Kym F. Faull - US grants

electrospray ionization mass spectrometry; biomedical equipment purchase;

DESCRIPTION (provided by applicant): Funds are requested to purchase a hybrid quadrupole-time-of-flight laser mass spectrometer (QTOF) for the UCLA Pasarow Mass Spectrometry Laboratory. This is a shared facility jointly supported by the Department of Chemistry and Biochemistry (College of Letters and Sciences) and the Department of Psychiatry and Biobehavioral Sciences (School of Medicine) that is available to the entire UCLA campus research and teaching enterprise. There is no instrument of this type on the UCLA campus. Because of their sensitivity and selectivity, so-called QTOF instruments have been proven to be indispensable for the rapid and reliable identification of proteins from a variety of sources including spots excised from polyacrylamide gel electrophoretograms. This instrument will be a central feature of the UCLA proteomics initiative which is now being established. An instrument of QTOF design with all accessory components necessary for routine protein identification, is essential for the continued productivity, success and competitiveness of a growing number of campus research programs. Reflecting this diversity of use, descriptions of the research of the major users of the instrument are provided.

[unreadable] DESCRIPTION (provided by applicant): Approximately 80% of chronic pancreatitis cases are associated with alcohol consumption. Analogous to liver fibrosis and hepatic stellate cells, there is increasing evidence for a key role in the activation of the pancreatic stellate cell in pancreatitis. As in the liver, the central gateway for this process may be NADPH oxidase. When the reactive oxygen species generated by NADPH oxidase overwhelm the innate anti-oxidant system in cells, then tissue injury can result. In susceptible individuals these injuries can lead to chronic activation of the inflammatory response and ultimately pancreatitis. Although much is known about the composition and activation of the NADPH oxidase system in neutrophils, little is known about the complex in other tissues. For example, this proposal contains the first report of the presence of components of this reactive oxygen species generating complex in activated pancreatic stellate cells. Current knowledge is limited by the available antibodies that target NADPH oxidase subunits, and mRNA experiments that show the presence of protein precursors. Because of these limitations and the importance of this system in alcoholic pancreatitis, we propose to identify the components of the NADPH oxidase complex from pancreatic stellate cells by state-of-the-art methods. The specific hypothesis we will be testing is that alcohol and growth factors alter the subunit composition of the NADPH oxidase system in pancreatic stellate cells, leading to a synergistic increase in the activity of the NADPH oxidase, an effect that mediates several pathologic responses in the pancreas. Pancreatic stellate cell cultures will be prepared from rat pancreas by established methods and treated with PDGF and ethanol, alone and in combination. Membrane fractions will be isolated from these cultures and analyzed by blue native two-dimensional gels. For the first dimension electrophoretic separation will be used with a non-denaturing buffer that enables separation of complexes. The second dimension utilizes SDS-PAGE denaturing conditions to separate the component parts of each complex isolated in the first dimension of the gel. In-gel tryptic digestion followed by microbore liquid chromatography coupled to tandem mass spectrometry with data dependant acquisition will be used to collect mass spectra and tandem mass spectra of the digest components. Data sets will be screened against human genomic databases of predicted or known open reading frame translations. Significant matches will be examined manually to confirm assignments. In this way components of the complex will be identified by mass and sequence information derived from in-gel digests. These data are essential for the successful development of targeted therapeutic agents for treatment of alcohol related pancreatitis. By far the most common cause of chronic pancreatitis is alcohol abuse, estimated to cause 70- 90% of the cases. The processes of chronic pancreatitis include inflammation and scarring with the loss of tissue. Of particular note, the processes of this condition continue even after cessation of alcohol intake, indicating that the processes are self-sustaining once a certain stage of the disease is reached. Adding to the morbidity and mortality of this disorder is the fact that patients with chronic pancreatitis are at a significantly increased risk for pancreatic cancer. Successful completion of the experiments described in this proposal are essential for the development of targeted therapeutic agents for treatment of alcohol related pancreatitis. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Funds are requested for a state-of-the-art triple quadrupole mass spectrometer (QqQ) and two essential ancillary attachments - an ultra-high pressure liquid chromatography (UPLC) system, and a High- Field Assymetric Waveform Ion Mobility Spectrometry (FAIMS) attachment. This equipment will be located in the Pasarow Mass Spectrometry Laboratory (PMSL) within the Semel (formerly Neuropsychiatric) Institute at UCLA, a campus-wide core mass spectrometry laboratory. The QqQ mass spectrometers currently in the PMSL are about 17 years old and these are obsolete by any criteria. In the intervening period there have been significant improvements in sensitivity, resolution, accuracy of m/z assignment, computer control, experimental flexibility, and detection dynamic range of these instruments. UPLC brings liquid chromatographic resolution close to that attainable with capillary gas chromatography, and is an important development for high sensitivity work, high throughput work, and for work with complex mixtures. There is no UPLC system on the UCLA campus. The FAIMS technology is a new development that for some compounds lowers the background from isobaric ions, and as such can provide a significant advantage for low molecular weight analyses of complex mixtures by LC/MS, as is intended for the new instrument. The research projects for which the equipment will be used involve quantitative and qualitative analyses of a diverse range of molecules, many but not all with relatively low molecular weights (generally less that 1500 Da), in a variety of milieu. Collectively these compounds are implicated in diverse phenomena including signal transduction, cellular communication, hormonal homeostasis, addictive processes, ageing, logical thought processing, immune responses and inflammation. The application includes descriptions of eighteen collaborating laboratories with mass spectrometric analysis essential to their research projects. These groups are expected to occupy approximately 90% of instrument time. The remaining instrument time will be used by other collaborating groups as needed. Funding support is in place for all the collaborating laboratories. The PMSL has the necessary infrastructure to maintain and support the new equipment. Professor Faull, the PI on this application and the Director of the PMSL, and Postdoctoral Research Fellow Christopher Ryan, PhD, will be responsible for the day-to-day operation and maintenance of the new equipment. The PI has a strong track- record for the effective use of equipment purchased through the NIH SIG mechanism. This application represents a critical component of an effort by the UCLA Semel Institute and School of Medicine to re-place obsolete equipment within the campus core laboratories with state-of-the-art instruments for cutting edge research in metabolomics and related fields. PUBLIC HEALTH RELEVANCE: The research for which the new equipment will be used is directly relevant to many aspects of human health and disease, including cancer, metabolic diseases, brain injury, viral infectivity, psychiatric diseases, and age-related neurological diseases including Alzheimer's and Parkinson's diseases. The frequency with which many of these diseases occur in Western society is rising as the population ages. Research equipment such as that requested is crucial for understanding the molecular basis of these diseases and continued progress on these important problems that significantly impact human health and well-being.

? DESCRIPTION (provided by applicant): Funds are requested for an Agilent 6550 hybrid quadrupole-time-of-flight mass spectrometer (QTOF) with an Agilent 1290 ultra-high pressure liquid chromatography system (UHPLC). This equipment will be located in the Pasarow Mass Spectrometry Laboratory (PMSL), a collaborative core laboratory within the Semel Institute at UCLA. The PMSL does not currently have an instrument suitable for liquid chromatography/mass spectrometry (LC/MS) metabolomics or proteomics experiments. Such an instrument is essential to support the range of research projects in which the PMSL is involved and for which it has earned high regard. Acquisition of the requested equipment will ensure that the PMSL and its collaborating laboratories remain competitive in their respective fields and continue to make progress on the many important and exciting research projects on which they are working. The projects for which the requested instrument will be used encompass basic, translational, and clinical sciences, involving cutting-edge research in molecular biomarker screening for a number of diseases including Alzheimer's Disease and related disorders, a variety of cancers, and iron deficiency anemia, among others. The compounds that are being identified and quantified in these experiments are implicated in diverse biological phenomena including signal transduction, cellular communication, hormonal homeostasis, the addictive process, aging, immune responses, and inflammation. This application includes descriptions of projects from 23 collaborating laboratories: 6 are designated as Major and the remainder as Minor Projects, the basis of this distinction being overall time-commitment of instrument and personnel, all equally important and exciting. These collaborations are expected to occupy approximately 90% of instrument time. The remaining instrument time will be available to other collaborating groups for short-term exploratory research. The PMSL has the infrastructure to maintain and support the new equipment backed by an outstanding track record fostering the application of contemporary mass spectrometry to address important biological problems in science and medicine. Professor Faull, the PI on this application and the Director of the PMSL, Professor Whitelgge, de facto deputy director of the PMSL, and Research Assistants Joseph Capri and Farbod Fazlollahi, will be responsible for the day-to-day operation and maintenance of the new equipment. Professors Faull and Whitelegge have an exceptional reputation for the effective use of equipment purchased through the NIH SIG mechanism. This application represents a critical component of the effort by UCLA, the David Geffen School of Medicine, and The Semel Institute, to equip collaborative campus core laboratories with state-of-the- art instruments for cutting-edge research in metabolomics, proteomics, and related fields.