Targeted Proteomics Core

Yale University, New Haven CT

Investigators

Linked publications & trials

Abstract

The Targeted Proteomics Core designs and implements targeted assays that have been used increasingly by Center investigators to quantitate and validate potential protein biomarkers initially identified from the protein profiling analyses carried out by the Discovery Proteomics Core (DPC); genomic studies, including the use of neuronal cell type-specific transcriptional and translational profiling; or the literature. These analyses will include relative and absolute quantitation of targeted protein levels or alterations in their post- translational modifications (PTMs) that have been implicated in model and other organisms as adaptive changes that occur in response to drugs of abuse. To address the huge level of cellular and sub-cellular heterogeneity in the central nervous system, another objective is to develop the targeted workflows needed to analyze subcellular organelles and sub-proteomes from the single types of neurons that define the circuits that underlie the actions and addictive properties of drugs of abuse. In Aim 1 we will develop high throughput Parallel Reaction Monitoring (PRM) assays to validate the most significantly differentially expressed proteins and PTMs identified by the Discovery Proteomics core. Highly sensitive PRM assays also will be built to examine the proteomes of specific neuronal sub-types and their organelles (e.g., nuclei, synaptic vesicles), sub-cellular fractions or partially enriched samples (e.g., PSD). Eukaryotic proteins are almost always assembled into multiprotein complexes in vivo and these complexes play important roles in neural signaling and synaptic plasticity. Aim 2 will leverage the data from our targeted assays by using existing and implementing improved proximity labeling (PL) technologies to identify protein interactomes in a cell type and subcellular manner. To accomplish this aim we will optimize workflows for carrying out BAR, Turbo-ID, and other PL approaches and work with the Biostatistics and Bioinformatics Core to improve PL data analysis pipelines. In Aim 3, we will broaden the utility of PL for neuroproteomics applications by developing new chemical probes for existing PL enzymes and alternate and orthogonal enzyme/probe pairs. This will involve a combination of probe synthesis, protein engineering, and yeast display based directed evolution. In Aim 4 we will continue to train Yale/NIDA Neuroproteomics Center members in PL and targeted mass spectrometric techniques including experimental design, sample preparation and handling, digestion protocols, and interpretation of PRM spectra and data so they can optimally utilize the advanced technologies available in the Center.