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

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.

Towards structure-focused glycoproteomics

“…( m/z 138 + m/z 168) and ( m/z 126 + m/z 144), referred as the GlcNAc/GalNAc ratio, generated using ion trap CID- or HCD-MS/MS, is reportedly able to discriminate between GlcNAc- and GalNAc-containing N - and O -glycopeptides [ 226 , 227 ]. In support, multiple glycoproteomics studies have applied this approach to distinguish extended α-linked GalNAc and β-linked GlcNAc O -glycopeptides [ 36 , 131 , 236 , 237 ], glycopeptides carrying either a single O -GlcNAc or O -GalNAc residue [ 238 ] or, more crudely, to simply classify O - and N -glycan types on peptides [ 41 , 197 ]. Furthermore, α2,3- and α2,6-sialyl linkage information was obtained by determining the signal intensity ratio of the HCD-MS/MS-derived oxonium ions specific for LacNAc ( m/z 204 + m/z 366) and NeuAc ( m/z 274 + m/z 292) following an elaborate signal normalisation process that considers both the glycan composition and the LacNAc structure (Galβ1,3GalNAc or Galβ1,4GlcNAc) [ 228 ].…”

Synthetic amyloid beta does not induce a robust transcriptional response in innate immune cell culture systems

“…First, the synthetic Aβ used here has known differences in structure, heterogeneity and toxicity compared with the Aβ found in human AD brains. Aβ isolated from AD patients comprises a complex mix of Aβ peptides with heterogeneous lengths and posttranslational modifications, 27,[56][57][58][59][60][61][62] . The use of Aβ preparations extracted from postmortem human brains using previously published extraction protocols 63 could give more insight into this hypothesis in future studies.…”

Progress of proteomics‐driven precision medicine: From a glycosylation view

“…Absolute quantitation, which can acquire the information of accurate concentrations of targeted glycopeptides, is crucial in many cases, such as the discovery of biomarkers. Some research groups have achieved accurate quantitation by successfully synthesizing isotope‐labeled standards 60,61 . Recently, Wang et al developed a chemoenzymatic strategy to produce standard glycopeptides with various glycoforms including 36 IgG Fc glycopeptides 62 .…”