Services

Small Molecule Mass Spectrometry Facility

The Small Metabolite Mass Spectrometry Core owns a triple quadrupole (QqQ) mass spectrometer (MS), the preferred design for quantitative mass spectrometry, also excellent for molecule identification by “mass fragmentography”. Each quadrupole (quad) section is an ion mass filter. Liquid entering the MS TurboIon Spray source is aerosolized and evaporated to produce gas-phase ions. The first quad stage can select and ana-lyze ions by their mass-to-charge ratio; the second can fragment them by collision with nitrogen gas; the third can analyze collision product ions. Either a sample syringe pump or a high performance liquid chromatogra-phy (HPLC) system supplies liquid to the MS source. The core also has HPLC systems with amperometric electrochemical (EC) and ultraviolet absorbance (UV) detectors. Finally, the core has a room for specimen stabilization and homogenization; it houses an automated freeze dryer with shelf cooling, and a dry-nitrogen atmosphere glove box with an analytical microbalance and a pH meter.

That these resources coexist in the same facility reflects the core’s most ambitious service: quantification of many small molecules from single tissue/fluid specimens. A web search of core facilities is unlikely to reveal another core that offers this.

Basic Services: The core also offers basic services that exploit some but not all of these resources.

• Small molecule (150-3000 amu) mass spectrometry. For reasonably pure products, e.g., of chemical synthesis or biochemical purification, the core offers MS mass determination (resolution 0.6 amu), and MS/MS structure elucidation (product-ion scans following collision-induced disintegration of a precursor ion), priced per determination.

• ‘Hyphenated’ HPLC services. If molecules are less pure or in a biological matrix, LC-MS, LC-MS/MS, LC-UV and LC-EC may be used to resolve them, determine their retention times, and quantify them. This service is priced per hour.

• Specimen banking. The core offers specimen banking regardless of whether the specimen will be used for core-based assessments, a client’s own assays, commercial tests, or any combination of these. A specimen is protected from oxygen and moisture at all times. For banking, a specimen must have been frozen in a flat-bottomed container that can accept a standard lyophilization stopper. To facilitate this the core can provide a mobile “cryo” cart with a nitrogen vapor bottle, numbered vials, and stoppers. The complete banking service includes (a) specimen spiking with a frozen cocktail of internal standards; (b) lyophilization in a programmable freeze dryer with controlled shelf and specimen temperatures, dry ni-trogen gas venting and automatic stoppering; (c) dry pulverization; (d) recovered mass determination; (e) pH determination; (f) transfer to gas-tight amber vials; (g) bar-code labeling; (h) desiccated vial storage at –80ºC, (j) computerized specimen tracking, and (k) weighed aliquot removal. In the future, the core may offer the capacity for low-power microscopic viewing, imaging and crude dissection of dried speci-mens before pulverization. Specimen banking fees are per specimen except for freeze-drying, which is calculated per shelf load (up to 80 shell vials or 40 serum bottles, all of the same height, and submitted at the same time).

Core Assays: The core offers or will offer the following assays, either singly, or as part of a set of multiple assays performed on single specimens. Small metabolite assays: These assessments generally require smaller specimen samples than do those that measure in vitro-freed adducts of DNA, protein and lipids.

• Thiols & Disulfides #1, free in tissue: (homocysteine, homocystine, cysteine, cystine, cysteinylglycine, methionine and oxidized and reduced glutathione). This is a complete profile of the most common monothiols and disulfides present in plasma and tissue extracts, by the LC-EC method of Melnyk et al., (1999) except with amperometric instead of coulometric detection.

• Thiols & Disulfides #2, total reduced: (total homocysteine, total cysteine, total glutathione, methionine and cystathionine). All disulfides are reduced by borohydride treatment prior to chromatography , . The assay otherwise is likethe same as #1 and its fee is reduced if both #1 and #2 are requested.

• Thiols & Disulfides #3, adenosyls: (S-adenosylmethionine, S-adenosylhomocysteine, adenosine). This assay was developed in the same laboratory3. The authors have also applied this method to solid tissues, e.g., liver, brain, whole Drosophila and C. elegans (Melnyk, personal communication).

• Energy Charge: (the adenine nucleotides ATP, ADP, and AMP). The core will select from a published HPLC-UV methods, e.g., those of Harr (1990) and Sibrowski et al. (1990) for stored red blood cells, and Viarengo et al. (1989) for tissue extracts .

• Nicotine and Flavin Adenine Dinucleotides: (NAD+, NADH, NADP+, NADPH, FAD+, FADH). These will be assayed by LC-UV (or LC-fluorescence, when the core can acquire a fluorescence detector).

• Gluconeogenic & Krebs Cycle Flux: This LC-MS analysis requires in vivo administration of [13C]-lactate prior to specimen collection, and thus may or may not be applicable to multianalyte analyses. The method is to be built upon original publications from the W. Lee laboratory , .

• Amino Acid Panel: The core will choose from among published LC-EC and LC-MS amino acid assays.

• Metabolic Fingerprinting: This methodology intentionally generates complex LC-MS or LC-EC-MS chromatographs of hundreds of metabolites in minimally preprocessed serum, plasma and urine samples, then applies multivariate statistical methods to extract from them a subset of pattern features that track with the experimental or clinical variable of interest. The core also plans to use this methodology for solid tissue sample extracts. Metabolic fingerprinting reveals strikingly high inter-individual pattern differ-ences. Because of this, the core strongly urges clients to design time-course studies, with repeated speci-men collections before and after a treatment, or clinical event.

• Metabolomics: This modern methodology differs from metabolic fingerprinting only in that metabolites are identified by mass fragmentography (MS/MS) for most chromatographic peaks. The data can then be analyzed by methods that exploit compiled metabolic pathway databases.