(Version 20230306) Version 4 (20230306) of the RKI MALDI-ToF mass spectra database is the third update of the original database (version 20161027, https://doi.org/10.5281/zenodo.163517). The RKI Database v.4 now contains a total of 11055 MALDI-ToF mass spectra from 1599 microbial strains of highly pathogenic (i.e. biosafety level 3, BSL-3) bacteria such as Bacillus anthracis, Brucella melitensis, Yersinia pestis, Burkholderia mallei / pseudomallei and Francisella tularensis as well as a selection of spectra of their close and distant relatives. The database can be used as a reference for the diagnosis of BSL-3 bacteria using proprietary and free software packages for MALDI-ToF MS-based microbial identification. The spectral data are provided as a zip archive (zenodo db 230306.zip) containing the original mass spectra in their native data format (Bruker Daltonics). Please refer to the pdf file (230306-ZENODO-Metadata.pdf) for information on cultivation conditions, sample preparation and details of the spectra acquisition. Please do not try to print this document (>1600 pages!). Version 20230306 of the RKI database contains for the first time a file in btmsp format (230306_v4_RKI_DB_BSL3.btmsp). This file was generated using the MALDI Biotyper software (Bruker Daltonics) and contains a total of 1599 main spectra from the BSL-3 database in the proprietary data format of the MALDI Biotyper software. *.btmsp files can be imported and used for identification with this software solution. Note that the btmsp file available in database version 4 is broken and cannot be imported. Please refer to updated database versions (4.1, or 4.2) to download valid btmsp files. The pkf files (230306_ZENODO_30Peaks_0.75.pkf, 230306_ZENODO_45Peaks_0.75.pkf) represent two versions of the MS peak list data in a Matlab compatible format. The latter data can be imported into MicrobeMS, a free Matlab-based software solution developed at the RKI. MicrobeMS can be used for the identification of microorganisms by MALDI-ToF MS and is available at https://wiki-ms.microbe-ms.com. The RKI mass spectrometry database is updated regularly. The author would like to thank the following individuals for providing microbial strains and species or mass spectra thereof. Without their help, this work would not have been possible. Wolfgang Beyer - University of Hohenheim, Faculty of Agricultural Sciences, Stuttgart, Germany Guido Werner - Robert Koch-Institute, Nosocomial Pathogens and Antibiotic Resistances (FG13), Wernigerode, Germany Alejandra Bosch - CINDEFI, CONICET-CCT La Plata, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Buenos Aires, Argentina Michal Drevinek - National Institute for Nuclear, Biological and Chemical Protection, Milin, Czech Republic Roland Grunow, Daniela Jacob, Silke Klee, Susann Dupke and Holger Scholz - Robert Koch-Institute, Highly Pathogenic Microorganisms (ZBS2), Berlin, Germany Jörg Rau - Chemisches und Veterinäruntersuchungsamt Stuttgart, Fellbach, Germany Jens Jacob - Robert Koch-Institute, Hospital Hygiene, Infection Prevention and Control (FG14), Berlin, Germany Martin Mielke - Robert Koch-Institute, Department 1 - Infectious Diseases, Berlin, Germany Monika Ehling-Schulz - Functional Microbiology, Institute of Microbiology, University of Veterinary Medicine, Vienna, Austria Armand Paauw - Department of Medical Microbiology, CBRN protection, Universitair Medisch Centrum Utrecht, TNO, Rijswijk, The Netherlands Herbert Tomaso – Friedrich-Löffler-Institut (FLI), Federal Research Institute for Animal Health, Jena, Germany Gabriel Karner - Karner Düngerproduktion GmbH, Research & Development, Neulengbach, Austria Rainer Borriss - Institute of Marine Biotechnology e.V. (IMaB), Greifswald, Germany Le Thi Thanh Tam - Division of Plant Pathology and Phyto-Immunology, Plant Protection Research Institute, Hanoi, Socialist Republic of Vietnam Xuewen Gao - College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Integrated Management of Crop Diseases and Pests, Nanjing, People’s Republic of China

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