Plant viruses are important pathogens that cause significant crop losses. A plant protein extraction protocol that combines crushing the tissue by a pestle in liquid nitrogen with subsequent crushing by a roller-ball crusher in urea solution, followed by RuBisCO depletion, reduction, alkylation, protein digestion, and ZipTip purification allowed us to substantially simplify the sample preparation by removing any other precipitation steps and to detect viral proteins from samples, even with less than 0.2 g of leaf tissue, by a medium resolution nanoLC-ESI-Q-TOF. The presence of capsid proteins or polyproteins of fourteen important viruses from seven different families (Geminiviridae, Luteoviridae, Bromoviridae, Caulimoviridae, Virgaviridae, Potyviridae, and Secoviridae) isolated from ten different economically important plant hosts was confirmed through many identified pathogen-specific peptides from a protein database of host proteins and potential pathogen proteins assembled separately for each host and based on existing online plant virus pathogen databases. The presented extraction protocol, combined with a medium resolution LC-MS/MS, represents a cost-efficient virus protein confirmation method that proved to be effective at identifying virus strains (as demonstrated for PPV, WDV) and distinct disease species of BYDV, as well as putative new viral protein sequences from single-plant-leaf tissue samples. Data are available via ProteomeXchange with identifier PXD022456.

Biomedical Research Center of the Slovak Academy of Sciences Institute of Virology Dúbravská cesta 9 845 05 Bratislava Slovakia

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Technická 5 166 28 Praha Czech Republic

Department of Computing and Control Engineering University of Chemistry and Technology Prague Technická 5 166 28 Praha Czech Republic

Department of Plant Protection Czech University of Life Sciences Kamýcká 129 165 00 Praha Czech Republic

Division of Crop Protection and Plant Health Crop Research Institute Drnovská 507 161 06 Praha Czech Republic

Faculty of Natural Sciences University of Ss Cyril and Methodius in Trnava Nám J Herdu 2 917 01 Trnava Slovakia

National Agricultural and Food Centre Research Institute of Plant Production 921 68 Piešťany Slovakia

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