A Strategy for Discovery and Verification of Candidate Biomarkers in Cerebrospinal Fluid of Preclinical Alzheimer’s Disease

Alzheimer’s disease (AD), a progressive neurodegenerative disease, is characterized by the accumulation of senile plaques, neurofibrillary tangles, and loss of synapses and neurons in the brain. The pathophysiological process of AD begins with a long asymptomatic phase, which provides a potential opportunity for early therapeutic intervention. Therefore, it is crucial to define putative biomarkers via reliable and validated methods for early diagnosis of AD. Here, we characterized candidate biomarkers by discovery proteomics analysis of cerebrospinal fluid (CSF), revealing that 732 and 704 proteins with more than one unique peptide were identified in healthy controls and preclinical AD patients, respectively. Among them, 79 and 98 proteins were significantly altered in preclinical AD for women and men, respectively, many of which have been demonstrated with consistent regulation pattern in patients with mild cognitive impairment or AD dementia. In-house developed 5-plex isotopic N,N-dimethyl leucine (iDiLeu) tags were further utilized to verify candidate biomarkers, neurosecretory protein VGF (VGF) and apolipoprotein E (apoE). By labeling peptide standards with different iDiLeu tags, a four-point internal calibration curve was constructed to allow for determination of the absolute amount of target analytes in CSF through a single liquid chromatography-mass spectrometry run.