Obesity now surpasses smoking as a negative health risk factor. Epidemiologic and genetic analyses have demonstrated excess weight is a causal factor for diabetes, coronary artery disease, hypertension, dyslipidemia, heart failure, multiple types of cancer, knee osteoarthritis, and other co-morbidities and complications. Expanded understanding of the pathophysiology of obesity is critically needed to reduce its impact on human health. We are proposing practical and impactful biomarker tools that allow researchers to decipher circulating obesity proteomic signatures from the complex background of serum or plasma. We will apply liquid chromatography-mass spectrometry (LC-MS) techniques using top-down (Aim 1) and bottom-up (Aim 2) workflows, which can be scaled to include other potential candidate markers (Aim 3). Aim 1 focuses on obesity-related peptide hormone processing using top-down targeted LC-MS multiplexed assays. We hypothesize that dysregulated processing of small molecular weight (MW) bioactive hormones is involved in obesity and alters their circulating concentrations. We have two approaches to quantify the processing of key obesity related bioactive peptides: i) top-down LC-MS analysis of endogenous peptide fragments generated during processing (initially) for insulin, IGF-1, glucagon, GLP-1, GIP, ghrelin, motilin, obestatin, and somastatin, with secondary targets approached later; and ii) a novel method to assess plasma proteolytic enzyme activity and the simultaneous generation/loss of peptide fragments focusing specifically on GLP-11-41, GIP1-42, proghrelin and somatostatin-28 and the production of low MW peptides generated from these. In Aim 2, we hypothesize that regulation of adipocytes, liver, gut, and other organs impact obesity via circulating proteins and vice versa. To address this, we will develop a bottom-up targeted multiplex assay for precise quantification of leptin, adiponectin, gastrin (and modified forms), resistin, and secretin, which are all involved in obesity. Additionally, we will include protein markers representing inflammation (CRP), cardiac disease risk (ApoB/ApoA1), diabetes (hemoglobin A1C), and kidney function (cystatin C) for the assessment of obesity as it relates to comorbid conditions. In Aim 3 we will produce additional multiplex assays associated with obesity assigned in consult with our experts and NIDDK. For each assay developed we will demonstrate analytical validity (following CPTAC guidelines) by appropriately evaluating performance characteristics (e.g., precision, accuracy, linearity, sensitivity, specificity, etc.). We will additionally work to increase transferability and access of the assays to the broader obesity-research community. In summary, the development of robust protein biomarker assays that represent broad obesity pathophysiology will assist researchers in further understanding obesity regulatory pathways, developing new treatments, monitoring the effects of lifestyle and genetics on individual phenotypes, and evaluate the potential for future clinical diagnostic biomarker utility.