4.5. Isobaric Tags for Relative and Absolute Quantification (iTRAQ) Labeling and Mass Spectrometry Analysis

For iTRAQ labeling, protein samples containing 100 μg protein each were diluted using a buffer containing 500 mM TEAB, 0.1% SDS, and the same protease inhibitor as described above at the same concentration to reduce urea concentration to below 1 M. Then the protein sample was processed following the instructions of the 8-plex iTRAQ labeling kit [21]. Protein tryptic digestion was conducted using sequence grade modified trypsin (Promega, Madison, WI, USA) after incubation at 37 °C for 16 h. The control samples were labeled with tags 113, 115, 117, and 118 and the treated samples with 114, 116, 119, and 121. After combining all the labeled samples, unbound tags and SDS were removed through cation exchange cartridge (AB SCIEX). Salts and other impurities were removed using reverse-phase (RP) solid-phase extraction procedure involving 1-cm³, 50-mg Sep-Pak C18 cartridges following the manufacturer’s instructions (Waters; Milford, MA, USA). Peptides were eluted in 500 μL 50% (v/v) acetonitrile with 0.1% trifluoroacetic acid (TFA). Samples were dried at reduced pressure using a CentiVac Concentrator (labConco, Kansas City, MO, USA).

The peptide samples were subjected to a first dimension of high-pH Ultra Performance Liquid. Chromatography (UPLC) separation using an Acquity UPLC System (Waters) coupled with a robotic fraction collector (Probot; Dionex, Sunnyvale, CA, USA) [21]. One hundred micrograms of the multiplexed sample were injected and fractionated into 48 fractions in a 96-well plate. The 48 fractions were concatenated to yield 22 samples as follows: samples 1–4 and 45–48 were combined to yield two 2nd dimension fractions (samples 1–4 not analyzed in 2nd dimension); then for the remaining samples (5–44), every 20th fraction was combined. For the low-pH second dimension, low-pH RP chromatography was employed. Dried samples were reconstituted with 15 μL of 2% acetonitrile with 0.5% formic acid. Nano-LC separations of tryptic peptides were performed as described previously. The eluent from the analytical column was delivered to the LTQ-Orbitrap Elite (Thermo-Fisher Scientific, Waltham, MA, USA) via a “Plug and Play” nano ion source (CorSolutions LLC, Ithaca, NY, USA). The mass spectrometer was externally calibrated across the m/z range from 375–1800 with Ultramark 1621 for the Fourier transform (FT) mass analyzer, and individual runs were internally calibrated with the background polysiloxane ion at m/z 445.1200025 as a lock mass [24,90,91].

The Orbitrap Elite was operated in the positive ion mode with nanosource voltage set at 1.7 kV and capillary temperature at 250 °C. A parallel data-dependent acquisition (DDA) mode was used to obtain one MS survey scan with the FT mass analyzer, followed by isolation and fragmentation of the 15 most abundant, multiply-charged precursor ions with a threshold ion count higher than 50,000 in both the LTQ mass analyzer and the high energy collisionally induced dissociation (HCD)-based FT mass analyzer at a resolution of 15,000 full width at half maximum (FWHM) and m/z 400. MS survey scans were acquired with resolution set at 60,000 across the survey scan range (m/z 375–1800). Dynamic exclusion was utilized with repeat count set to 1 with a 40 s repeat duration; exclusion list size was set to 500, 20 s exclusion duration, and low and high exclusion mass widths set to 1.5. Fragmentation parameters were set with isolation width at 1.5 m/z, normalized collision energy at 37%, and activation Q at 0.25. Activation time for HCD analysis was 0.1 min. All data were acquired using XCalibur 2.1 (Thermo-Fisher Scientific) [18,24]. Proteins were identified using the MS data to query the switchgrass annotated database (http://www.phytozome.net/) via Mascot v2.3.02 (Matrix Sciences, Boston, MA, USA). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [92] partner repository with the dataset identifier PXD004675 and 10.6019/PXD004675.