Large-scale phosphoproteome analysis in seedling leaves of Brachypodium distachyon L
In this study, we carried out the first large-scale phosphoproteome analysis of seedling leaves in Brachypodium accession Bd21 using TiO2 microcolumns combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MaxQuant software. A total, 1,470 phosphorylation sites in 950 phosphoproteins were identified. Of the 950 phosphoproteins identified, 127 contained 3 to 8 phosphorylation sites. The phosphoproteins and phosphosites identified in our study expanded our knowledge of protein phosphorylation modification in plants, especially in monocot.
Sample Processing Protocol
Seeds of Brachypodium distachyon Bd21 were surface sterilized in 5% sodium hypochlorite for 5 min, and rinsed 4 times in sterile distilled water. Seeds were submerged in water for 12 h at room temperature, and then transferred to wet filter paper for 24 h to germinate at room temperature (22–25 ºC). Uniformly germinated seeds were selected and grown in three plastic pots containing Hoagland’s solution; the Hoagland’s solution was changed every two days. At the three leaf stage, all three leaves were collected and frozen at –80 ºC. Culture of the Bd21 seedlings was repeated three times and the leaves were collected independently. Total proteins were extracted from seedling leafs according to the method of Wang et al. with minor modifications. Approximately 400 mg fresh leaves of each sample were ground into fine powder in liquid nitrogen. The ground powder was suspended in 4 mL SDS buffer (30% sucrose, 2% SDS, 100 mM Tris-HCl, pH 8.0, 50 mM EDTA-Na2, 20 mM DTT) and 4 mL phenol (Tris-buffered, pH 8.0) in a 10 mL tube, and 1 mM phenylmethanesulfonyl fluoride (PMSF) and PhosSTOP Phosphatase Inhibitor Cocktail (Roche, Basel, Switzerland), were added to inhibit the activities of proteases and phosphatases. The mixtures were thoroughly vortexed thoroughly for 30 s and the phenol phase was separated by centrifugation at 14,000 × g and at 4 ºC for 15 min. The upper phenol phase was pipetted to fresh 10 mL tubes and four volumes of cold methanol plus 100 mM ammonium acetate were added, and the mixture was stored at –20 ºC for at least 30 min. After centrifugation at 14,000 × g and at 4 ºC for 15 min, the supernatant was carefully discarded and the precipitated proteins were washed twice with cold methanolic ammonium acetate (100 mM) and ice-cold 80% acetone, respectively. Finally the pellet was vacuum-dried and then dissolved in lysis buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS and 65 mM DTT) over 3 h at 4 ºC. The protein mixtures were harvested by centrifugation at 14,000 × g and 4 ºC for 15 min to remove insoluble materials. The concentrations of the extracted protein mixtures were determined with a 2-D Quant Kit (Amersham Bioscience, Buckinghamshire, UK) using BSA (2 mg/mL) as standard, and the final protein solution was stored at –80 ºC for later use. Extracted protein mixtures were directly reduced with dithiothreitol (DTT), alkylated with iodoacetamide, and subsequently digested with endoproteinase Lys-C and trypsin as previously described. The enrichment procedure for phosphopeptides was performed as reported by Wu et al. with modifications. The TiO2 beads (GL Sciences, Tokyo, Japan) were incubated in 400 μL loading buffer containing 65% Acetonitrile (ACN) / 2% trifluoroaceticacid (TFA) / saturated by glutamic acid. A total of 2 mg of tryptic peptides were dissolved in 600 μL loading buffer, and incubated with an appropriate amount of TiO2 beads. After washing with 600 μL wash buffer (65% ACN / 0.1% TFA), phosphopeptides were eluted twice with 300 μL elution buffer (500 mM NH4OH / 60% ACN) and the eluates were dried down and reconstituted in 0.1% formic acid (FA) / H2O for MS analysis. Enriched phosphopeptides were separated on a self-packed C18 reverse phase column (75 μm I.D., 150 mm length) (Column Technology Inc., Fremont, CA), which directly connected the nano electrospray ion source to a LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Pump flow was split to achieve a flow rate at 1 μL/min for sample loading and 300 nL/min for MS analysis. The mobile phases consisted of 0.1% FA (A), and 0.1% FA and 80% ACN (B). A five-step linear gradient of 5% to 30% B in 105 min, 35% to 90% B in 16 min, 90% B in 4 min, 90% to 2% B for 0.5 min and 2% B for 14.5 min was performed. The spray voltage was set to 2.0 kV and the temperature of the heated capillary was 240 ºC. For data acquisition, each MS scan was acquired at a resolution of 60,000 (at 400 m/z) with lock mass option enabled and was followed by a data-dependent top 10 MS/MS scans using collision induced dissociation (CID). The threshold for precursor ion selection was 500 and mass window for precursor ion selection was 2.0 Da. The dynamic exclusion duration was 120 s, repeat count was 1 and repeat duration was 30 s. The analyzer for the MS scans was Orbitrap and for the MS/MS scans LTQ (37% relative collision energy). Three biological replicates were performed independently from sample collection to phosphopeptide identification using LC-MS/MS.
Data Processing Protocol
The raw files were processed with MaxQuant (version 126.96.36.199) and searched against the B. distachyon protein database (31,029 entries in total) in Phytozome (http://www.phytozome.net/search.php; version 9.1) concatenated with a decoy of reversed sequences. The following parameters were used for database searches: cysteine carbamidomethylation was selected as a fixed modification; methionine oxidation, protein N-terminal acetylation, and phosphorylation on serine, threonine and tyrosine were selected as variable modifications. Up to two missing cleavage points were allowed. The precursor ion mass tolerances were 7 ppm, and fragment ion mass tolerance was 0.5 Da for MS/MS spectra. The false discovery rate (FDR) was set to < 1.0% for both peptide and protein identifications, the minimum peptide length was set to 6. Phosphorylation site localization was based on PTM scores that assign probabilities for each of the possible sites according to their site-determining ions. In this study, MaxQuant (version 188.8.131.52) was used to calculate PTM scores and PTM localization probabilities. Potential phosphorylation sites were then grouped into three categories depending on their PTM localization probabilities, namely class I (localization probability, P ≥ 0.75), class II (0.75 > P ≥ 0.5) and class III (P < 0.5). A false discovery rate (FDR) of 1% was used for phosphorylation sites identification.
Lv DW, Li X, Zhang M, Gu AQ, Zhen SM, Wang C, Li XH, Yan YM. Large-scale phosphoproteome analysis in seedling leaves of Brachypodium distachyon L. BMC Genomics. 2014 May 16;15:375 PubMed: 24885693