More high molecular mass proteins were observed on the 2 D gels when this optimized extraction protocol was adopted. Inhibitors,Modulators,Libraries In Figure 2, the 2 D gel electrophoresis images of the control transformant and the Yap1p overex pressing transformant are shown. By using the SYPRO Ruby staining method, more than 2,000 pro tein spots were detected on each 2 D gel. This number is higher than what has been achieved by silver staining, for which only a few hundred spots were detected. The 2 DE analyses were performed in triplicate to allow statistical analysis, and Students t test was used to deter mine if the relative change in protein expression was sta tistically significant. Based on this analysis, protein spots that were significantly up regulated upon Yap1p overex pression were identified on the 2 D gels.
In total, 78 such spots were detected on the 2 D gels. Typical Inhibitors,Modulators,Libraries exam ples are shown in Figure 2C and D. These spots were further analyzed Brefeldin_A by MALDI MS and LC MS MS, result ing in identification of 55 unique proteins, while LC MS MS was used for analysis of a few spots for which MALDI MS analysis did not give satisfactory results. Interestingly, some of the proteins were identi fied in more than one spot on the 2 D gels. Comparative proteome analysis of S. cerevisiae The 55 proteins that were identified are listed in Table 1 and the relative quantity is indicated. Of the averaged total spot volumes of the 55 identified proteins, 16 changed significantly at 99% confidence level, 33 changed significantly at the 95% confidence level, and 6 changed significantly at 90% confidence level.
The identified proteins were divided into differ ent Inhibitors,Modulators,Libraries categories, namely enzymes involved in carbon metab olism and proteins involved in pathways other than carbon metabolism, such as protein biosynthesis, cell cycle and growth regulation, etc. It is noteworthy that 16 proteins that play a role in carbon metabolism were up regulated in the Yap1p overexpressing yeast transformant. These proteins include ten glycolytic enzymes, four enzymes involved in conversion of pyruvate to ethanol, and two enzymes that are involved in the pentose phosphate pathway. Based on image analysis, we observed that the Inhibitors,Modulators,Libraries combined spot volumes of all identi fied enzymes involved in carbon metabolism enzymes increased about 1. 5 fold in the Yap1p overexpressing transformant.
Eight proteins involved in stress response were identi fied that were significantly more abundant in the Yap1p overexpressing transformant. These proteins include seven heat shock and chaperone proteins and one per oxiredoxin. Compared to the control transfor mant, most of the heat shock and chaperone proteins showed more than 2 fold increase in the Yap1p overex pressing transformant. Moreover, 13 proteins involved in protein biosynthesis and 10 proteins involved in cell cycle and growth regulation were identified on the 2D gels.