Pdquest

For comparative proteomic analysis 2D gels were scanned and analyzed using the PDQUEST software package from BioRad (version 8.0.1). All detected proteins were identified by mass spectrometry (see below), selected for the comparative analysis and added to the master image.

Two-DE was performed with 17 cm (linear, pH 4–7) immobilized pH gradient (IPG) gel strip (Bio-Rad), according to Kim et al. [41 (link)]. A total of 1200 μg yellow seedling protein was loaded onto IPG strip using active rehydration (13 h with 50 V), and the isoelectric focusing (IEF) was performed at 17 °C with a voltage gradient of 250 V for 0.5 h, 1000 V for 1 h, 10,000 V for 5 h, then continued for a total of 60,000 Vh. The focused strip was equilibrated for 15 min with equilibration solution (6 M urea, 0.375 M Tris-HCl, 20% (v/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS)) containing 2% (w/v) DTT, then was equilibrated for another 15 min with equilibration solution containing 2.5% (w/v) iodoacetamide. Equilibrated strip was then sealed on the top of 12% SDS-PAGE gel for electrophoresis. The gel was visualized with 0.1% coomassie brilliant blue (CBB) R-250, and scanned with a high precision scanner (ScanMaker 9700XL, Microtek, Shanghai, China) at a resolution of 600 dpi. Spot analysis was performed using PDQuest (version 8.0.1, Bio-Rad).

Quantitative analysis of the digitized images was carried out using the PDQuest (version 7.0, Bio-Rad Laboratories, Hercules, CA, USA) software according to the protocols provided by the manufacturer. The quantity of each spot was normalized by the total valid spot intensity. Protein spots were selected for significant variation in expression that deviated more than two folds in their expression level compared with the control or normal sample.

Protein separation, image analysis and quantitation of branchial cilia and sperm flagella were performed as described [18 (link)]. Branchial cilia or sperm flagella were suspended in a lysis buffer with an IPG buffer (final concentration 0.5%), and applied to an IPG strip (pH 3–10). Isoelectric focusing was carried out on the Ettan IPGphor III (GE Healthcare) at a programed voltage [18 (link)]. Two-dimensional SDS–PAGE was performed using 10% polyacrylamide gels for separating gels. Gels were stained with Coomassie Brilliant Blue R-250 and were scanned using a GS-710 Calibrated Imaging Densitometer (Bio-Rad). Images were analyzed by PDQuest Basic version 8.0 software (Bio-Rad). Spot peaks were automatically detected and manually added or removed. Unique protein spots were assigned with special spot numbers (SSP). The relative quantity (ppm) of a protein spot was expressed as the ratio to the total quantity of all protein spots. At least three gels were analyzed to obtain reproducible 2DE pattern. Comparison and matching of the spot pattern in 2D gels were performed by PDQuest (BioRad). The proteomic identification of protein components in branchial cilia and sperm flagella was carried out by the combination of 2DE and peptide mass finger printing with MALDI-TOF/MS (Bruker Daltonics), as described [18 (link)].

Twenty samples were randomly selected from each group for 2-DE. Each sample corresponded to one gel. Protein extracts from control and patient serum were processed in parallel to ensure maximal comparability. Albumin was removed from serum samples as described previously [25] (link). In short, serum was precipitated by addition of ice-cold acetone containing 10% (w/v) trichloroacetic acid, and subsequently washed with acetone four times. Total protein concentration was determined using a Bradford Protein Assay Reagent Kit (Thermo Scientific). 2-DE was performed as reported previously [26] (link). A protein load of 1.3 mg protein was applied to 17 cm IPG Strips (pH 4–7, GE Healthcare). Focusing was performed according to the protocol provided by Bio-Rad Laboratories. The second-dimensional separation was performed on 12.5% SDS-polyacrylamide gels. After fixation, gels were stained using colloidal Coomassie Blue (CCB) with slight modifications [27] (link). Gels were scanned, and analysis for differences in protein patterns was performed using PDQuest (Version 7.1, Bio-Rad).

Gels were fixed for 1 h in 50% v/v methanol, 10% v/v acetic acid, followed by staining with SYPRO Ruby (BioRad, Hercules, CA, USA) overnight. The background stain was removed by incubation in 10% v/v acetic acid, 7% v/v methanol, for 1 h. Gels were stored in water at 4 °C. Gel images were obtained using a high-resolution scanner (Typhoon FLA9000, GE Healthcare). The resulting gel image files were analyzed by image software (PD Quest, BioRad). Spot intensity levels were normalised by expressing the intensity of each protein spot in a 2-D gel as a proportion of the total protein intensity detected for the entire gel. The analysis was carried out on independent biological triplicates of gels for each sample (treated or not CPEX, and either without stress or under OS conditions). Proteins that showed 2-fold change in expression or higher were taken into account. The differentially regulated spots among the various samples were considered potentially significant by the Student′s t-test (<0.05).