Q exactive hf benchtop orbitrap mass spectrometer

Isolated peptides were desalted using SDB-RPS StageTips as it was described earlier (27 (link)). LC-MS/MS analysis was performed using the Q Exactive HF benchtop Orbitrap mass spectrometer (Thermo Fisher Scientific) which was coupled to the Ultimate 3000 Nano LC System (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). The HPLC system was configured in a trap-elute mode. Peptide solution were loaded on an Acclaim PepMap 100 (100 μm × 2 cm) trap column and separated on an Acclaim PepMap 100 (75 μm × 50 cm) column (both from Thermo Fisher Scientific). Correlation of MS/MS spectra with peptide sequences was made using PEAKS Studio 8.0 build 20160908 software (28 (link)). Peptide lists generated by the PEAKS Studio were searched against the Homo sapiens Uniprot FASTA database (154257 species, version July 2016) and with methionine oxidations and asparagine/glutamine deamidations as variable modifications. The false discovery rate (FDR) for peptide-spectrum matches was set to 0.01 and was determined by searching a reverse database. Peptide identification was performed with an allowed initial precursor mass deviation up to 10 ppm and an allowed fragment mass deviation of 0.05 Da.

Peptides were separated on Acclaim PepMap 100 C18 (75 μm × 50 cm; Thermo Fisher Scientific). Reverse‐phase chromatography was performed with an Ultimate 3000 Nano LC System (Thermo Fisher Scientific), which was coupled to the Q Exactive HF benchtop Orbitrap mass spectrometer (Thermo Fisher Scientific) by a nanoelectrospray source (Thermo Fisher Scientific). Peptides were analysed, identified, and quantified as described in Methods S1. Taking into account that iTRAQ quantification usually underestimates the amount of real fold changes between two samples (Ow et al., 2009), differential protein screening (determined by the ratio in the PVY‐infected samples and untreated control [mock‐inoculated] plants at 22 °C) was performed using a fold change ratio >1.20 (for up‐regulated DEPs) or <0.83 (for down‐regulated DEPs) (p < .05, one‐way analysis of variance).