In-Depth Site-Specific O‑Glycosylation Analysis of Glycoproteins and Endogenous Peptides in Cerebrospinal Fluid (CSF) from Healthy Individuals, Mild Cognitive Impairment (MCI), and Alzheimer’s Disease (AD) Patients

Site-specific
O-glycoproteome mapping in complex biological systems
provides a molecular basis for understanding the structure–function
relationships of glycoproteins and their roles in physiological and
pathological processes. Previous O-glycoproteome analysis in cerebrospinal
fluid (CSF) focused on sialylated glycoforms, while missing information
on other glycosylation types. In order to achieve an unbiased O-glycosylation
profile, we developed an integrated strategy combining universal boronic
acid enrichment, high-pH fractionation, and electron-transfer and
higher-energy collision dissociation (EThcD) for enhanced intact O-glycopeptide
analysis. We applied this strategy to analyze the O-glycoproteome
in CSF, resulting in the identification of 308 O-glycopeptides from
110 O-glycoproteins, covering both sialylated and nonsialylated glycoforms.
To our knowledge, this is the largest data set of O-glycoproteins
and O-glycosites reported for CSF to date. We also developed a peptidomics
workflow that utilized the EThcD and a three-step database searching
strategy for comprehensive PTM analysis of endogenous peptides, including
N-glycosylation, O-glycosylation, and other common peptide PTMs. Interestingly,
among the 1411 endogenous peptides identified, 89 were O-glycosylated,
and only one N-glycosylated peptide was found, indicating that CSF
endogenous peptides were predominantly O-glycosylated. Analyses of
the O-glycoproteome and endogenous peptidome PTMs were also conducted
in the CSF of MCI and AD patients to provide a landscape of glycosylation
patterns in different disease states. Our results showed a decreasing
trend in fucosylation and an increasing trend of endogenous peptide
O-glycosylation, which may play an important role in AD progression.