Nanolc ultra

Mass spectrometry measurements were performed on a LTQ OrbiTrap Velos mass spectrometer (Thermo Fisher) coupled to a NanoLC-ultra (Eksigent) using electrospray ionization. A 15-cm capillary column, which was heated to 50°C and packed with 15 cm C18 beads with a diameter of 3 μm and a pore size of 100 Å was used for LC separation. Peptides were loaded on the column with a flow rate of 300 nL/min for 20 min and eluted with a flow rate of 300 nL/min for 65 min by an increasing gradient from 3% acetonitrile to 50% acetonitrile. The FT OrbiTrap was used for obtaining full scans at a range of 300–1,700 mass/charge, followed by MS/MS scans of the 20 highest parent ions. Dynamic exclusion was enabled at a duration of 45 s.

The 1st dimension of peptide separation was conducted using an Eksigent nanoLC Ultra and ChiPLC-nanoflex (USA) in TrapElute configuration. Subsequently, the samples were loaded on a 200 μm × 0.5 mm column and eluted on an analytical 75 μm × 15 cm column (ChromXP C18-CL, 3 μm). A gradient formed by mobile phase A (2% acetonitrile, 0.1% formic acid) and mobile phase B (98% acetonitrile, 0.1% formic acid) was used to separate 2 and 5 μl of the sample at a 0.3 μl/min flow rate. The following gradient elution was used for peptide separation: 0 to 5% of mobile phase B in 1 min, 5 to 12% of mobile phase B in 15 min, 12 to 30% of mobile phase B in 114 min, 30 to 90% of mobile phase B in 2 min, 90% for 7 min, 90 to 5% in 3 min and finally held at 5% of mobile phase B for 13 min. The tandem MS analysis was performed using a 5600 TripleTOF system (AB SCIEX, USA) under Information Dependent Acquisition (IDA) mode. The mass range of 400–1800 m/z and accumulation times of 250 millisec per spectrum were chosen for precursor ion selection. MS/MS analysis was performed on the 20 most abundant precursors (accumulation time: 100 millisec) per cycle with 15 s dynamic exclusion. Recording of MS/MS was acquired under high sensitivity mode with rolling collision energy and adjusted capillary electrophoresis (CE) when iTRAQ reagent use was selected.

The total protein was extracted by a modified phenol extraction method as described by Desjardin et al. (2012) [67 (link)]. The protein concentration was determined using 2D-QUANT kit (Amersham Biosciences, Little Chalfont, UK), then it was digested by trypsin (Promega, Madison, WI, United States). Desalting and concentration of the peptides were implemented before the operation of HPLC by NanoLC-Ultra (Eksigent, Dublin, OH, USA) coupled with an MS analysis by Qexactive PLUS (Thermo Fisher, Waltham, MA, USA). LC-MS analyses were performed on 3 independent batches: dry seeds, 16h-imbibed seeds and 30 h-imbibed seeds.

Peptide separation was performed using an Eksigent nanoLC Ultra and ChiPLC-nanoflex (Eksigent, Dublin, CA) in TrapElute configuration. Samples were loaded on a 0.5 mm × 200 μm column and eluted on an analytical 15 cm × 75 μm column (ChromXP C18-CL, 3 μm). A gradient formed by mobile phase A (2% (v/v) ACN, 0.1% (v/v) formic acid) and mobile phase B (98% (v/v) ACN, 0.1% (v/v) formic acid) was used to separate 2 μL of the sample. The flow rate was set at 0.3 μL/min. The following gradient elution was used for peptide separation: 0 to 5% of mobile phase B in 1 min, 5 to 12% of mobile phase B in 19 min, 12 to 30% of mobile phase B in 40 min, 30 to 90% of mobile phase B in 2 min, 90 to 90% in 7 min, 90 to 5% in 3 min and finally held at 5% of mobile phase B for 13 min. The tandem MS analysis was performed using a TripleTOF 5600 system (SCIEX) under Information Dependent Mode. For precursor ions selection, the mass range of 400–1800 m/z and accumulation times of 250 ms per spectrum were chosen. MS/MS analysis was performed on the 20 most abundant precursors with accumulation time of 100 ms per cycle. The dynamic exclusion time was at 15 s. High sensitivity mode with rolling collision energy was used to acquire the MS/MS spectra.

The LC–MS/MS measurements (Gillet et al., 2012 ) (SWATH‐MS acquisition) were conducted with an Eksigent NanoLC Ultra and TripleTOF 4600 tandem‐mass spectrometer or an Eksigent nanoLC 415 and TripleTOF 6600 mass spectrometer (AB Sciex, U.S.A.). The trap column used for nanoLC was a 5.0 mm × 0.3 mm ODS column (L‐column2, CERI, Japan) and the separation column was a 12.5 cm × 75 μm capillary column packed with 3 μm C18‐silica particles (Nikkyo Technos, Japan). The detailed settings for the LC–MS/MS measurements are shown in Table S5. The SWATH acquisition was performed three times for each sample. Data analysis of the SWATH acquisition was performed using the DIA‐NN software with default settings (Demichev et al., 2020 (link)). The library for SWATH acquisition was obtained from the SWATH atlas {http://www.swathatlas.org, the original data are in Midha et al. (2020 (link))}. Only the proteins detected in all three measurements for both samples were used for the fold change calculation. The obtained protein intensities were averaged by using an in‐house R script. The p‐value was determined by Welch's t‐test and corrected by the Benjamini‐Hochberg method for multiple comparisons, using the “p.adjust” function in R (for Windows, version 4.1.2).