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Goodlett Laboratory Research

Young Ah Goo prepares I-CAT samples for quantitative expression analysis via MS/MS.
Young Ah Goo prepares I-CAT samples for quantitative expression analysis via MS/MS.		   Jenny Chen labors to aquire high quality MS/MS spectra on our LTQ-FT (right) which are then analyzed by SEQUEST(TM) on our analysis cluster (left).
Jenny Chen labors to aquire high quality MS/MS spectra on our LTQ-FT (right) which are then analyzed by SEQUEST(TM) on our analysis cluster (left).
Comparing two MS runs over scan number (i.e. chromatographic time) and m/z.
Comparing two MS runs over scan number (i.e. chromatographic time) and m/z.		   Identifying an unknown peptide via MS/MS using SEQUEST(TM).
Identifying an unknown peptide via MS/MS using SEQUEST(TM).

Following are funded research areas of interest in our laboratory: 1. Mass spectrometry core for biodefense regional center of excellence funded by NIAID, 2. Parallel peptide tandem mass spectrometry funded by the NCI, 3. Androgen receptor in prostate growth and cancer funded by NIDDK, 4. Mechanisms of prostate cancer prevention by lycopene and 5. Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) for biological research funded by the NCRR. More detail is provided below.

  1. Mass spectrometry core for biodefense regional center of excellence. The mass spectrometry (MS) core will generate global profiles of protein levels, characterize post-translational modifications (PTMs), define chemical structures of small biological molecules like lipopolysaccharides (LPS), integrate data from global protein and mRNA profiles to produce new hypotheses for testing, develop software for analysis of disparate global data sets and refine analytical methods. Specific aims 1 and 2 are experimental in nature, whereas specific aim 3 is computational. Specific aims 1 and 3 will generate novel hypothesis for testing the long-term goal of which is to further our understanding of protein regulatory networks.

  2. Parallel peptide tandem mass spectrometry. It is the aim of this research to develop the software tools necessary to interpret tandem mass spectra produced by collision induced dissociation (CID) of peptides in parallel rather than in series, as is commonly practiced in shotgun proteomics, and to prove it's advantages over serial CID on samples of increasing complexity from peptide standards to proteins extracted from a medulloblastoma primary cell line lysate. Our proposed technology is referred to as shotgun CID and shotgun tandem mass spectrometry (MS/MS) to distinguish it from serial tandem MS and multiplex MS in a Fourier transform-ion cyclotron resonance-mass spectrometer (FT-ICR-MS). It is the general aim of this combined R21-R33 proposal to provide a cost effect competitor to the advantages of multiplex MS in FT-ICR-MS. Our approach will use lower resolution and mass accuracy time-of-flight (TOF) mass analyzers and a continuously alternating data acquisition scheme of parent ion measurement followed fragment ion measurement throughout the chromatographic introduction of sample. Proteins will be identified by a combination of mass mapping of parent peptide ions across the entire chromatographic time, unique chromatographic constraints and their combined fragment ions.

    Once developed the technology will be applied to better understand primary brain tumors which are the leading cause of cancer-related death in children. Medulloblastoma is a primitive neuroectodermal tumor that typically arises from the cerebellar vermis and shows variable degrees of arrested neural differentiation. The cerebellum requires endogenous retinoids for proper control of neuronal apoptosis and differentiation during development. In embryonal carcinoma and neuroblastoma cells, retinoids induce neural differentiation and cell-cycle arrest. Retinoids have recently been shown to induce extensive apoptosis and neuronal differentiation in medulloblastoma cell lines and freshly resected medulloblastoma cells. Together with Dr Jim Olson of the Fred Hutchinson Cancer Research Institute who is organizing a Phase III clinical trial on the effects of 13-cis retinoic acid in medulloblastoma therapy, we will develop shotgun CID within a model that seeks to: 1) facilitate identification of markers associated with retinoid-responsiveness in medulloblastoma cell lines and 2) define key components of the retinoic acid pathway modulated by treatment in retinoid-responsive medulloblastoma cells. This work will be done with medulloblastoma cell lines and primary cells lines derived from retinoid-sensitive and resistant tumors.

  3. Androgen receptor in prostate growth and cancer. The androgen receptor (AR) is a member of the nuclear receptor super family, a large group of ligand-activated transcription factors crucial to the development of prostate cancer and functions as part of a multi-protein complex which binds to the promoter regions of androgen responsive genes via specific DNA sequences called androgen response elements (AREs). Identification and characterization of the constituents of the AR transcription complex is essential to the development of strategies for controlling prostate cancer. Although a number of proteins have been shown to interact with the AR from studies using the yeast two-hybrid system and chromatin immunoprecipitation (ChIP), the AR transcription complex has yet to be purified and directly examined. This proposal will explore the technical feasibility of capturing the AR transcription complex using immobilized promoter DNA segments and applying a novel mass spectrometry (MS)-based proteomic method to examine these captured AR complexes.

  4. Mechanisms of prostate cancer prevention by lycopene. Lycopene is known to concentrate in hormonally regulated cells such as prostate, ovary, breast and testis. Several studies have shown a positive correlation with lycopene consumption and lower risk of developing prostate cancer. It is known to be a powerful antioxidant and it has been suggested therein lies the apparent protective advantage. A better understanding of lycopene uptake by these cells selectively may allow design of therapeutics that target hormonally regulated cells. These experiments involve conducting ICAT experiments in various prostate cancer cells lines some of which concentrate lycopene and have an active androgen receptor (AR) and some of which do not concentrate lycopene and have no or a dysfunctional AR.

  5. FTICRMS for biological research. Funded to provide access to Seattle area biotechnology and biomedical community to FTICRMS instrumentation. NIH funded users from the FHCRC, UW and ISB supported this application. We are expecting delivery of the Thermofinnigan LTQ-FT in April 2004.