The Targeted Proteomics Core designs and implements targeted assays that have been used increasingly by Center investigators to rapidly quantitate and validate potential protein biomarkers related to their studies of neuronal signaling and the actions of drugs of abuse. By utilizing our in-house targeted proteome workflows for both MRM and data-independent (DIA) analysis, we propose to collaborate with NIDA Center investigators, and the Discovery Proteomics and Bioinformatics and Biostatistics Cores, to accurately quantitate either protein expression or their modifications in selected sub-groups of interest. Fully consistent with the overall goals of the Center, targeted proteomic approaches will be used in an innovative manner to systematically address the inherent challenges of single cell type analysis through the ability to design MRM and DIA assays for subcellular organelles and sub-proteomes. Targeted proteomic approaches will also be used to leverage RNA-seq data from selected brain regions or neuronal cell types, for example, in directed analysis of specific proteins or groups of related proteins that are selectively expressed or regulated in a specific cell type. Such targeted approaches will be unique to the TPC and will have a major impact on the ability of Center investigators to carry out innovative research using state-of-the-art approaches to study the action of drugs of abuse. Specifically, we will leverage our optimized MRM workflow by developing highly sensitive MRM assays to examine the proteomes of specific organelles (e.g., nuclei, mitochondria, synaptic vesicles), sub-cellular fractions or partially enriched samples from single types of neuronal cells. We will also expand our current rat/mouse brain targeted MRM PSD proteome assay from 112 proteins to 200 proteins. In addition, we will integrate and expand high resolution DIA acquisition into our next generation large scale targeted proteome assays directed at sub-proteomes from specific types of neurons. Other projects include implementation of a DIA assay to quantify as many as possible of the ~400 proteins that have been identified as being members of the human phosphoinositide-binding proteome. DIA analysis has the potential to change the proteomic landscape since each sample only needs to be run once, and then all peptide fragments can retrospectively be identified and quantified. To advance these studies, we will build a library of peptide biomarkers that will be posted publically on our YPED database and that can be used by multiple investigators in the Center and elsewhere who analyze basic signal transduction mechanisms in the brain, or who study the effects of exposure to drugs of abuse on these signaling mechanisms. Importantly, we will continue to train Neuroproteomics Center members in mass spectrometric techniques including experimental design, sample preparation (e.g. tissues, cells) and handling, digestion protocols, interpretation of MRM and DIA spectra so they can optimally utilize the advanced technologies available in the Center.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Center Core Grants (P30)
Project #
5P30DA018343-15
Application #
9700628
Study Section
Special Emphasis Panel (ZDA1)
Project Start
Project End
Budget Start
2019-06-01
Budget End
2021-05-31
Support Year
15
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Yale University
Department
Type
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
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