Ltq orbitrap mass spectrometry

The entire LC-MS/MS procedure was performed at Yonsei Proteome Research Center (Seoul, South Korea). Briefly, LC was performed with an Easy n-LC 1000 system (Thermo Fisher Scientific, Rockford, IL, USA). A C18-nanobore column (150 mm × 0.1 mm, 3-μm pore size, Agilent) was used for peptide separation. LTQ-Orbitrap mass spectrometry (Thermo Fisher, San Jose, CA, USA) was used to identify and quantify peptides. Xcalibur (version 2.1, Thermo Fisher Scientific, Waltham, MA, USA) was used to generate peak lists. The peak lists were examined by searching the National Center for Biotechnology Information database using the MASCOT search engine (http://www.matrixscience.com, Matrix Science, Boston, MA, USA). The acquired data were compared to the whole database with search parameters set as follows: enzyme, trypsin; allowance of up to one missed cleavage peptide; mass tolerance ±0.5 Da and MS/MS tolerance ±0.5 Da; modifications of methionine oxidation and cysteine carbamidomethylation when appropriate, with auto hits allowed and only significant hits to be reported. The proteins were identified on the basis of two or more peptides whose ion scores exceeded the threshold, P < 0.05, which indicated the 95% confidence level for these matched peptides.

The initial analysis of the main flavonoids in PFF was conducted as previously described by Cho et al. [29 (link)] and Chao et al. [30 (link)]. Briefly, each purified sample was dissolved in methanol (1 mg/mL), then sample films were dotted on potassium bromide crystal tablets by the capillary. The compressed tablets were scanned by Fourier transform infrared spectrometry (Bruker, Rheinstetten, Germany) in a frequency range of 4000–400 cm⁻¹, and the infrared absorption spectra of each purified sample were recorded. Simultaneously, they were also diluted and scanned from 200 to 600 nm using UV-Vis spectrophotometry (Thermo Fisher Scientific, Waltham, MA, USA). Furthermore, liquid chromatography–mass spectrometry (LC-MS) analysis was performed using LTQ Orbitrap mass spectrometry (Thermo Fisher Scientific, Waltham, MA, USA).

Total protein extracted from N. benthamiana leaves infected with BBWV2-53/37-Flag was separated by 10% SDS-PAGE. A gel fragment corresponding to the molecular weight of VP53-Flag (~53 kDa) was excised from the gel and subjected to in-gel digestion using trypsin followed by LC-MS/MS analysis. The entire LC-MS/MS analysis was performed at Yonsei Proteome Research Center (Seoul, South Korea). Briefly, LC was performed using an Easy n-LC 1000 system (Thermo Fisher Scientific, USA). Peptide separation was performed using a C18-nanobore column (150 mm × 0.1 mm, 3-μm pore size, Agilent, USA). LTQ-Orbitrap mass spectrometry (Thermo Fisher, USA) was used to identify and quantify peptides. Xcalibur (version 2.1, Thermo Fisher Scientific, USA) was used to generate peak lists. The peak lists were identified by searching the National Center for Biotechnology Information database using the MASCOT search engine (http://www.matrixscience.com, Matrix Science, USA).