Computational protocol: Proteomic analysis of developing rye grain with contrasting resistance to preharvest sprouting

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Protocol publication

[…] Two sets of rye recombinant inbred lines (RILs), 20 PHS-resistant lines (0 % sprouting in spikes after 7 days of water spray at maturity) and 20 PHS-susceptible lines (90–100 % sprouting in spikes after 7 days of water spray at maturity), were grown on the experimental field of the West Pomeranian University of Technology, Szczecin in 2011. These lines represented F10 generation of RILs developed from the 541 × Ot1-3 intercross described in earlier studies (Masojć et al. ; Masojć and Kosmala ). Two spikes per line were immersed in liquid nitrogen at the 25th day after anthesis (25 DPA) and kept at −30 °C for 1 month. Frozen kernels with green pericarp and milky endosperm (10 per line) were threshed out from spikes and freeze-dried in an Alpha 1-2 LD plus freeze drier (Christ®). Dry kernels of the PHS-resistant group of lines were bulked and ground in a Retsch MM 200 mill. Kernels from the PHS-susceptible group of lines were bulked and ground in the same mill. Two bulked samples of the rye flour representing 20 PHS-resistant and 20 PHS-susceptible lines were kept in a freezer prior to protein extraction.The proteomic work involved: (1) the analyses of rye grain protein abundance in the bulked samples of the resistant and the susceptible rye inbred lines using 2-DE, (2) MS identification of proteins which were differentially accumulated between the analysed bulks. The protocol for proteomic research was the same as that described in detail by Masojć and Kosmala ().Protein extraction was performed according to the method described by Hurkman and Tanaka (). This method was shown earlier to be useful to extract the proteins from different species and its use resulted in high-quality 2-D maps with proteins derived from different cell components, including membranes, chloroplasts, mitochondria and cytoplasm (Kosmala et al. , ; Winiarczyk and Kosmala ; Bocian et al. ). Briefly, the powdered tissue was homogenised with 500 μl of extraction buffer (0.7 M sucrose, 0.5 M Tris, 30 mM HCl, 50 mM EDTA, 2 % DTT, 0.1 M KCl). An equal volume of water-saturated phenol was then added and the sample was incubated for 5 min at 4 °C, followed by 10 min of vortexing and centrifuging at 8,700 × g for 30 min. The phenol phase (upper) was recovered and re-extracted with an equal volume of extraction buffer. After centrifuging, the proteins from the phenol phase were precipitated by the addition of five volumes of cold 0.1 M ammonium acetate in methanol and then incubated at −20 °C at least overnight. The sample was then centrifuged at 20,500 × g at 0 °C for 30 min, the precipitate washed twice with the cold ammonium acetate in methanol and once in cold acetone, and dried. The pellet was finally dissolved in 100 μl of the sample solution (7 M urea, 2 M thiourea, 2 % NP-40, 2 % IPG buffer pH range 4–7 or 3–10, 40 mM DTT). The protein concentration was determined by the use of a 2-D Quant Kit (GE Healthcare). The aliquots of proteins extracted from 25 mg of rye flour were mixed with rehydration solution (7 M urea, 2 M thiourea, 2 % NP-40, 0.5 % IPG buffer pH range 4–7, 0.002 % bromophenol blue, 18 mM DTT) to the final volume of 450 μl and used for 2-DE, performed according to Hochstrasser et al. (). In the first dimension, isoelectric focusing (IEF), 24-cm Immobiline DryStrip gels with linear pH range 4–7 were used. This range was selected as the standard condition for resolving the proteins after the pre-selection performed with a relatively broad pH range (3–10) (data not shown) and followed our earlier work (Masojć and Kosmala ). Rehydration and focusing was carried out in Ettan IPGphor II (GE Healthcare) at 50 μA per strip at 20 °C, applying the following programme: 12 h of rehydration at 0 V and 9 h of focusing at 1 h/500 V, 2 h/1,000 V and 6 h/8,000 V. After IEF, the strips were equilibrated for 15 min in SDS equilibration buffer solution (6 M urea, 75 mM Tris–HCl pH = 8.8, 29.3 % glycerol, 2 % SDS, 0.002 % bromophenol blue, 65 mM DTT), followed for 15 min with the same buffer but containing 135 mM iodoacetamide instead of DTT. After equilibration, the proteins were separated in the second dimension, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using 13 % polyacrylamide gels (1.5 × 255 × 196 mm) at 4 W/gel for 30 min and then at 17 W/gel for 4 h. Rainbow™ Molecular Weight Marker (GE Healthcare) was used as a standard to determine the molecular weights (MWs) of proteins for particular spots. Following electrophoresis, the gels were stained with colloidal Coomassie Brilliant Blue G-250, using the modified method of Neuhoff et al. (). Total separated protein spots on the gels were scanned by an ImageScanner III (GE Healthcare) and subjected to processing by the LabScan 6.0 program (GE Healthcare). Spot detection and image analyses (normalisation, spot matching, accumulation comparison) were performed with the ImageMaster 2D Platinum software package (GE Healthcare). To compensate for subtle differences in sample loading, gel staining and destaining, the abundance of each protein spot was normalised as a relative volume (% Vol). The % Vol of each spot was automatically calculated by the ImageMaster software as a ratio of the volume of a particular spot to the total volume of all the spots present on the gel. The extraction procedure and electrophoretic separation were performed twice (technical replicates), and the % Vol for the spots from the two replicated gels were then used to calculate the means, which were used to make comparisons between the analysed rye lines.The protein spots which showed at least 2-fold (p < 0.05) differences in protein abundance between two analysed lines (quantitative analysis) together with protein spots present only in one of the analysed lines (qualitative analysis) were subjected to MS analysis and identification. Protein spots were excised from the gel and analysed by liquid chromatography coupled to the mass spectrometer in the Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences (Warsaw, Poland). Samples were concentrated and desalted on an RP-C18 pre-column (Waters) and further peptide separation was achieved on a nano-Ultra Performance Liquid Chromatography (UPLC) RP-C18 column (Waters, BEH130 C18 column, 75 μm i.d., 250 mm long) of a nanoACQUITY UPLC system, using a 45-min linear acetonitrile gradient. The column outlet was directly coupled to the electrospray ionisation (ESI) ion source of an Orbitrap-type mass spectrometer (Thermo), working in the regime of data-dependent MS to MS/MS switch. An electrospray voltage of 1.5 kV was used. Raw data files were pre-processed with Mascot Distiller software (version 2.3, MatrixScience). The obtained peptide masses and fragmentation spectra were matched to the National Center Biotechnology Information (NCBI) non-redundant database with a Viridiplantae filter (1,032,142 sequences) using the Mascot search engine (Mascot Daemon v. 2.3, Mascot Server v. 2.2.03, MatrixScience). The following search parameters were applied: enzyme specificity was set to trypsin, peptide mass tolerance to ±40 ppm and fragment mass tolerance to ±0.8 Da. The protein mass was left as unrestricted and mass values as monoisotopic, with one missed cleavage being allowed. Alkylation of cysteine by carbamidomethylation as fixed and oxidation of methionine was set as a variable modification.Protein identification was performed using the Mascot search probability-based MOWSE score. The ions score was −10* log(P), where P was the probability that the observed match was a random event. The Mascot-defined threshold, which indicated identity or extensive homology (p < 0.05), was 40 or less, therefore, an ion score of 40 was taken as a threshold for analysis. The proteins with the highest Multidimensional Protein Identification Technology (MudPIT) scores and/or the highest number of peptide sequences were selected. If more than one reliable identification appeared in the single spot, they were all indicated. However, in these cases, it was impossible to evaluate the abundance of particular proteins present in the spot and the total protein abundance was shown. Further research to estimate detailed protein contents of such multi-protein spots would be required according to, for example, the method described by Ishihama et al. (). When the protein was identified as “predicted protein”, its amino acid sequence was blasted using the blastp algorithm. The protein with the highest score was then selected as the functional homologue of the “predicted protein”. […]

Pipeline specifications

Software tools ImageMaster 2D, Mascot Distiller, Mascot Server, BLASTP
Application MS-based untargeted proteomics
Organisms Secale cereale, Triticum aestivum, Oryza sativa