1. Two AB Sciex 4000 Q-Trap hybrid triple quadrupole linear ion trap mass spectrometer with Shimadzu automated HPLC systems for small molecule quantitation by selective reaction monitoring mode tandem mass spectrometry. Each instrument is equipped with a Parker Balston gas generator providing N2, zero and dry air. Each instrument is interfaced with a computer running ABSciex Analyst Software for instrument control, data acquisition and analysis. Both instruments are equipped with a ABSciex TurboV electrospray ionization (ESI) sources that can also be used for atmospheric pressure chemical ionization (APCI). We also have an ABSciex photoionization source that can be used with either instrument. Both instruments can be operated with individual Shimadzu HPLC systems comprising two pumps with solvent switching valves, a column oven with automated column switching, a controller and an autosampler which in one case is refrigerated. A valve between the column and ion source is used to avoid unnecessary solvent introduction into the source which minimizes source contamination. Both of these instruments can be used for conventional HPLC ESI MS/MS assays for quantitation of molecules of interest using standard selective reaction monitoring approaches.

2. Advion Nanomate Chip based nano ESI source for direct infusion mass spectrometry. One of our ABSciex 4000 Q-Trap instruments is adapted for use with an Advion nanomate chip-based robotic nano-ESI source. The Advion Nanomate is mounted on a platform in front of the instrument that allows it to be used interchangeably with the other instrument ion sources. In brief, the Advion TriVersa Nanomate consists of an integrated liquid handling platform that uses disposable tips to aspirate samples from a multiwell plate. The tip containing the sample is then connected to a nano-ESI chip which is in front of the curtain plate/interface heater of the nanospray-ESI ion source. This microfluidics chip contains an array of nanoelectrospray nozzles etched in a silicon wafer. Pressurization of the tip delivers the sample into the nozzle at a controlled flow rate. The instrument is controlled by a computer running a dedicated software package that allows specification of variables including spray volumes, spray pressure, sample temperature and the sequence of sample analysis. This configuration is used for metabolite analysis by flow infusion introduction of samples into the ion source and either targeted analysis by multiple precursor ion and neutral loss scans or unbiased analysis using survey scans to trigger MS/MS scans for every sample component within a specified mass range and ion intensity. The ability of the Advion nanomate to handle small sample volumes precisely and, because each sample uses a unique tip and ESI nozzle, to avoid sample to sample carry-over makes it uniquely well suited to this application.

3. AB Sciex 4000 Q-Trap hybrid triple quadrupole linear ion trap mass spectrometer/ABSciex Flashquant Vacuum MALDI source for high throughput quantitation of small molecules and tissue imaging mass spectrometry. One of our ABSciex 4000 Q-Trap instruments is adapted for use with an ABSciex Flashquant vacuum MALDI ion source. This is an orthogonal MALDI source in which the laser can be scanned across a 96 or 384 well target plate. The primary application of this source and instrument configuration is for small molecule quantitation by MALDI tandem mass spectrometry in selected reaction monitoring mode. This instrument configuration can also be used for tissue imaging mass spectrometry in which the two dimensional distribution of molecules of interest is monitored using MALDI mass spectrometry.

4. ABSciex 5600 “Triple TOF” hybrid quadrupole time of flight mass spectrometer. The instrument is serviced by a Parker Balston gas generator and is operated with an Eksigent micro flow HPLC system which incorporates a low dispersion ESI electrode for capillary HPLC and automated flow infusion of samples. This system can also be operated with a Shimadzu HPLC system with the same configuration as listed above. This new instrument offers significantly higher levels of mass accuracy and mass resolution at fast scan speeds making it a uniquely powerful tool for targeted and unbiased metabolite profiling and metabolism studies using stable isotope tracers and for applications where selected ion monitoring approaches using our triple quadrupole instruments lack the necessary specificity to overcome interference from other sample components. It also enables highly sensitive and selective quantitation of molecules of interest using selected ion monitoring strategies and integration based on high resolution product ion spectra which overcomes interference that can be a problem caused by the lower resolution of triple quadrupole instruments.

5. Data Analysis Software. Instruments are controlled using ABSciex Analyst Software which can be installed on additional computers for off-line data analysis and processing. We use ABSciex MultiQuant software for relative and absolute quantitation using mass labeled internal standards or off line calibration as needed. AB Sciex Markerview software can be used for statistical comparisons of components identified in data sets from targeted and unbiased metabolomics experiments. AB Sciex Lipidview software can be used to identify many lipid species from data sets obtained using targeted or unbiased profiling approaches. Data can also be exported in a tab delimited format for off line analysis using the large scale statistical approaches and component/metabolite identification strategies23; 24 described in the Quantitative Biology Component section.

6. Agilent 6890 gas chromatograph/ electron capture detector and Agilent 5975 inert mass selective detector. This instrument also has dual headspace autosamplers, can operate in dual column mode and is connected to a computer running Agilent Chemstation software for instrument control and data acquisition and analysis. This instrument is used primarily for routine analysis of PCBs by well documented EPA compliant methods particularly in environmental samples where unbiased identification of PCBs cannot be accomplished using our HPLC ESI MS/MS methods.