Nanolc 425 system

Peptides samples were analyzed by LC-MS/MS on AB SCIEX TRIPLE TOF 5600+ mass spectrometer and separated using NanoLC 425 system (Eksigent, Dublin, CA, USA) in a trap-elute configuration, including a trapping column C18, 5 μm, 300 μM ID, 25 mm long and analytical column Eksigent 5C18-CL-120, 300 μM ID, 150 mm length, connected to DuoSpray ion source (AB Sciex, Framingham, MA, USA). In the analysis, 5 μL of the peptides samples were loaded and cleaned on the trap column at 40 μL /min using Solvent A (0.1% formic acid) and eluted using a gradient from 5 to 80% Solvent B (0.1% formic acid in acetonitrile) over 105 min at 5 μL per min flow with column temperature of 55 °C. Each sample was run in triplicate. Ionization was achieved via electro spray ionization in positive ion mode with the ion spray voltage at 5500 V and source temperature at 200 °C. The TRIPLE TOF 5600+ was operated in DIA SWATH-MS mode, 64-variable-windows. The MS1 survey scan was acquired from 400–1250 m/z, MS2 spectra were acquired in high-sensitivity mode from 100–2000 m/z. The accumulation time was set to 0.25 s, and the ion scan was sampled at 45 ms time windows in high sensitivity mode, resulting in a final time of 5 s cycle. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [103 (link)] partner repository with the dataset identifier PXD040040.

The peptide samples were subjected to LC–MS/MS analysis using an AB SCIEX TRIPLE TOF 5600+ mass spectrometer and separated with a NanoLC 425 system (Eksigent, Toronto, Canada). The setup included an analytical column (Eksigent 5C18-CL-120, 300 μM ID, 150 mm length) connected to DuoSpray ion source (AB Sciex, Toronto, Canada). A volume of 5 μL of the peptide samples was loaded using Solvent A (0.1% formic acid) and eluted with a gradient from 5% to 90% Solvent B (0.1% formic acid in acetonitrile) over 90 min at a flow rate of 5 μL/min, with a column temperature of 55 °C. Each sample was analyzed in triplicate.
Electrospray ionization in positive ion mode was used, with an ion spray voltage of 5500 V and a source temperature of 200 °C. The TRIPLE TOF 5600+ was operated in DIA SWATH-MS mode with 64 variable windows. The MS1 survey scan ranged from 400 to 1250 m/z, while the MS2 spectra were acquired in high-sensitivity mode from 100 to 2000 m/z. The accumulation time was set to 0.049 s, and the ion scan was sampled in 55 ms time windows in high-sensitivity mode, resulting in a cycle time of 3.5 s.
The mass spectrometry proteomics data were deposited into the ProteomeXchange Consortium via the PRIDE [79 (link)] partner repository with the dataset identifier PXD043257. Reviewer account: Username: [email protected]; Password: M0KNo3iI.

Total protein extracts from fresh and dry plants (prepared as described above) were analyzed in triplicate using LC-MS/MS. For each sample, 45 µg of total proteins was loaded into SDS-PAGE until separation on the gel was reached. Stacked protein bands were excised, and the proteins were reduced, alkylated, and destained prior to digestion with trypsin enzyme (sequencing mass grade, Promega). The extracted peptides were analyzed with an Ekspert nanoLC™ 425 system (Eksigent, SCIEX, Redwood City, CA, USA) coupled to a TripleTOF^® 6600 MS (Sciex) [9 (link)]. Peptide identification was carried out by searches in a homemade Haberlea RNA-Seq contigs database (1605312 seq) and Boea RNA-Seq contigs database (68670 seq) [14 (link),19 (link),55 (link)] and the UniProt Viridiplantae database released in March 2019 (7072838 sequences), as recently described in [9 (link)]. The MS proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE [72 (link)] partner repository with the dataset identifier PXD030496.