Custom main spectra (MSP) library for the identification of Phytophthora species by MALDI-TOF MS

MALDI-TOF MS is a relatively new technology which has revolutionized the way microorganisms are identified. The method relies on the reproducible detection of microbial protein mass patterns obtained from whole cells, cell lysates, or crude bacterial extracts. Microbial MALDI-TOF mass spectra can be regarded as snapshots of the protein composition of the individual strains studied. The protein biomarkers are typically high-abundance proteins with housekeeping functions, such as ribosomal proteins. For microbial identification mass spectra are analysed by pattern-matching approaches by which mass spectra from the bacteria under study are matching against validated databases of microbial reference spectra. The availability of high-quality and comprehensive spectral databases has been considered of paramount importance for attaining accurate identification results. Samples: Culture of 43 selected strains belonging to 26 species of Phytophthora and as an outgroup one species belonging to the genus Pythium were obtained form the Czech Collection of Phytopathogenic Oomycetes (CCPO), https://www.vukoz.cz/index.php/en/collections/collection-of-phytopathogenic-oomycetes-of-rilog Data collection and processing: Ethanol/Formic acid extraction sample preparation procedure according to Bruker instructions for use was used. Bruker Bacterial Test Standard (BTS, Bruker, DE) solution was used for calibration. Each spot was measured three times by MALDI-TOF MS Autoflex Speed (Bruker, DE) and flexControl 3.4 (Build 135) with a standard MALDI Biotyper method (MBT_FC.par). Ion source 1 was 19.38 kV, ion source 2 was 18.18 kV, and detection was set from 2 to 20 kDa. For each sample at least 24 spectra were measured, and each spectrum was collected by 2000 shots in 200 steps. For each measurement, the spectra were manually inspected by flexAnalysis 3.4 Compass 1.4 (Bruker Daltonics, DE) and then MSP spectra were processed by standard MALDI Biotyper MSP creation method using MALDI Biotyper Version 3.1 (Build 66).This work was supported by the internal grant agency of Czech University of Life Sciences Prague (CIGA) Project no. 20182019 and the European Regional Development Fund-Project Centre for the investigation of synthesis and transformation of nutritional substances in the food chain in interaction with potentially harmful substances of anthropogenic origin: comprehensive assessment of soil contamination risks for the quality of agricultural products (No. CZ.02.1.01/0.0/0.0/16_019/0000845). The authors are grateful for support and assistance provided by the Research Infrastructure METROFOOD-CZ supported by the Ministry of Education, Youth and Sports of the Czech Republic under Project No. LM2018100 and the Ministry of the Environment of the Czech Republic under Project No. IP–00027073–VUKOZ