ABSTRACT In amyloidosis and neurodegenerative disorders, proteins undergoing structural changes have high propensity to form insoluble deposits. These proteins convert from soluble functional states to highly organized fibrillary aggregates, termed amyloid fibrils, becoming toxic to neurons. As the most common form of dementia, Alzheimer’s disease (AD) is characterized as amyloid plaques composed of aggregated amyloid-β (Aβ) and neurofibrillary tangles formed by hyperphosphorylation and aggregation of tau. Given that the accumulation of protein aggregates is the common feature of progressive neurodegeneration diseases, we hypothesize that AD may be associated with misfolding of some unknown proteins in an independent or synergistic way with Aβ. The primary goal of this proposal is to explore the novel concept of “conformational biomarkers” and their utility for AD diagnosis and monitoring disease progression. We will develop cutting-edge mass spectrometry (MS)-based proteomics technologies to discover, characterize, and evaluate potential conformational biomarkers of AD using cerebrospinal fluid (CSF). Different from classical concentration-based disease biomarkers, conformational biomarkers reflect the changes in protein structure, directly revealing its function during the disease progression. We aim to address the following questions: (1) Will some unknown aggregation-prone proteins play important roles in Alzheimer’s disease? How to probe the structural transition of these misfolding proteins in complex proteomes? (2) How to evaluate the validity of putative conformational biomarkers resulting from non-targeted CSF analysis? (3) How to establish correlation between these putative protein conformational biomarkers and pathological features in Alzheimer’s disease? Can we use them to monitor disease progression or degree of cognitive impairment? To address these questions, we propose the following specific aims: Specific Aim 1 – To perform global proteomic and post-translational modification analysis of CSF samples obtained from age-matched cognitively- healthy subjects, asymptomatic preclinical individuals, and patients with mild cognitive impairment (MCI) and dementia, to probe protein structural transitions and dynamic changes in AD. Specific Aim 2 – To validate the conformational biomarkers with targeted quantitative proteomic approaches in CSF and plasma samples. Specific Aim 3 – To evaluate and establish the correlation between conformational biomarkers and pathological features including AD-related clinical, cognitive and neuroimaging measures. Collectively, our proposed experiments will be the first system-wide, large-scale analysis of protein structure changes in AD. The newly discovered and validated conformational biomarkers would be invaluable in exploring molecular mechanism and designing therapeutic targets in Alzheimer’s disease.