newer version of the 5973N and is used for the measurement of low-level analytes, such as amino acids in tissue fluid, tRNA and dialysate samples. [D5]glycerol and [13C3]glycerol levels in plasma and very-low-density lipoprotein (VLDL) fractions also are monitored. The 5975C has both EI and CI sources, the latter being able to run in both positive and negative ion modes.
Tandem mass spectrometers
Applied Biosystems API 5000
Waters Quattro Micro
Thermo TSQ Quantum Ultra
Thermo TSQ Quantum
Thermo LTQ Orbitrap
Applied Biosystems API 5000.
The API 5000 is a triple quadrupole instrument with both TurboIonSpray and atmospheric pressure ionization inlets. The system is optimized for high LC flow rates (0.2 to 1.5 mL per minute) and is coupled to a Cohesive TX2 high-performance liquid chromatography (HPLC) system, which has two LC systems in parallel. This allows for more efficient utilization of the mass spectrometer.
Analyses include high-throughput assays for [d4]cortisol and metabolites, 8-oxo deoxyguanosine, [U-13C]palmitoyl carnitine in tissue, [U-13C]palmitate in plasma, and [ring-13C6]phenylalanine in proteins isolated by 2-D gel electrophoresis.
Waters Quattro Micro.
This triple quadrupole system has both EI and CI capabilities and is used for routine amino acid enrichment measurements in plasma and low-level enrichment levels of [ring-13C6]phenylalanine in proteins isolated by 2-D gel electrophoresis.
Thermo TSQ Quantum Ultra.
The Quantum Ultra is a highly sensitive LC-based triple quadrupole mass spectrometer that is used for metabolomic quantitative profiling of plasma and tissue samples by utilizing multiple reaction monitoring.
Two assays recently implemented are the analysis of 45 amino acids and their amino metabolites from plasma and the measurement of ceramide and its metabolites in tissues and cells. Both assays utilize an electrospray ionization source coupled to a Waters Acquity Ultra-Performance Liquid Chromatography (UPLC) system. Compared with conventional LC systems, the UPLC system offers superior resolution combined with shorter run times.
Thermo TSQ Quantum.
This instrument has been converted from a GC-based version of the Quantum to an LC system and uses a Waters Acquity UPLC system for sample introduction. Assays currently being performed with this instrument include the monitoring of low levels of enrichment of [15N2]lysine and [ring-13C6]phenylalanine in plasma and tissue extracts. Concentration profiles of nine free fatty acids in plasma are also measured together with [U-13C]palmitate enrichments in plasma.
The Agilent 6220 time-of-flight (TOF) mass spectrometer is a new LC-based instrument that offers accurate mass (< 2 parts per million, internal) and high-resolution (20,000) capabilities. It is being used for qualitative differential metabolomic profiling experiments.
Plasma or serum samples are analyzed for changes in metabolite intensity on two columns of different polarities (C18 and HILIC) utilizing a Waters Acquity UPLC system. Both positive and negative ions are monitored in separate runs. The profiles obtained are then compared using principal component analysis software to discover metabolites that are either upregulated or downregulated. Both Mass Profiler Professional and Simca-P software packages are being utilized for this analysis.
Thermo LTQ Orbitrap.
This mass spectrometer offers sub-parts per million mass accuracy with resolution of up to 100,000. It is used in conjunction with the Waters Acquity UPLC system to identify metabolites that are differentially expressed in the metabolomics profiles obtained on the Agilent 6220. Both accurate mass and MS/MS data in conjunction with spectral databases are used for possible identification.