Abstract
As the body fluid that directly interchanges with the extracellular fluid of the 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 the analyses of human CSF samples 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 that 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.
Highlights
As the body fluid that directly interchanges with the extracellular fluid of the central nervous system (CNS), cerebrospinal fluid (CSF) serves as a rich source for CNSrelated disease biomarker discovery
We have developed an enhanced integrated large-scale site-specific glycoproteomic approach for in-depth CSF N-glycoproteome analysis, including sequential hydrophilic interaction liquid chromatography (HILIC) and boronic acid enrichment for improved N-glycopeptide coverage, intact N-glycopeptide characterization enabled by electron-transfer and higher-energy collision dissociation (EThcD), and automated false discovery rate (FDR)-based large-scale data analysis by Byonic
The current knowledge of site-specific protein glycoforms in CSF is quite limited due to the inherent structural complexities of intact glycopeptides and less well-developed analytical technologies compared with other posttranslational modification (PTM) studies
Summary
In Brief An exploratory glycosylationbased biomarker study has been conducted for in-depth mapping of an overall glycosylation landscape and site-specific alteration in glycoproteome collected from cerebrospinal fluids (CSF) in healthy control and Alzheimer’s disease (AD) subjects. We have developed an enhanced integrated large-scale site-specific glycoproteomic approach for in-depth CSF N-glycoproteome analysis, including sequential hydrophilic interaction liquid chromatography (HILIC) and boronic acid enrichment for improved N-glycopeptide coverage, intact N-glycopeptide characterization enabled by electron-transfer and higher-energy collision dissociation (EThcD), and automated false discovery rate (FDR)-based large-scale data analysis by Byonic. 2893 intact N-glycopeptides from 511 N-glycosites and 285 N-glycoproteins were identified in CSF, representing the largest reported site-specific CSF N-glycoproteome dataset so far This developed strategy was applied to N-glycoproteome analysis of CSF samples from AD patients, allowing us to conduct a glycosylation pattern comparison between healthy control and AD. This work lays a foundation for more in-depth investigation of the functional roles of these glycosylated proteins in AD progression
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