The Neuroproteomics Core of The Scripps Research Institute Alcohol Research Center (TSRI:ARC) will provide mass spectrometry based bioanalytical services to meet the specific needs of the Center at Large. Given the breadth of work to be conducted in this translational research center, it is understandable that not all personnel will have expertise in specialized areas of analytical chemistry. The first goal of the Core is to provide access to proteomic methods such as protein expression analysis, phosphoproteomics, protein interactions and biorthogonal chemistry for discovery of molecular mechanisms in animal models of alcohol drinking and dependence to Center investigators. Personalized consultation with investigators on issues related to experimental design, sample collection, sample handling, and data interpretation will be provided. The second goal of the Core is to advance methods to perform single neuron proteomic measurements in combination with electrophysiology measurements to study AUD. In all these experiments core personnel will work closely with ARC investigators to solve technical problems and assess and analyze the data.
Expected outcomes/impact. We will use mass spectrometry-based strategies to analyze the proteome in AUD models in rats and mice. Methods to assess proteomic alterations in precise regions of the brain will identify protein pathways disrupted by alcohol use and addiction and when possible these measurements will be combined with electrophysiology measurements to provide physiological context to protein changes. We will apply methods developed for single cell proteomics to neurons to distinguish the contributions of single neurons from those of collective circuits which will improve our understanding of the pathways perturbed in AUD.
Specific Aim 1
Proteomic analysis of protein and posttranslational modification analysis from treated and control brain tissue will be performed. Well established methods for the analysis of proteomes will be used to analyze brain tissue or tissue punches from select regions of brains using electrophysiology to guide sampling as needed. Global analysis of protein expression will be performed, and proteins newly synthesized in response to perturbations will be analyzed using bioorthogonal chemistry, on either a global or cell specific scale. Posttranslational modifications such as phosphorylation or acetylation can be measured from tissue punches.
Specific Aim 1a. Single cell proteomic methods will be used to measure protein changes in specific neuronal circuits in conjunction with patch clamp electrophysiology.
Specific Aim 2
Protein-protein interactions will be determined for key proteins involved in AUD. Affinity Purification Mass Spectrometry (APMS) will be used to pull down proteins and their interactors to identify regulatory elements, molecular pathways and to understand the protein networks involved physiological responses of AUD.
Significance and value to the ARC. The core will provide state of the art proteomic methods to discover molecular mechanisms of AUD. A unique aspect of these measurements will be an ability to guide proteomic analyses using electrophysiology and mass spectrometry-based proteomics to identify the molecular mechanisms that underlie changes in neural circuits. Single cell RNA-SEQ measures gene expression in neurons, but gene expression profiles fail to account for rates of protein synthesis, degradation, proteostasis, post translational modification, protein interactions and enzymatic activity, all of which are critical cellular functions accomplished by proteins. We have established methods to measure proteomes in tissue punches after electrophysiology measurements and these methods have been extended to measurements in single neurons. We will apply these methods to studies of AUD to measure proteomes, phosphoproteomes, protein interactions, and newly synthesized proteins.