Isotopic N,N-dimethyl Leucine Tags for Absolute Quantification of Clusterin and Apolipoprotein E in Alzheimer's Disease

Alzheimer's disease (AD) is the most common form of dementia and one of the leading causes of death in the United States. In the past decades, extensive efforts have been devoted to biomarker discovery for early diagnosis and treatment of AD. Herein, this study aims to quantify clusterin (CLU) and apolipoprotein E (APOE) in blood samples from AD patients and evaluate these two proteins as potential biomarkers in AD diagnosis. In-house synthesized 5-plex isotopic N,N-dimethyl leucine (iDiLeu) tags were used to label target peptide standards at different concentrations to construct standard curves. Our study revealed that the levels of CLU and APOE exhibited clear differences in male vs. female AD groups but not in male vs. female non-AD groups. In contrast, the levels of serum CLU and APOE did not show statistically significant differences in the AD groups and non-AD groups. Principal component analysis (PCA) with CLU and APOE showed some separation between the AD and non-AD participants. Significance: Dissecting CLU and APOE heterogeneity in AD pathogenesis may therefore facilitate delineating the pathological relevance for sex-related pathways, leading to personalized medicine in the future. Collectively, this study introduces a cost-effective absolute quantitative proteomics strategy for target protein quantitation and lays the foundation for future investigation of CLU and APOE as potential biomarkers for AD. SIGNIFICANCE STATEMENT: As blood-based biomarkers for AD diagnosis are cost-effective and introduce less invasiveness, discovery and validation of biomarkers in the blood samples of AD patients have become a hot topic in Alzheimer's and dementia research. Thus far, amyloid β (Aβ), total-tau and phosphorylated tau (p-tau) in blood show great accuracy and specificity in diagnosis of AD. However, the underlying mechanism of AD pathology remains to be elusive and complex. Besides these well studied proteins, many other proteins, such as clusterin (CLU) and apolipoprotein E (APOE) have also been found to be related to AD development. It has been implicated that these two proteins are involved in Aβ clearance and deposition. In this study, we measure the absolute concentrations of these two proteins in blood and shed some light on the potential roles of CLU and APOE in AD pathology. Dissecting CLU and APOE heterogeneity in AD pathogenesis may therefore facilitate delineating the pathological relevance for specific pathways between different genders, leading to personalized medicine in the future. Collectively, this study introduces a cost-effective absolute quantitative proteomics strategy for target protein quantitation and lays the foundation for future investigation of CLU and APOE as potential biomarkers for AD.