Danielsen, Heidi Nolsøe

Dueholm, Morten Simonsen

Hansen, Susan Hove

Herbst, Florian-Alexander

Nielsen, Per Halkjær

English

    Aalborg UniversitetDirect identification of amyloids by label-free quantitative LC-MS combined with formicacid treatmentDanielsen, Heidi Nolsøe; Hansen, Susan Hove; Herbst, Florian-Alexander; Nielsen, PerHalkjær; Dueholm, Morten SimonsenPublication date:2016Document VersionPublisher's PDF, also known as Version of recordLink to publication from Aalborg UniversityCitation for published version (APA):Danielsen, H. N., Hansen, S. H., Herbst, F-A., Nielsen, P. H., & Dueholm, M. S. (2016). Direct identification ofamyloids by label-free quantitative LC-MS combined with formic acid treatment. Abstract from MEWE andBiofilms IWA Specialist Conference , Copenhagen, Denmark.General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            ? Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            ? You may not further distribute the material or use it for any profit-making activity or commercial gain            ? You may freely distribute the URL identifying the publication in the public portal ?Take down policyIf you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access tothe work immediately and investigate your claim.Downloaded from vbn.aau.dk on: November 30, 2020IWA Specialist Conference Microbial Ecology and Water Engineering 2016:  A joint conference of the MEWE and Biofilm specialist groups 1 Direct identification of amyloids in biofilms by label-free quantitative LC-MS combined with formic acid treatment H. N. Danielsen, S. H. Hansen, F.-A. Herbst, P. H. Nielsen, M. S. Dueholm* Aalborg University, Department of Chemistry and Bioscience, Fredrik Bajers Vej 7H, 9220 Aalborg, Denmark, *md@bio.aau.dk Keywords: Amyloid; label-free quantitative LC-MS; biofilm; extracellular polymeric substances Introduction Extracellular polymeric substances (EPS) determine the structural and functional properties of biofilms. Detailed knowledge of the key EPS components may therefore provide the theoretical background required for the development of new biofilm control strategies.   Amyloids represent a fascinating class of highly ordered fibrillar protein polymers, which are characterized by a cross- quaternary structure and the ability to self-assemble from their monomeric counterparts in a nucleation-dependent process (Fandrich, 2007). Although, amyloids were first discovered as malicious entities associated with protein misfolding disorders, it is now well-recognized that amyloids are also used for a wide range of beneficial purposes by organisms from all domains of life due to their exceptional properties (Dueholm et al., 2013a; Blanco et al., 2012).   Histological staining of biofilms with amyloid-specific dyes and amyloid conformation-specific antibodies has revealed that functional amyloids are widespread among microorganisms in biofilms that originate from nature and engineered systems (Larsen et al., 2007). They are therefore believed to play important roles in biofilm ecology. Despite their omnipresence in biofilms, few amyloids have been purified from biofilm-associated bacteria and investigated in depth. However, the few amyloids which have been studied so far have already provided an astonishing demonstration of how amyloids are exploited in biofilms with roles ranging from fimbriae and other cell appendages for adhesion and biofilm formation to structural components of cell envelopes and spore coats (Dueholm et al., 2013a; Blanco et al., 2012). They may even be used as cytotoxins (Maji et al., 2009) and as reservoirs for quorum-sensing signaling molecules (Seviour et al., 2015).   The identification of novel functional amyloids is key to understand the many roles of amyloids in biofilm structure and function. Isolation of amyloids is, unfortunately, not a straightforward task. The insolubility and extreme stability of most functional amyloids exclude them from traditional protein analyses such as SDS-PAGE (Dueholm et al., 2013b). Many functional amyloids are furthermore highly adhesive and therefore bind to pipette tips and other consumables. Large sample volumes and high productivity of amyloids are therefore required for successful purification. We here demonstrate a novel label-free proteomics technique that allow the direct identification of functional amyloid candidates based on the structural stability in the presence of increasing formic acid concentration.  Material and Methods The method is described in Figure 1.1. Biofilm samples are resuspended in buffer and lysed by freeze/thaw cycles or sonication. The samples are then divided into aliquots, which are lyophilized and treated with concentration of formic acid ranging from 0-100%. The samples are lyophilized again and subjected to a short run SDS-PAGE, yielding a narrow gel band containing all proteins. The protein bands are excised and in-gel digested with trypsin. They are then analysed by label-free quantitative LC-MS.  Amyloid proteins require a high concentration of formic acid to depolymerize and they are therefore only observed in samples treated with high concentration of formic acid. IWA Specialist Conference Microbial Ecology and Water Engineering 2016:  A joint conference of the MEWE and Biofilm specialist groups 1 Figure 1.1: Direct identification of amyloids by label-free quantitative LC-MS and formic acid treatment.  Results and Conclusions The new method was evaluated using the well-described functional amyloid model systems, Escherichia coli curli (Chapman et al., 2002) and Functional amyloids of Pseudomonas (Fap) (Dueholm et al., 2010). The method showed amyloid-specific profiles for the major (CsgA) and minor (CsgB) curli subunit as well as for the major (FapC) and minor (FapB) Fap subunits. No other proteins showed similar profiles. The method was also evaluated on the TasA system of Bacillus subtilis (Romero et al., 2010). We where here able to identify TasA, however, we also observed multiple false positives, which we believe originate from amyloid coated spores, which are first lysed during the formic acid treatment. Finally, we analysed a complex sample (biofilm from a wastewater treatment plant). Again, we were able to identify amyloid candidates. These will be the focus of future studies. The possibility to directly identify amyloid proteins in complex samples is a game changer in the field of functional amyloids. We believe that it will be used to uncover many new amyloid systems in the future, which will have implications in technical and medical settings.  References Blanco LP, Evans ML, Smith DR, Badtke MP, Chapman MR. (2012). Diversity, biogenesis and function of microbial amyloids. Trends Microbiol 20: 66–73. Chapman MR, Robinson LS, Pinkner JS, Roth R, Heuser J, Hammar M, et al. (2002). Role of Escherichia coli curli operons in directing amyloid fiber formation. Science (80- ) 295: 851–855. Dueholm MS, Nielsen PH, Chapman M, Otzen D. (2013a). Functional amyloids in bacteria. In: Amyloid fibrils and prefibrillar aggregates, Otzen, DE (ed), Wiley-VCH Verlag GmbH & Co. KGaA, pp 411–438. Dueholm MS, Petersen S V, Sønderkaer M, Larsen P, Christiansen G, Hein KL, et al. (2010). Functional amyloid in Pseudomonas. Mol Microbiol 77: 1009–1020. Dueholm MS, Søndergaard MT, Nilsson M, Christiansen G, Stensballe A, Overgaard MT, et al. (2013b). Expression of Fap amyloids in Pseudomonas aeruginosa, P. fluorescens, and P. putida results in aggregation and increased biofilm formation. Microbiologyopen 2: 365–382. Fandrich M. (2007). On the structural definition of amyloid fibrils and other polypeptide aggregates. Cell Mol Life Sci 64: 2066–2078. Larsen P, Nielsen JL, Dueholm MS, Wetzel R, Otzen D, Nielsen PH. (2007). Amyloid adhesins are abundant in natural biofilms. Environ Microbiol 9: 3077–3090. Maji SK, Perrin MH, Sawaya MR, Jessberger S, Vadodaria K, Rissman RA, et al. (2009). Functional amyloids as natural storage of peptide hormones in pituitary secretory granules. Science (80- ) 325: 328–332. Romero D, Aguilar C, Losick R, Kolter R. (2010). Amyloid fibers provide structural integrity to Bacillus subtilis biofilms. Proc Natl Acad Sci U S A 107: 2230–2234. Seviour T, Hansen SH, Yang L, Yau YH, Wang VB, Stenvang MR, et al. (2015). Functional Amyloids Keep Quorum-sensing Molecules in Check. J Biol Chem 290: 6457–6469. Cell lysis Freeze/thaw Treat with formic acid Bacterium  Amyloid DNA and RNA  Protein contamination SDS-PAGE In-gel digest Label-free Quantitative MS Bioinformatic analyses 100% formic acid 0% formic acid 

Direct identification of amyloids by label-free quantitative LC-MS combined with formic acid treatment

1Morten Simonsen DueholmFrom: Danmarks Mikrobiologiske Selskab <info@dmselskab.dk>Sent: Monday, 13 March 2017 14.06To: Morten Simonsen DueholmSubject: Symposium on Microbiological applications of mass spectrometry in clinical and environmental microbiology and general assembly in DMS on March 23 Newsletter 13 March 2017                                                                 www.dmselskab.dk   General Assembly & Symposium   On Thursday 23 March DMS will host its general assembly in Copenhagen followed by a symposium on the topic "Microbiological applications of mass spectrometry in clinical and environmental microbiology."   The general assembly and symposium takes place at Universitetsparken 15, Building 1, Auditorium A,2100 København Ø (see map).   PROGRAMME 12:00 DMS General Assembly (in Danish)   13:00 Introduction and welcome   13:10 Application of mass spectrometry for studying bacterial interactions  Jakob Herschend, Department of Biology, University of Copenhagen   13:40 Rapid MALDI-TOF identification of environmental bacteria Preben Nielsen, Novozymes A/S   14:10 Break – coffee and cake   14:40 MALDI-TOF MS – the view from the diagnostic laboratory Niels Nørskov-Lauritsen, Department of Clinical Medicine, Aarhus University   15:10 Direct identification of amyloids by label-free quantitative LC-MS combined with formic acid treatment Morten Simonsen Dueholm, Center for Microbial Communities, Aalborg University   15:40- Social networking - beer   On behalf of the Danish Microbiological Society   Carsten Suhr Jacobsen Chairman of DMS       Indkaldelse til ordinær generalforsamling (in Danish) DMS afholder generalforsamling den 23. marts i Auditorium A, Universitetsparken 15, 2100 Kbh Ø. Bestyrelsen har stillet to forslag til vedtægtsændringer, som ses af punkt 4.1 og 4.2.    Dagsorden 1. Valg af dirigent 2. Beretning om selskabets virksomhed i det forløbne år 3. Fremlæggelse af det reviderede regnskab til godkendelse 4. Behandling af eventuelle forslag fra bestyrelsen eller medlemmerne:  1. Der stilles forslag om, at punkter, der skal behandles ved generalforsamlingen, skal være bestyrelsen i hænde senest 2 uger før generalforsamlingen. 2. Endvidere stilles forslag om, at bestyrelsen kan optage op til 2 suppleanter. 5. Valg af bestyrelse 6. Valg af revisorer 7. Eventuelt   Save the date!   The 2017 DMS Congress takes place on 13 November. We look forward to welcoming you at Eigtveds Pakhus in Copenhagen.    

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Direct identification of amyloids by label-free quantitative LC-MS combined with formic acid treatment

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