Twenty-five faculty members with an established history of studies of the molecular actions of psychostimulants and psychotropic drugs, as well as of other basic aspects of neurobiology, are working with the W.M. Keck Foundation Biotechnology Resource Laboratory to continually strengthen the Neuroproteomics Center. The theme of the Center, "Proteomics of Altered Signaling in Addiction" brings exceptionally strong Yale programs in proteomics and signal transduction in the brain together with neuroscientists from nine institutions across the country to identify adaptive changes in protein signaling that occur in response to substances of abuse. In addition, the Center provides training in proteomics technologies, sponsors an active pilot projects program directed at acquiring 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, 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. The Center includes five cores: Protein Profiling and Analysis; Protein Post-translational Modifications; Targeted Proteomics; Bioinformatics and Biostatistics, and Administration.
The behavioral adaptations that accompany drug addiction are believed to result from both short and long term adaptive changes in brain reward centers. 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). One area of very active biotechnology research is the development and continued optimization of the workflows, visualization, and other tools that are being used to develop Targeted Proteome Assays (TPAs). Each 90 min TPA uses scheduled LC-MRM to determine the relative concentrations of 50-200 pre-selected protein biomarkers in tissue extracts or other biological samples (e.g., human urine) of interest. The first scheduled MRM assay that has been released is the Rat/Mouse Brain Assay (MBA/112) that interrogates the relative level of expression of 112 proteins from 24 different protein classes. Methods are also continually being improved to better apply these technologies to the brain and to studies of the actions of drugs of abuse. Specific goals of the research supported by the Center include analysis of the actions of opiates; the psychostimulants, cocaine and amphetamine; and nicotine on the neuroproteome. Other studies focus on the proteomic changes that occur both pre- and post-synaptically following the actions of drugs of abuse.