The entire constituent of intracellular signaling molecules expressed in the organisms under investigation represents an extremely complex analytical challenge with a vast number of components varying dramatically in size and concentration and expressed in a highly heterogeneous manner within the CNS of these organisms. To address these challenges, and to provide our users with a battery of state-of-the-art protein identification techniques, this core proposes the use and development of techniques capable of molecular interrogation in three separate areas. We will (1) identify signaling molecules by a variety of """"""""Bottom Up"""""""" approaches including mass fingerprinting, automated MS/MS sequencing, and accurate mass measurement, (2) characterize identified intercellular signaling molecules by intact protein analysis to identify post-translational modifications that are common among intercellular signaling molecules and correct for possible errors in the genomic databases due to signal peptide cleavage etc., and (3) localize intercellular signaling molecules by a variety of MS imaging techniques including current state-of-the-art and development of proposed """"""""stretched sample"""""""" and """"""""capillary array"""""""" methods that have the potential to enhance resolution and sensitivity of imaging methods to a level needed by our users. The needs for intercellular signaling molecule identification of our users are quite different from """"""""shotgun"""""""" proteomics of unicellular organisms in sample size and complexity. With the combined expertise and facilities in the groups associated with the proposed center, a unique opportunity exists to identify and characterize intercellular signaling molecules at their sources.
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