The goal of this application is to identify candidate biomarkers which, in combination, may constitute a lingering biosignature that is informative of cocaine abuse history over a protracted period of time. The neurochemical and behavioral manifestations of chronic cocaine abuse are known to endure for a considerable duration, and given the role of neuropeptides and other secreted proteins in compulsive behaviors, drug abuse, and addiction, we hypothesize that the proteome of biophases that can be obtained, by minimally-invasive means, may contain families of biomarkers that reflect the well-established neurochemical alterations and constitute a peripherally-accessable cocaine abuse biosignature. Our approach is to apply recent inter-related analytical advances in sample processing, nano-scale liquid chromatography (nano-LC), and mass spectrometry (MS) that enhance the suitability of nano-LC/MS approaches for the analysis of highly complex proteomic samples such as tissues or bodily fluids. These advances provide: (i) highly quantitative recovery of tissue or sample proteins, (ii) highly reproducible, selective, and sensitive expression profiling of proteins, (iii) quantification of significantly greater numbers of proteins, and (iv) highly sensitive, multiplexed quantification of specific proteins of interest and (v) their PTM status. In the context of a well-controlled and validated rat model of cocaine abuse and withdrawal, samples will be obtained from naive- and drug-withdrawn animals both before and during a relapse of cocaine administration. Proteomes of select neural centers and blood will be contrasted to assemble lists of abuse-selective biomarker candidates, and their importance will be ranked by the commonality among replicates, the magnitude of change, and bioinformatic criteria relating them plausibly as drug-abuse responsive. In the second phase, the top-prioritized candidates will be confirmed and absolute quantification will be performed;matched plasma samples will be interrogated for these candidate biomarkers to identify promising constituents of a biosignature. Finally, using a rat model of addiction involving self- administration, we will attempt to refine biosignature candidates in terms of use/abuse vs. dependence/addiction, to determinate the exclusivity of these markers for drug addiction. By employing these linked analytical technologies and a well-validated animal model, we aim to investigate, identify, and validate potential biosignatures of cocaine abuse and relapse, based upon differential comparison of proteins in peripheral blood samples, which could aid in diagnosis, evaluation of therapy, and monitoring of recovery. PUBLIC HEALTH RELEVANCE: Chronic use of drugs of abuse represents a scourge to users and society. The ability to obtain information about use patterns beyond the period of time in which drug or metabolites are detectable in blood would have broad applications in diagnosis, estimation of prognosis, personalization of treatment, evaluation of drug effects, and detection of relapse. We propose to combine novel analytical advances in liquid chromatography, mass spectrometry (LC/MS), and sample preparation, along with a series of validated animal models, to interrogate samples that are obtainable by minimally-invasive means, such as blood, to achieve the elusive goal of identifying families of persistent biomarkers, comprising a biosignature, of chronic cocaine abuse and addiction.