This application is to secure funds to purchase an AB SCIEX QTRAP 5500 PRO triple quadrupole (QQQ) mass spectrometer coupled with an Eksigent nanoLC to carry out analyses based on multiple- reaction monitoring (MRM). MRM is an extremely sensitive, quantitative, and reproducible method for monitoring the levels of targeted, low abundance proteins in a complex biological sample. Conceptually it is similar to performing quantitative immunoblots but without the need to tag or raise antibodies against the targeted proteins. In the long term, this instrument will enable many research projects for which quantitative protein science is a core component. This application comprises eight specific projects. The Deshaies laboratory will quantify the levels of 64 proteins from the cullin-RING ubiquitin ligase (CRL) network in yeast bearing mutations in the Nedd8/Rub1 conjugating and deconjugating enzymes. This analysis will provide insights into the molecular logic of this key regulatory system which has been implicated in the pathogenesis of cancer. The Rothenberg laboratory will study a defined set of transcription factors in mice that will provide fundamental insight in T-cell differentiation. In addition, the Davidson laboratory will study a defined set of transcription factors in sea urchins that will delineate the spatial specifications of gene expression in sea urchin embryos. The Kennedy laboratory will use MRM to quantify synaptic regulatory proteins. The Hess lab will use mass spectrometry to quantify select acetylated histone peptides. The Chan laboratory will employ MRM to evaluate specific changes in the mitochondrial proteome in which cycles of mitochondrial fission and fusion or mitophagy are disrupted. The Patterson lab will study a set of 14 target proteins in an established mouse model of maternal immune activation, a condition known to increase the risk of schizophrenia in the offspring of affected women by 3-7 fold. Insights in the molecular pathways that trigger this pathophysiology will suggest new targets for cures. The Varshavsky lab will quantify a set of previously defined substrates of the N-end rule pathway in budding yeast using MRM. This will provide molecular insight into the neurological deficits that result when the orthologous gene is mutated in humans. The availability of a state-of-the-art QTRAP 5500 mass spectrometer will enable these Caltech researchers to employ targeted MRM to quantitatively monitor changes in proteomes in response to a variety of chemical, genetic, and environmental perturbations. All of the projects relate to critical subcellular processes and many of the genes and proteins to be studied are targets of human therapeutics or are causative agents in human diseases including neurodegeneration, schizophrenia, and cancer. Thus, the proposed studies have the potential to have a great impact on human health.
The pathways that are targeted in the studies described in this application are either deregulated in cancer, the target of anti-cancer drugs in development, or are known to be compromised by genetic mutations in specific cases of inherited neurodegeneration and neuropathy, mental retardation and schizophrenia. Using a newly-developed resource for performing a powerful analytical method known as multiple reaction monitoring (MRM) the applicants seek to quantify proteins that are targets of the pathways under investigation. These efforts may yield markers to monitor development, disease onset/progression or suggest new strategies for the development of therapeutics to mitigate the pathologies that arise when these pathways are compromised by genetic mutation.
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