Decyder 6

The labelled protein samples were mixed with the internal standard (Supplementary Table S1 at JXB online), adjusted to a total volume of 450 μl with rehydration buffer [7M urea, 2M thiourea, 4% CHAPS, 1% DTT, and 1% immobilized pH gradient (IPG) buffer, pH 4–7], and used for isoelectric focusing (IEF). IEF was performed on an IPG strip holder with 24cm, linear gradient IPG strips with pH 4–7 (GE Healthcare) and then on an Ettan DALT System (GE Healthcare) according to the manufacturer’s instructions. The images were analysed using DeCyder 6.5 (GE Healthcare). Spots reproducible in 24 of 30 images were used to identify protein abundance change by two-way analysis of variance (ANOVA) (P<0.05). The gels used for 2D-DIGE analyses were stained with Coomassie Brilliant Blue (CBB) to observe highly abundant spots. Then, additional gels with 1mg of internal standard proteins were stained with CBB for spots that could not be determined from 2D-DIGE gels.

Data of scanned DIGE gels were imported into DeCyder 6.5 software (GE Healthcare) and processed in DIA module for separate analysis of each gel (intra gel analysis), comprising the assignment of dye tag to images (internal standard: Cy2, control: Cy3 or Cy5, diseased: Cy5 or Cy3 due to reverse labeling), spot detection and normalization of Cy3 and Cy5 labeled spot abundances to the internal standard as well as comparison of spot abundances between control and ERU data sets. To avoid detection of “false” spots, inclusion and exclusion criteria for spot detection were set at: spot slope >2, spot volume <30000, threshold 2.5. After automatic detection and matching of spots by software analysis was verified manually and corrected if necessary. In BVA module, standardized protein spot abundances from each gel data set generated in the experiment were compared (inter gel analysis), enabling detection of protein abundance differences between groups. Detected differences in protein abundance were considered significant at p<0.05 (Student's t test) and inclusion criterion for further analysis was a fold change of >1.5. Differentially abundant protein spots from digital DeCyder map were located on matching silver stained gels and excised for subsequent identification by mass spectrometry (MALDI/TOF-TOF; ABI 4700 Proteomics Analyzer, Applied Biosystems, Darmstadt, Germany).

The in-gel fluorescence of different experimental setups were detected at three different emission wavelengths (Cy5: 670 mm, Cy3: 580 nm, Cy2: 520 nm) using a Typhoon Trio (GE Healthcare). Spots in the multiplexed gel images were detected by differential in-gel analysis (DIA) included in the DeCyder 6.5 software package (GE Healthcare) according to the manufactures instructions. Detection of spot boundaries followed by normalization of spot volumes revealed regulated proteins. For biological variance analysis (BVA) Cy2 images were merged resulting in vectors used for Cy5 and Cy3 image alignment. Quantification of each matched spot was accomplished by comparing the spot volumes ratios of their respective Cy5 and Cy3 fluorescence in the biological replicates. The level of significance among the replicates was compiled by student’s t-test set to 0.05. Since we and others observed molecular weight shifts resulting by minimal labeling [40 (link)], gels were post-stained with Flamingo Pink (Bio Rad) according to the manufactures instructions. The post-stained gels were analyzed using Image Master 6.0 (GE Healthcare). Respective differential spots resulting from the BVA were selected and picked by Ettan spotpicker (GE Healthcare) equipped with a 2 mm picking head according to the manufacturer’s instructions. Corrected punching of the spots was verified by rescanning.

The individual images of Cy2-, Cy3-, and Cy5-labeled proteins of each gel were obtained using Typhoon FLA9000 imager (GE Healthcare) with the wavelengths of 488 nm (Cy2), 532 nm (Cy3), and 633 nm (Cy5), respectively. The analysis of images was performed through DeCyder 6.5 software (GE Healthcare) to identify the different expression levels of proteins displayed. t-test P value and Average Ratio (control group/experimental group) were used to select differentially expressed protein spots. Protein expression value with an Average Ratio > 1.2 or Average Ratio < −1.2 and P < 0.05 was considered to be statistically significant.

For 2-D DIGE analysis, a mixture of Cy2-, Cy3-, or Cy5-labeled protein was mixed with 2-D DIGE buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.4% DTT, 0.5% IPG buffer) and then loaded on an immobilized pH gradient (IPG; Amersham Biosciences, Uppsala, Sweden) for isoelectric focusing (Tang et al., 2008 (link)). The running conditions were as follows: rehydration for 14 h at 20°C, followed by holding at 100, 300, 600, and 1000 V for 1 h at each step; then the voltage was raised to 10,000 V linearly and held until reaching a total value of 120,000 V-h. Second-dimension electrophoresis was performed using 12.5% SDS-polyacrylamide gel (Gan et al., 2013 (link)).
The images of Cy2-, Cy3-, and Cy5-labeled proteins were acquired by a Typhoon 8600 scanner (GE Healthcare, UK) using difference wavelength and analyzed by DeCyder 6.5 software (GE Healthcare, UK). Spot detection was performed by the DIA (differential in-gel analysis) module. After removing the artifact spots by manual editing, the images were further analyzed by the DeCyder BVA (biological variation analysis) module. For each treatment, images from at least three biological replicates were used for statistical analysis.

The labeled samples were loaded onto Immobiline Dry Strips (24 cm, linear pH gradient from pH 4–7, GE Healthcare). The IPG strips were rehydrated at 40 V for 5 h, followed by 100 V for 6 h, and IEF was subsequently conducted for a total of 69.8 kVhr on a Multiphor II System (GE Healthcare). After completion of the IEF analysis, the strips were equilibrated and applied to 12.5% polyacrylamide gels. The SDS-PAGE was performed using Ettan™ Daltsix equipment (GE Healthcare) at 15°C. All electrophoresis procedures were performed in the dark and ran in duplicate. The gels were scanned using a Typhoon™ Variable Mode Imager (GE Healthcare) at a resolution of 100 µm, followed by silver staining. The gel images were analyzed using DeCyder 6.5 software (GE Healthcare). The biological variation analysis mode (BVA) was used to detect the significant between-group differences across all gels. Data was statistically analyzed using Student's t-test, and p<0.05 was considered significant.