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Protein thermostability is typically assessed by calorimetric or spectroscopic techniques that monitor different complementary signals during unfolding. The CalFitter webserver has already proved integral to deriving invaluable energy parameters by global data analysis. Here, we introduce CalFitter 2.0, which newly incorporates singular value decomposition (SVD) of multi-wavelength spectral datasets into the global fitting pipeline. Processed time- or temperature-evolved SVD components can now be fitted together with other experimental data types. Moreover, deconvoluted basis spectra provide spectral fingerprints of relevant macrostates populated during unfolding, which greatly enriches the information gains of the CalFitter output. The SVD analysis is fully automated in a highly interactive module, providing access to the results to users without any prior knowledge of the underlying mathematics. Additionally, a novel data uploading wizard has been implemented to facilitate rapid and easy uploading of multiple datasets. Together, the newly introduced changes significantly improve the user experience, making this software a unique, robust, and interactive platform for the analysis of protein thermal denaturation data. The webserver is freely accessible at https:\/\/loschmidt.chemi.muni.cz\/calfitter.<\/jats:p>","DOI":"10.1093\/nar\/gkac378","type":"journal-article","created":{"date-parts":[[2022,5,17]],"date-time":"2022-05-17T19:30:40Z","timestamp":1652815840000},"page":"W145-W151","source":"Crossref","is-referenced-by-count":15,"title":["CalFitter 2.0: Leveraging the power of singular value decomposition to analyse protein thermostability"],"prefix":"10.1093","volume":"50","author":[{"given":"Antonin","family":"Kunka","sequence":"first","affiliation":[{"name":"Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University ,\u00a0 Brno ,\u00a0 Czech Republic"},{"name":"International Centre for Clinical Research, St. Anne's University Hospital Brno ,\u00a0 Brno ,\u00a0 Czech Republic"}]},{"given":"David","family":"Lacko","sequence":"additional","affiliation":[{"name":"Faculty of Information Technology, Brno University of Technology ,\u00a0 Brno ,\u00a0 Czech Republic"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3139-3700","authenticated-orcid":false,"given":"Jan","family":"Stourac","sequence":"additional","affiliation":[{"name":"Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University ,\u00a0 Brno ,\u00a0 Czech Republic"},{"name":"International Centre for Clinical Research, St. Anne's University Hospital Brno ,\u00a0 Brno ,\u00a0 Czech Republic"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7848-8216","authenticated-orcid":false,"given":"Jiri","family":"Damborsky","sequence":"additional","affiliation":[{"name":"Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University ,\u00a0 Brno ,\u00a0 Czech Republic"},{"name":"International Centre for Clinical Research, St. Anne's University Hospital Brno ,\u00a0 Brno ,\u00a0 Czech Republic"}]},{"given":"Zbynek","family":"Prokop","sequence":"additional","affiliation":[{"name":"Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University ,\u00a0 Brno ,\u00a0 Czech Republic"},{"name":"International Centre for Clinical Research, St. Anne's University Hospital Brno ,\u00a0 Brno ,\u00a0 Czech Republic"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3659-4819","authenticated-orcid":false,"given":"Stanislav","family":"Mazurenko","sequence":"additional","affiliation":[{"name":"Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University ,\u00a0 Brno ,\u00a0 Czech Republic"},{"name":"International Centre for Clinical Research, St. Anne's University Hospital Brno ,\u00a0 Brno ,\u00a0 Czech Republic"}]}],"member":"286","published-online":{"date-parts":[[2022,5,17]]},"reference":[{"key":"2022070423594473300_B1","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1146\/annurev-biochem-061516-045115","article-title":"Protein misfolding, amyloid formation, and human disease: a summary of progress over the last decade","volume":"86","author":"Chiti","year":"2017","journal-title":"Annu. 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