The Yale/NIDA Neuroproteomics Center, which was founded 8/23/2004, brings exceptionally strong Yale programs in proteomics and signal transduction in the brain together with neuroscientists from nine other institutions across the U.S. to identify adaptive changes in protein signaling that occur in response to substances of abuse. Twenty-three faculty with established records of highly innovative research into the molecular actions of psychoactive addictive drugs, as well as of other basic aspects of neurobiology, will work together in a unique synergy with the Keck Foundation Biotechnology Laboratory to continually strengthen the Yale/NIDA Neuroproteomics Center. The main goal of the Center, whose theme is “Proteomics of Altered Signaling in Addiction”, is to use cutting edge proteomic technologies to analyze neuronal signal transduction mechanisms and the adaptive changes in these processes that occur in response to drugs of abuse. With Co-Directors Drs. Angus Nairn (Psychiatry) and Kenneth Williams (Mol. Biophys. & Biochem.) in the Administration Core, the Center includes Discovery Proteomics (DPC) and Targeted Proteomics (TPC) technology cores. Biophysical technologies from the DPC extend protein profiling analyses into the functional domain while lipid analyses from the DPC very positively leverage proteome level analyses to provide an increasingly biological systems level approach. A Bioinformatics and Biostatistics Core, which includes high performance computing and the Yale Protein Expression Database (YPED), provides essential support that positively leverages the value of each of the proteomic technology cores. The Pilot Research Project Core is a cornerstone in the Center’s efforts to encourage strong mentoring relationships that will help attract and train future outstanding scientists. A major goal of the Center’s pilot research project grants is to help enable investigators to acquire the preliminary data needed to bring novel and highly promising research ideas relevant to the Center's theme to the point where they can successfully compete for NIH and other grant support. In addition, the Center provides training and is constantly striving to improve existing and develop new proteomics technologies that can be applied to biological questions related to the actions of drugs of abuse.
Behavioral adaptations that accompany drug addiction are believed to result from both short and long-term adaptive changes in brain reward centers. Thus, exposure to drugs of abuse regulates intracellular signaling processes that alter gene expression, protein translation, and protein post-translational modifications. Repeated exposure to drugs of abuse leads to stable alterations in these signaling systems that are critical for the changes in brain chemistry and structure of the addicted brain. To date, molecular studies of drugs of abuse have elucidated some of the transcriptional changes that occur in the addicted brain. However, little is known about the effects of drugs of abuse on the neuronal proteome. The Center is, through its highly interdisciplinary and collaborative organization, bringing together faculty from across the country with complementary expertise to gain a far deeper insight into how drugs of abuse alter expression and post-translational modification of proteins on a global scale. Proteomics technologies that are being used include isobaric tags for relative and absolute quantitation (iTRAQ), label-free quantitation (LFQ) LC/MS, stable isotope labeling by amino acids in cell culture (SILAC), multi-dimensional protein identification technology (MudPIT), phosphoproteome enrichment and profiling, and targeted quantitation of pre-selected proteins and their post-translational modifications using multiple reaction monitoring (MRM) and Parallel Reaction Monitoring (PRM). The Center’s research goals include analysis of the actions of cannabis, cocaine, nicotine, and opioids on these intracellular signaling pathways in brain reward areas and development of methods that enable proteomic analysis of the molecular changes that are occurring in the single types of neurons that define the circuits that underlie the actions and addictive properties of drugs of abuse. To begin to address the challenge of immense cellular heterogeneity in neurological samples several new approaches will be introduced into the DPC. These will include laser capture (LCM), fluorescence-based (FACS and FANS), or immuno-affinity methods, in conjunction with transgenic and viral tools to isolate cell type- and organelle-specific proteomes. A central feature of these methods is the use of unique transgenic mouse lines that express fluorescent proteins as affinity tags on specific proteins in specific neuronal cell types. Targeted and data-independent MS analyses of signaling proteins implicated in the actions of drugs of abuse will be used to analyze the impact of substance abuse on the neuroproteome. The DPC will also employ several novel approaches for enrichment of low abundance proteins and peptides and their post-translationally modified (PTMs) proteoforms including hexapeptide combinatorial ligand libraries (CPLL), “DigDeAPr” differential trypsin digestion (Fonslow et al, 2013), and denaturing size exclusion (SEC) and hydrophilic interaction (HILIC) chromatography to enrich for membrane proteins. In addition, motif-based enrichment, “Top-Down” MS/MS, and other approaches will be used to study protein post-translational modifications. A major initiative led by the Bioinformatics and Biostatistics Core will be to develop novel methods for deep integration of genomic, transcriptomic, and proteomic data with brain region and cell type-specificity.