PROJECT SUMMARY
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.
