Synthetic standard aided quantification and structural characterization of amyloid-beta glycopeptides enriched from cerebrospinal fluid of Alzheimer\u2019s disease patients

Jonas Nilsson,Lisa Gilborne,Göran Larson,Fredrik Noborn,Sherif Ramadan,Gunnar Brinkmalm,Xuefei Huang,Johan Svensson,Anders Wallin,Kaj Blennow,Mohamed A Abo-Riya

doi:10.1038/s41598-019-41897-5

Abstract

An early pathological hallmark of Alzheimer’s disease (AD) is amyloid-β (Aβ) deposits in the brain, which largely consist of up to 43 amino acids long Aβ peptides derived from the amyloid precursor protein (APP). We previously identified a series of sialylated Tyr-10 O-glycosylated Aβ peptides, 15–20 residues long, from human cerebrospinal fluid (CSF) and observed a relative increase of those in AD vs non-AD patients. We report here on the synthesis and use of an isotopically double-labeled Aβ1-15 glycopeptide, carrying the core 1 Galβ3GalNAcα1-O-Tyr-10 structure, to (1) identify by HCD LC-MS/MS the definite glycan core 1 structure of immunopurified and desialylated Aβ glycopeptides in human CSF and to (2) establish a LC-MS/MS quantification method for desialylated Aβ1-15 (and Aβ1-17) glycopeptides and to (3) compare the concentrations of these Aβ glycopeptides in CSF from 20 AD patients and 20 healthy controls. Although we unambiguously identified the core 1 structures and Tyr-10 attachment sites of the glycopeptides, we did not observe any quantitative differences, determined through both peptide and oxonium ion fragments, of the desialylated Aβ1-15 or Aβ1-17 glycopeptides between the AD and non-AD group. The new quantitative glycoproteomic approach described, using double-labeled glycopeptide standards, will undoubtedly facilitate future studies of glycopeptides as clinical biomarkers but should also embrace sialylated Aβ standards to reveal specific sialylation patterns of individual Aβ glycopeptides in AD patients and controls.

Highlights

10 attachment sites of the glycopeptides, we did not observe any quantitative differences, determined through both peptide and oxonium ion fragments, of the desialylated Aβ1-15 or Aβ1-17 glycopeptides between the Alzheimer’s disease (AD) and non-AD group
We previously developed a method based on liquid chromatography – tandem mass spectrometry (LC-MS/MS) with electron capture dissociation (ECD) and collision induced dissociation (CID) fragmentation techniques, which enabled the identification of several glycosylation sites of endogenous peptides originating from amyloid precursor protein (APP) in human cerebrospinal fluid (CSF)[8]
We previously found that the relative concentration of the natively occurring sialylated Tyr O-glycosylated Aβ peptides were increased in CSF samples from AD patients relative to those of non-AD controls (n = 6 + 7), indicating that the presence of a Tyr-10 glycan might have an impact on the metabolic pathway of APP and become a candidate biomarker for AD8

Summary

Introduction

10 attachment sites of the glycopeptides, we did not observe any quantitative differences, determined through both peptide and oxonium ion fragments, of the desialylated Aβ1-15 or Aβ1-17 glycopeptides between the AD and non-AD group. An increasingly important strategy for investigating glycoproteins is to undertake glycoproteomic analyses where glycopeptides, either natively occurring or produced by protease digestions, are structurally characterized by mass spectrometry[4,5,6,7] To this end, we previously developed a method based on liquid chromatography – tandem mass spectrometry (LC-MS/MS) with electron capture dissociation (ECD) and collision induced dissociation (CID) fragmentation techniques, which enabled the identification of several glycosylation sites of endogenous peptides originating from APP in human cerebrospinal fluid (CSF)[8]. In addition to O-glycosylations at several Ser/Thr residues of APP, we identified a series of glycopeptides uniquely O-glycosylated at Tyr-10 in shorter Aβ peptides, for instance Aβ1-15 and Aβ1-17 (DAEFRHDSGYEVHHQKL) We later expanded this glycosylation to additional mammals by the detection of similar Aβ1-15 glycopeptides in feline CSF9. When added to the CSF prior to the Aβ immunopurification (IP) protocol, the compound 1 served as an internal standard for LC-MS/MS based identification and quantitation of desialylated Aβ1-15 and Aβ1-17 O-glycopeptides

Methods

Results

Conclusion