Isobaric tags for relative and absolute quantification analysis

Total protein was isolated from 1 g of 2-week-old seedlings growing on solid Murashige and Skoog medium as described above following a previously described protocol (Vélez-Bermúdez et al. 2016). Protein treatment, protease digestion, and labeling prior to liquid chromatography (LC)–mass spectrometry (MS) (LC-MS) analysis were performed as described previously (Lan et al. 2011) with minor modifications. Protein concentrations were measured using a Pierce 660 nm protein Assay kit (Thermo Scientific). Proteins in 8 M urea, 50 mM Tris-HCl, pH 8.5, were reduced in 10 mM DTT for 1 hr at 37° and Cys were alkylated in 50 mM iodoacetamide at room temperature for 30 min in the dark. The protein solution was then diluted to contain 4 M urea with 50 mM Tris-Cl, pH 8.5; digested with 250 units/ml Benzonase (Sigma-Aldrich, St. Louis, MO) at room temperature for 2 hr; followed by Lys-C (Wako, Osaka, Japan) digestion [1:200 weight by weight (w/w)] at room temperature for 4 hr. The protein solution was further diluted to contain <2 M urea with 50 mM Tris-Cl, pH 8.0, and incubated with modified trypsin (1:50 w/w; Promega, Madison, WI) at 37° overnight. The digested solution was acidified with 10% trifluoroacetic acid, desalted using an Oasis HLB cartridge (Waters Associates, Milford, MA), and dried using a SpeedVac. For phosphoproteome analysis, prior to isobaric tags for relative and absolute quantification (iTRAQ) labeling, phosphopeptides were enriched from digested proteins (3.5 mg) using TiO₂ affinity chromatography (Titansphere Phos-TiO; GL Sciences) according to the method described by the vendor.

Dissolution of dried peptides in dissolution buffer and labeling with iTRAQ reagents (Multiplex kit; AB Sciex) were performed according to the manufacturer’s instructions. Tryptic peptides from two different samples, prp4ka-4 and the wild-type T line, were labeled with iTRAQ 116 and 117 reagents, respectively. For the phosphoproteome analysis, the prp4ka-4 and T-line samples were labeled with 114 and 115 reagents, respectively, after the phosphopeptides were enriched using TiO₂ affinity chromatography. The labeling reactions with iTRAQ reagents were incubated for 1 hr at room temperature. Following the reaction, solutions from different iTRAQ labels were combined and further fractionated on a strong cation-exchange (SCX) (PolySulfoethyl A, 4.6 × 200 mm, 5 µm, 200 Å; PolyLC) HPLC. The SCX chromatography was performed with an initial equilibrium buffer A containing 10 mM KH₂PO₄, 25% acetonitrile, pH 2.65, followed by a 0–15% buffer B (1 M KCl in buffer A, pH 2.65) gradient for 20 min, a 15–30% buffer B gradient for 10 min, a 30–50% buffer B gradient for 5 min, a 50–100% buffer B gradient for 1 min, and 100% buffer B for 5 min. The flow rate was 1 ml/min. The chromatography was recorded with absorbance 214 nm UV light. Fractions (0.5 min/fraction) were collected and pooled into 16 final fractions. Fractions were desalted using an Oasis HLB Cartridge (Waters) prior to LC-MS/MS analysis. Enriched phosphopeptide sample was fractionated using hydrophilic interaction liquid chromatography (HILIC) (TSKgel Amide-80 HR, 4.6 × 250 mm, 5 μm; Tosoh). The HILIC was performed in solvent containing acetonitrile and 0.1% trifluoroacetic acid with decreasing acetonitrile gradients: 90–85% in 5 min, 85–60% in 50 min, and 60–0% in 5 min, at flow rate of 0.5 ml/min. Ten fractions were collected for LC-MS/MS analysis.

Peptides in each fraction were redissolved in 0.1% formic acid and the LC-MS/MS was performed using the Q Exactive Mass Spectrometer equipped with the Dionex UltiMate 3000 RSLCnano LC system or the LTQ-Orbitrap Fusion Lumos Mass Spectrometer equipped with the EASY-nLC system. A C18 capillary column (Acclaim PepMap RSLC, 75 μm × 250 mm; Thermo Scientific) was used to separate peptides with a 120-min linear gradient (from 3 to 35%) of solvent B (0.1% formic acid in acetonitrile) at a flow rate of 300 nl/min on the LC system. The MS was operated in the data-dependent mode with the top 10 (Q Exactive) or top 20 (Fusion Lumos) ions (charge states ≥2) for MS/MS analysis following an MS survey scan for each acquisition cycle. The selected ions were isolated in the quadrupole and subsequently fragmented using higher-energy C-trap dissociation (HCD) and then analyzed in the Orbitrap cell. The MS was set as follows on Q Exactive: mass-to-charge ratio range of 380–1800, resolving power of 70,000, automatic gain control (AGC) target of 3e⁶, and maximum ion trap (IT) of 30 ms. For the Fusion Lumos Mass Spectrometer, the MS was set as follows: resolving power of 120,000, AGC target of 4e⁵, maximum IT of 50 ms. The MS/MS was set as follows on Q Exactive: resolving power of 17,500, AGC target of 1e⁵, maximum IT of 200 ms, and HCD collision energy (NCE) of 30%. For the Fusion Lumos Mass Spectrometer, the MS was set as follows: resolution power of 15,000, AGC target of 5e⁴, maximum IT of 100 ms, and HCD NCE of 35%. For phosphopeptides, the HCD was set at 30 with 10% stepped NCE on Q Exactive, or at 35% NCE with 5% stepped NCE on the Fusion Lumos Mass Spectrometer.

Peptide identification was performed using the Proteome Discoverer software (version 2.1; Thermo Scientific) with the Sequest HT and Mascot (version 2.5; Matrix Sciences) search engines. MS data were searched against the AtRTD2 translation (Zhang et al. 2017) database. Search conditions were set as follows: full trypsin digestion, two maximum missed cleavage allowed, precursor mass tolerance of 10 ppm, fragment mass tolerance of 20 mmu, dynamic modifications of oxidation (M), protein N-terminal acetylation, iTRAQ4plex (Y), static modifications of carbamidomethyl (C), and iTRAQ4plex (N terminus and K). The peptide spectrum matches (PSMs) were validated using the Percolator validator algorithm, which automatically conducted a decoy database search and rescored PSMs using q-values and posterior error probabilities. All PSMs were filtered with a q-value threshold of 0.05 (5% false discovery rate, FDR) or 0.01 (1% FDR) for proteome or phosphoproteome analysis, respectively. A q-value threshold of at least 0.01 was finally used to filter protein FDR for the proteome analysis. For comparative peptide:protein quantification, the ratios (114:115 and 116:117) of iTRAQ reporter ion intensities in MS/MS spectra of PSMs were used to calculate the fold changes between samples.