LC-ELECTROSPRAY QUADRUPOLE ION TRAP MASS SPECTROMETER

University Of Tennessee Health Sci Ctr, Memphis TN

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Abstract

Funds are requested to purchase a liquid chromatograph-electrospray quadrupole ion trap mass spectrometer (LC-ESI-Q-IT MS). The LC-ESI- Q-IT MS is selected because it possesses several critical operating features: l) high detection sensitivity (pmol-fmol), 2) on-line coupling of HPLC to MS, 3) excellent MST capabilities, and 4) high throughput and automation. This instrument will be shared by a group of NIH-supported researchers at the University of Tennessee, Memphis, and at the St. Jude Children's Research Hospital, Memphis. The requested LC-ESI-Q-IT MS is needed to complement the current MALDI-TOF instrument and to provide an alternative to MALDI-TOF analysis. The LC-ESI-Q-IT instrument will be employed in the following research programs: Proteomics: (i) studies of the alterations of opioid neuropeptidergic systems in human cerebrospinal fluid (CSF), that are associated with idiopathic low back pain; (ii) analysis of the opioid systems in human pituitary tumor tissue; and (iii) investigation of the proteases involved in the activation of human macrophages for enhanced killing of microbes and tumor cells. Quantification: (i) neuropeptides in human pituitary and in human CSF; (ii) corticotropin-releasing hormone in the microdialysates of mouse CSF (in the investigation of the effects of nicotine on neuronal functions); (iii) neuropeptide Y and orexins in rat hypothalamus (for the study of the nicotine-induced changes in food intake stimulators); and (iv) synthetic opioid peptide analogs in ovine plasma (in the study of pain-reducing drugs for use in obstetrics). Structures of biologically active compounds: (i) bacterial cell wall constituents (lipopolysaccharides and glycopeptides) involved in macrophage activation; and (ii) blood-derived phospholipid growth factors. Protein characterization: (i) enzymes involved in arachidonic acid release in blood vessels; (ii) wild-type and mutant forms of thiopurine methyltransferase (for the investigation of the genetic polymorphism of TPMT); and (lii) mismatch repair proteins (in the study of their involvement in the determination of thiopurine cytotoxicity). Successful completion of these research programs will further the understanding in several areas related to human health, such as the role of neuropeptides in disease, effects of smoking.on brain functions, ability of the immune system to fight infection, and the use of thiopurines for the treatment of childhood cancer.