In-depth site-specific analysis of N-glycoproteome in human cerebrospinal fluid (CSF) and glycosylation landscape changes in Alzheimer's disease (AD).

As the body fluid that directly interchanges with the extracellular fluid of central nervous system (CNS), cerebrospinal fluid (CSF) serves as a rich source for CNS-related disease biomarker discovery. Extensive proteome profiling has been conducted for CSF, but studies aimed at unraveling site-specific CSF N-glycoproteome are lacking. Initial efforts into site-specific N-glycoproteomics study in CSF yield limited coverage, hindering further experimental design of glycosylation-based disease biomarker discovery in CSF. In the present study, we have developed an N-glycoproteomic approach that combines enhanced N-glycopeptide sequential enrichment by hydrophilic interaction chromatography (HILIC) and boronic acid enrichment with electron transfer and higher-energy collision dissociation (EThcD) for large-scale intact N-glycopeptide analysis. The application of the developed approach to human CSF sample enabled identifications of a total of 2893 intact N-glycopeptides from 511 N-glycosites and 285 N-glycoproteins. To our knowledge, this is the largest site-specific N-glycoproteome dataset reported for CSF to date. Such dataset provides molecular basis for a better understanding of the structure-function relationships of glycoproteins and their roles in CNS-related physiological and pathological processes. As accumulating evidence suggests defects in glycosylation are involved in Alzheimer's disease (AD) pathogenesis, in the present study, a comparative in-depth N-glycoproteomic analysis was conducted for CSF samples from healthy control and AD patients, which yielded a comparable N-glycoproteome coverage but a distinct expression pattern for different categories of glycoforms, such as decreased fucosylation in AD CSF samples. Altered glycosylation patterns were detected for a number of N-glycoproteins including alpha-1-antichymotrypsin, ephrin-A3 and carnosinase CN1 etc., which serve as potentially interesting targets for further glycosylation-based AD study and may eventually lead to molecular elucidation of the role of glycosylation in AD progression.