It is the specific aim of the R21 phase of this proposal 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 its 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-ICRMS). It is the general aim of this combined R21-R33 proposal to provide a cost effective 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. Primary brain tumors 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.
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