Single‐molecule Analysis of Protein Glycosylation Patterns for Early Detection of Alzheimer’s Disease Biomarkers

Abstract

AbstractBackgroundIn recent years, it has become apparent that protein glycosylation plays a variety of roles in the development of Alzheimer’s disease (AD). For instance, characteristic glycosylation patterns of tau and APP have been found in AD patient samples, and O‐GlcNAcylation of alpha‐synuclein (aSyn) has been indicated as a therapeutic anti‐AD target. By extension, the presence of specific glycoforms in patient samples could constitute an important biomarker for early detection purposes (Zhou et al., 2023, Alzheimer’s Dement., https://doi.org/10.1002/alz.13024). However current state‐of‐the‐art methods using mass spectrometry (MS) typically detect proteins starting at millions of copies, thus they lack the sensitivity to perform detection in early disease stages. Moreover, they are prohibitively expensive for routine clinical analysis.MethodWe have developed an ultra‐sensitive protein analysis method that can be used to localize post‐translational modifications in full‐length proteins, including glycans, at single‐molecule resolution (Filius et al., 2021, Nano letters, https://doi.org/10.1021/acs.nanolett.1c00725). Using Förster resonance energy transfer (FRET), a fluorescence‐based distance measurement approach with sub‐nanometer resolution, our method can determine both presence and approximate location of glycans on the protein. Contrary to MS, it does not require complex sample preparation, such as enzymatic digestion or peptide enrichment. We validated this approach by detecting O‐GlcNAcylation on aSyn.ResultBased on measured fluorescence intensities and without using prior information on aSyn’s O‐GlcNAcylation tendencies, we deduced that molecules are doubly O‐GlcNAcylated. Using a calibration construct, we then inferred that the modified residues are in between residues 42 and 78. This is in accordance with MS measurements on bulk samples, which indicate that double glycosylation had taken place at Thr54 and Thr64 respectively. Our results demonstrate for the first time that O‐GlcNAc can be localized on full‐length proteins with single molecule sensitivity.ConclusionUsing our highly sensitive protein analysis approach, we have demonstrated the detection and location of glycans on full‐length aSyn at single‐molecule resolution. We believe that the high resolution of our method will allow for the detection of biomarkers in patient samples in early stages of disease. In future work, we will expand the proof‐of‐concept presented here for such usage by detecting AD‐associated tau and APP glycoforms.