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

www.nature.com/scientificreports OPEN Received: 12 October 2018 Accepted: 20 March 2019 Published: xx xx xxxx Synthetic standard aided quantification and structural characterization of amyloid-beta glycopeptides enriched from cerebrospinal fluid of Alzheimer’s disease patients Jonas Nilsson 1,2, Gunnar Brinkmalm 3, Sherif Ramadan 4,5, Lisa Gilborne1, Fredrik Noborn1,2, Kaj Blennow3,6, Anders Wallin3, Johan Svensson7, Mohamed A. Abo-Riya5, Xuefei Huang4 & Göran Larson 1,2 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 Tyr10 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. The amyloid precursor protein (APP) is a type I transmembrane glycoprotein that undergoes proteolytic processing along two major pathways: the amyloidogenic and the non-amyloidogenic pathway1. In the amyloidogenic pathway, two enzymes (β- and γ-secretase) cleave APP into several Aβ peptide variants. The 42-amino acid long Aβ peptide, Aβ1-42, is considered to have a direct link to Alzheimer’s disease (AD) since it is a major constituent 1 Laboratory of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden. 2Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. 3Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. 4Departments of Chemistry and Biomedical Engineering, Institute for Quantitative Health, Science and Engineering, Michigan State University, East Lansing, MI, USA. 5Chemistry Department, Faculty of Science, Benha University, Benha, Qaliobiya, 13518, Egypt. 6Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden. 7Department of Internal Medicine, Institute of Medicine Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. Correspondence and requests for materials should be addressed to X.H. (email: ) or G.L. (email: goran. ) Scientific Reports | (2019) 9:5522 | https://doi.org/10.1038/s41598-019-41897-5 1 www.nature.com/scientificreports/ www.nature.com/scientificreports of amyloid plaques and has been shown to induce neuropathological changes, such as neuronal and synaptic loss2. There is also a range of presumably less toxic Aβ peptides released during APP processing, for instance Aβ1-38, 1-39 and 1-40. In the non-amyloidogenic pathway, APP is cleaved by α-secretase in the middle portion of the Aβ sequence resulting in shorter peptides, which are thought to protect from amyloid deposition in the brain. In general, the proteolytic destiny and lifetime of proteins are not only governed by the availability and specificity of proteases but also by the presence of glycans at specific glycosites, which may affect the cleavage efficiency of nearby proteolytic sites. Such an effect has been reported to be biologically valid for the shedding of other transmembrane glycoproteins where the cleavage site is in close proximity to the membrane3. 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 spectrometry4–7. 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. This was the first known example of mammalian Tyr glycosylation of an extracellular protein, but several examples have since then been identified indicating that Tyr glycosylation is widely occurring, although scarce compared to Ser/Thr glycosylations10,11. 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 thus become a candidate biomarker for AD8. The Tyr O-glycosylation of Aβ were mainly composed of NeuAcHex(NeuAc)HexNAc-O-Tyr and NeuAcNeuAcHex(NeuAc)HexNAc-O-Tyr structures (SA2 and SA3, see Fig. 2B). For mucin-type O-glycosylation of Ser and Thr, the HexNAc-O- core residue is composed of GalNAcα1-O-, but given the novelty of the Tyr attachment residue, the precise structure of the HexHexNAc-O-Tyr structure was initially uncertain. Therefore, we synthesized GalNAcα1-O-Tyr and GalNAcβ1-O-Tyr derivatized Aβ1-15 glycopeptides and found that the α-form shared diagnostic fragment similarities in their CID-based MS/MS analyses, thus assigning the native structure to be α-linked12, similar to the mucin-type O-glycosylation. Additionally, we presumed that the hexose was a galactose (Gal) since this is the classical hexose found linked to GalNAc and also the only known hexose to which N-acetylneuraminic acid (NeuAc) is linked in mammalian glycoproteins. Recently, we showed tha (...truncated)