Discovery Proteomics Core

Yale University, New Haven CT

Investigators

Linked publications & trials

Abstract

The Discovery Proteomics Core (DPC) uses cutting edge and robust sample preparation, state-of-the-art proteomic and complementary biophysical, and lipidomics technologies to analyze adaptive changes in neuronal signal transduction mechanisms that occur in response to drugs of abuse. Aim 1 uses Data Dependent Acquisition (DDA) and Data-Independent Acquisition (DIA) 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, FACS, FANS or single cell dispensing. We will continue to build project specific spectral libraries to support DIA assays as well as implement library-free searches. 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 FAIMS based data acquisition for glycosylation and “Middle-down” analyses, and use Parallel Reaction Monitoring by the 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 use new tools and databases to improve analysis and interpretation of large scale, quantitative MS/proteomics data sets. To improve peptide identification from searching databases with MS/MS spectra, we will implement a CHIMERYS search mechanism for DDA and DIA data; and we will collaborate with the Biostatistics and Bioinformatics Core to enable MS/MS searches of RNA-sequencing-predicted proteomes. In Aim 5 we will purify recombinant proteins and exosomes from plasma or cell culture media for our investigators and use Nanoparticle Tracking Analysis, Dynamic Light Scattering, and Asymmetric Flow Field-Flow-Fractionation coupled with Multiangle Static Light Scattering to characterize size distribution and to fractionate the purified exosomes. In Aim 6 we will monitor localization and metabolism of phosphoinositide lipids, a major class of signaling lipids, in brain circuits involved in addiction and provide methodology for studying the role of intracellular protein-mediated lipid transport in the control of these circuits. In Aim 7, we will provide training in experimental design, sample preparation, and use of software for analysis and interpretation of MS data, biophysical, and phosphoinositide analyses. The DPC 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.