Templating S100A9 amyloids on Ab fibrillar surfaces revealed by charge detection mass spectrometry, microscopy, kinetic and microfluidic analyses† Jonathan Pansieri, ‡ a Igor A. Iashchishyn, ‡ a Hussein Fakhouri, b Lucija Ostoji ´ c, a Mantas Malisauskas, a Greta Musteikyte, c Vytautas Smirnovas, c Matthias M. Schneider, d Tom Scheidt, d Catherine K. Xu, d Georg Meisl, d Tuomas P. J. Knowles, de Ehud Gazit, af Rodolphe Antoine b and Ludmilla A. Morozova-Roche * a The mechanism of amyloid co-aggregation and its nucleation process are not fully understood in spite of extensive studies. Deciphering the interactions between proinflammatory S100A9 protein and Ab 42 peptide in Alzheimer's disease is fundamental since inflammation plays a central role in the disease onset. Here we use innovative charge detection mass spectrometry (CDMS) together with biophysical techniques to provide mechanistic insight into the co-aggregation process and differentiate amyloid complexes at a single particle level. Combination of mass and charge distributions of amyloids together with reconstruction of the differences between them and detailed microscopy reveals that co-aggregation involves templating of S100A9 fibrils on the surface of Ab 42 amyloids. Kinetic analysis further corroborates that the surfaces available for the Ab 42 secondary nucleation are diminished due to the coating by S100A9 amyloids, while the binding of S100A9 to Ab 42 fibrils is validated by a microfluidic assay. We demonstrate that synergy between CDMS, microscopy, kinetic and microfluidic analyses opens new directions in interdisciplinary research. Introduction In spite of the key clinical importance of amyloid formation, the mechanisms of co-aggregation of different amyloid species remain elusive. Amyloid formation is a widespread phenom- enon routed in the generic property of polypeptide chains to self-assemble into cross-b-sheet containing superstructures 1,2 and manifested in numerous amyloid diseases 3,4 and functional amyloids. 5,6 Comorbidity of these diseases was reported to be linked to the co-aggregation of amyloidogenic proteins. 7,8 In Alzheimer's disease (AD), the amyloid-neuroinammatory cascade is manifested in co-aggregation of Ab with proin- ammatory S100A9 protein, which leads to intracellular and extracellular amyloid assembly, amyloid plaque depositions and cellular toxicity. 9 S100A9 co-aggregates with Ab also in traumatic brain injury, which is considered as a potential precursor state for AD. 10 The amyloid self-assembly of Ab was well described by the involvement of secondary nucleation pathways promoted by Ab amyloid surface. 11 In contrast, S100A9 undergoes nucleation-dependent autocatalytic amyloid growth. 12 There is a genuine unmet need to understand the architecture and mechanism of self-assembly leading to the formation of hetero-aggregates composed of various amyloid polypeptides. Since amyloids formed by individual polypeptides are highly polymorphic, 13–15 their co-aggregates add up to the complexity and heterogeneity of amyloid mixture. This complex problem has been addressed previously in a number of studies – the co-assembly of Ab 40 and Ab 42 was investigated by global kinetic analysis 16 and FTIR, 17 self-sorted supramolecular nano- brils by in situ real-time imaging, 18 co-aggregates of wild-type a-synuclein with the familial mutant variant by dual-colour scanning for intensely uorescent targets 19 and Ab 42 peptide a Department of Medical Biochemistry and Biophysics, Ume˚ a University, SE-90187 Ume˚ a, Sweden. E-mail: ludmilla.morozova-roche@umu.se b Institut Lumi` ere Mati` ere, UMR 5306, Universit´ e Claude Bernard Lyon 1, CNRS, Univ Lyon, F-69100 Villeurbanne, France c Institute of Biotechnology, Life Sciences Center, Vilnius University, Vilnius, Lithuania d Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Lenseld Road, Cambridge CB2 1EW, UK e Cavendish Laboratory, Department of Physics, University of Cambridge, JJ Thompson Ave, CB3 0HE Cambridge, UK f School of Molecular Cell Biology and Biotechnology, Tel Aviv University, Tel Aviv 69978, Israel † Electronic supplementary information (ESI) available: Experimental and computational details, 10 supplementary gures and 1 table. See DOI: 10.1039/c9sc05905a ‡ Authors with equal contribution. Cite this: Chem. Sci. , 2020, 11, 7031 All publication charges for this article have been paid for by the Royal Society of Chemistry Received 21st November 2019 Accepted 16th June 2020 DOI: 10.1039/c9sc05905a rsc.li/chemical-science This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 7031–7039 | 7031 Chemical Science EDGE ARTICLE Open Access Article. Published on 17 June 2020. Downloaded on 8/31/2021 11:22:13 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue