The Discovery Proteomics Core (DPC) uses cutting edge sample preparation, state-of-the-art proteomic and complementary recombinant protein expression, biophysical, and lipidomics technologies to analyze adaptive changes in neuronal signal transduction mechanisms that occur in response to drugs of abuse.
Aim 1 uses discovery proteomics and Data-Independent Acquisition (DIA) from the Targeted Proteomics Core (TPC) to identify proteins whose expression or posttranslational modifications (PTMs) are altered in cell-based systems, tissue from discrete brain regions, single cell types and their organelles isolated with LCM or FACS, or protein complexes isolated by proximity labeling or other approaches. To improve peptide identification from searching databases with MS/MS spectra, we will use DIA and ETD-DIA and we will collaborate with the Biostatistics and Bioinformatics Core (BBC) to enable MS/MS searches of RNA-sequencing-predicted proteomes. We will build peptide MS/MS spectral libraries to support DIA assays.
Aim 2 uses immunological and chemical approaches to enrich for peptides and proteins containing PTMs to facilitate their identification. In collaboration with TPC, the DPC will integrate PTM analyses into DIA, including use of ETD for glycosylation and ?Middle-down? analyses, and use Parallel Reaction Monitoring by TPC to validate differentially expressed proteins and PTMs.
Aim 3 uses ?Middle-down? analyses to identify the multiple PTMs that occur in individual proteoforms such as the combinatorial epigenetic changes in histone modification.
In Aim 4 we will collaborate with the BBC to streamline data output to improve visualization of quantitative protein PTM changes, and will improve existing and design new tools to carry out more advanced data analyses.
In Aim 5 we will overexpress and purify recombinant proteins for our investigators and use isothermal microcalorimetry, static/dynamic light scattering, circular dichroism, surface plasmon resonance, stopped-flow, and asymmetric flow field-flow-fractionation to extend protein profiling into the functional domain by quantitatively determining the thermodynamics and kinetics that underlie protein:protein and protein:ligand interactions.
In Aim 6 we will quantify phosphoinositide lipids in brain circuits involved in addiction and provide innovative cell-free assays for studying lipid transport proteins that function at membrane contact sites. In collaboration with TPC, we will build a DIA assay for the phosphoinositide interactome.
In Aim 7, we will provide training in experimental design, sample preparation, and use of softwares for analysis and interpretation of data from MS, biophysical, and phosphoinositide analyses. The DPC will collaborate with Center investigators and will ensure that the Center's research is supported by the most advanced instrumentation and biotechnologies. By taking a holistic approach the DPC will provide Center investigators with the broad range of tools and training needed to identify, and then to understand why certain proteins and their PTMs and phosphoinositide effectors are differentially expressed following exposure to drugs of abuse.
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