Computational protocol: Proteomic comparison reveals the contribution of chloroplast to salt tolerance of a wheat introgression line

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[…] Two dimensional gel electrophoresis was conducted according to Wang et al.. The iso-electronic focus (IEF) was conducted using 17 cm pH4-7 linear pH gradient IPG strip (Bio-Rad). The protein solution containing 600 μg proteins was diluted with lysis solution to 375 mL, and evenly coated in a channel of the rehydration tray (Bio-Rad). The IPG strip was carefully placed on the protein solution to avoid producing bubbles, covered by mineral oil, and rehydrated at 20 °C overnight. The rehydrated IPG strip was washed quickly by ultrapure H2O, slightly removed surface water with humid filter paper, transferred into the IEF groove (Bio-Rad), and covered with mineral oil. IEF was conducted on a Bio-Rad PROTEAN IEF cell at 20 °C with the following procedure: 500 V for 1 h, 1000 V for 1 h, linear increase to 3000 V within 3 h, linear increase to 8000 v within 6 h, stay at 8000 V up to 100 kVh. After IEF, the strip was equilibrated by soaking in 2% DTT in equilibration buffer (6 M urea, 2% SDS, 0.05 M Tris-HCl pH 8.8, 20% glycerol) for 20 min followed by 2.5% iodoacetamide in equilibration buffer in dark at room temperature. The equilibrated IPG strip was placed on the top edge of 10% SDS-PAGE gel to avoid producing bubbles, and sealed by 1% low melting point agarose. The SDS-PAGE was done on the PROTEAN II cell (Bio-Rad) at constant current setting of 5 mA/gel for 2 h followed with 15 mA/gel until the bromophenol blue tracking dye arrived at the bottom edge of the gel. After SDS-PAGE, the proteins in the 2-DE gel were visualised by Coomassie brilliant blue R-250 staining. Gel image was digitalized with a gel scanner (Powerlook 2100XL, UMAX), and analyzed with PDQuest™ software package (Bio-Rad). The analysis was based on total densities of gels with the parameter of percent volume, and a significant difference in expression of a spot (namely differentially expressed protein, DEP) was declared if the mean abundance varied more than two fold using t-test.The mass spectometry analysis followed Wang et al.. DEPs detected in gels were manually excised. After washing with Millipore pure water for three times, the gel pieces were destained with 50 mM ammonium bicarbonate (ABC)/50% acetonitrile (ACN) for several times until the blue staining was thoroughly disappeared, and then dehydrated with ACN. The gel pieces were reduced with DTT (10 mM in 50 mM ABC) for 60 min at 56 °C, and alkylated with iodoacetamide (55 mM in 50 mM ABC) for 45 min at room temperature in dark. Finally, the gel pieces were washed with 50 mM ABC for three times, dehydrated with ACN, and vacuum dried at −40 °C using a FREEZONE 6 freeze dry system (LABCONCO, USA). For in-gel digestion, the dry gel pieces were rehydrated in 5 μL trypsin solution (containing 20 ng/mL SIGMA proteomics grade porcine-modified trypsin in the buffer: 40 mM ABC solution and 9% ACN) for 1 h at 47 °C, covered with 15 μL trypsin buffer, and incubated at 37 °C for 16 h. The digested supernatant was transferred into a new tube. The peptides in gel pieces were successively extracted with 5% trifluoroacetic acid (TFA)/50% ACN on a sonication bath for 5 min twice and ACN for 10 min. The extract was combined with the digestion supernatant. The supernatant was vacuum dried at −40 °C, and the pellet was resuspended with 3 μL 0.5% TFA/30% ACN and desalted on a Ziptip C18 micro column (Millipore). Peptide aliquot (0.5 mL) was plotted onto metal plate and covered with 0.5 mL of matrix solution containing 10 mg/mL w/v α-cycano-4-hydroxycinnamic acid (CHCA) in 0.1% TFA/50% ACN. After air-dry, MALDI-TOF analysis was conducted using 4700 plus MALDI TOF-TOF™ analyzer (Applied Biosystems, USA). Peaklist-generating and peak-picking for MS data were conducted with GPS explorer (Applied Biosystems, 2006). Parameters and thresholds used for peak-picking were S/N threshold > 20, resolution > 10000 and means of calibrating each spectrum as external calibration. MS data were used to derive protein identity using the MASCOT search engine (http://www.matrixscience.com) applied to the NCBInr, MSDB and SwissProt databases. MS acceptance criterion is probability/E-value based scoring. The search parameters were one trypsin mis-cleavage permitted, MH + mass values and monoisotopic. [...] The sequence of each selected DEP was used as a tBLASTn query against the wheat EST database in NCBI. All matching ESTs were assembled to get a unigene using CAP3 software, and a pair of specific primers designed from this assembly was used to amplify a full-length cDNA. The PCR procedure consisted of a 5 min denaturation at 95 °C, followed by 35 cycles of 94 °C for 30 s, 58 °C for 50 s and 72 °C for 60 s, with a final extension of 72 °C for 10 min. The amplicon was inserted into the pMD18-T vector (Takara), following the supplier's instructions, and submitted for sequencing. […]

Pipeline specifications

Software tools PDQuest 2-D, Mascot Server, TBLASTN, CAP3
Databases Full Length cDNA
Applications MS-based untargeted proteomics, Amino acid sequence alignment
Organisms Triticum aestivum, Arabidopsis thaliana
Chemicals alpha-Linolenic Acid