Phosphoproteome Analysis of Platelets

The phosphorylated and total proteins in gels were stained with the Pro-Q Diamond phosphoprotein stain or SYPRO-Ruby protein stain, respectively, following the protocol provided by the manufacturer (Thermo Fisher Scientific) [27 (link)]. Protein samples prepared from the control or platelets incubated with glucose for 2 h were separated by 2DE (n = 3 per group). Since the gel staining method for detecting phosphorylated proteins requires a lot of time and effort, only three samples each in the control and loaded-glucose for 2 h were examined. The gels were first stained with Pro-Q Diamond phosphoprotein gel stain to detect phosphoproteins, and visualized using the Typhoon 9400 imager. The gels were then stained with SYPRO-Ruby protein gel stain to reveal the total proteome before being visualized. The analysis process was carried out by matching all gels from the control or glucose-incubated platelets to determine quantitatively the effect of glucose using PDQuest™ 8.0 Advanced 2D Analysis software (Bio-Rad Laboratories, Hercules, CA, USA). The relative abundance of phosphoproteins was calculated in the Pro-Q Diamond images and the SYPRO Ruby images, as described previously [27 (link)]. The Mann–Whitney U test was used for statistical analysis when comparing the values of the two groups using the JMP 16 software.
To determine the position of phosphoproteins in the total protein, phosphoprotein spots were matched in gels stained by the Pro-Q Diamond and SYPRO Ruby stains. The matched spots were further matched with the spots in 2D-DIGE gels. Subsequently, phosphoprotein spots detected qualitatively by Pro-Q Diamond staining, combined with SYPRO Ruby staining and also detected quantitatively by 2D-DIGE with a statistically significant difference (p < 0.05), were selected. Selected spots were analyzed using MALDI-TOF/TOF MS to identify the proteins, as described above.