Development of Blood-Based Phosphopeptide Biomarker for Diagnosis of Alzheimer’s Disease

Abstract

Neurodegenerative diseases, such as Alzheimer's disease (AD), are characterized by the large-scale death of brain cells, with AD accounting for approximately 70% of dementia cases. The hallmark features of AD include amyloid plaques and neurofibrillary tangles resulting from abnormal phosphorylation of tau protein. Current diagnostic methods, including MRI and PET scans, are limited by high costs and accessibility issues, increasing the interest in fluid-based biomarkers for AD diagnosis. Markers such as amyloid-beta 1-42, 1-40, and phosphorylated tau variants have been identified in cerebrospinal fluid (CSF), indicating potential for blood-based biomarkers. However, there is a scarcity of markers reflecting neurodegenerative changes in AD. Postmortem analyses have identified AD-specific phosphorylated proteomes beyond tau, highlighting the need for diverse biomarkers in bodily fluids. For quantifying phosphorylated peptide using immunoassays like Simoa, ELISA, and MSD, despite their high sensitivity, face limitations in simultaneously analyzing multiple biomarkers and are prone to false positives. In contrast, Immobilized Metal Ion Affinity Chromatography (IMAC) offers an antibody-free approach, enriching phosphorylated proteins effectively. Mass spectrometry-based quantification provides high sensitivity for low-concentration samples and enables multiplex analysis, making it particularly suitable for blood sample analysis in biomarker discovery. This study integrates Fe-IMAC enrichment and high-resolution Liquid Chromatography-Mass Spectrometry (LC-MS) to identify phosphorylated peptide biomarkers related to AD in plasma samples. Phosphorylated proteins, including tau (MAPT) and other AD brain-related proteins, were screened in amyloid-positive AD plasma. Quantitative differences in these candidates were evaluated across group-pooled plasma samples along the AD continuum. Finally, the potential of these phosphorylated peptides as biomarkers was validated by analyzing 40 individual plasma samples from an independent cohort within the AD continuum.