Development of a proteomic capability using 2-D electrophoresis

In order to view and analyse entire sets of proteins, it was decided that the proteomic technique used would be 2-D electrophoresis. In order to enable the proteomic capability that is the central technique to the study, efforts were made in the first instance to investigate different strategies for preparation of protein samples and gel running conditions. This section examines briefly some of problems that were encountered, and decisions made. Some of the gel images have been selected to illustrate the progress made over a significant time period of this study.

3.5.1.1 Sample preparation and lEF

One of the main problems encountered with the isoelectric focusing step of 2-DE was the inability to increase the voltage to over 5000V for prolonged periods. W ith 7cm pH 3-10 IPG strips this value is normally achieved during lEF. This was particularly notable when running samples from the isolation of HCV proteinase using the cation exchanger SP Sepharose HP. This inability to reach the required voltage results in proteins not separating to their relevant isoelectric points. Another point that needs consideration is that over focusing within the lEF gel can result in proteins migrating to points other than their isoelectric point. Running conditions for lEF were then an integral part in producing 2-D gels in which protein spots could be seen clearly and concisely.

It was found that levels of salt within samples could adversely effect protein migration by affecting the voltage. Samples from the isolation of HCV proteinase had

a concentration of approximately 0.35M NaCI. Therefore samples were exchanged into the high urea buffer with no salt. This allowed the required voltage level (>5000V) to be achieved. Figure 3.5.1.1 shows good focusing and resolution of proteins following the buffer exchange.

kD 100 70 40 30 20 10 10 pi

Figure 3.5.1.1. Proteins from the separation of HCV proteinase. Proteins were exchanged into rehydration buffer containing no salt. Gel was stained using coomassie brilliant blue.

3.5.1.2 Pre-cast NuPAGE gels versus normal SDS-PAGE

Initially the second dimension of 2-DE was run using gels that were poured ‘in house’. These gels were found to be ideal for viewing ladders within ID SDS-PAGE, but for the reproducibility required in 2-DE it was decided to use pre-cast NuPAGE gels that gave more normalisation to the 2-DE process. Protein samples run using pre-cast gels ran well, but unfortunately gave a smearing effect across the gel when staining with coomassie brilliant blue (Figure 3.5.1.2). This was found to be due to artifacts within the gel (personal communication with Invitrogen).

Following investigation of different gel types, it was decided to use the NuPAGE gels in most cases in order to get reproducibility, but investigate different staining techniques in order to decrease or rid gel images of the smearing effect.

kD 100 70 40 30 20 10 10 pi

Figure 3.5.1.2. Proteins from the separation of HCV proteinase using pre-cast NuPAGE gel for the second dimension. Proteins were stained using coomassie brilliant blue. The blue box indicates the smearing effect found with NuPAGE gels when stained with coomassie brilliant blue.

3.5.1.3 Investigation of protein stains

Following the investigation of different gel types, it was decided to investigate different staining techniques in order to eliminate smearing effects that had been found when previous gels had been stained with coomassie brilliant blue. The stains investigated were sypro orange (Molecular Probes, Netherlands), colloidal coomassie (Invitrogen Life Sciences), and silver staining (Invitrogen Life Sciences). Sypro staining is a comparable stain to silver staining that can detect up to 1-2ng of protein. Due to problems with the equipment for visualisation of sypro orange, it was

decided to use silver staining over sypro orange. After considering sample preparation and protein concentration upon loading, it was decided to use silver stain. Silver staining is up to 100 times more sensitive than coomassie brilliant blue (Ausubel et al., 1992). Colloidal coomassie was discarded as the smearing effect experienced with coomassie brilliant blue was still present (Figure 3.5.1.3a)

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Figure 3.5.1.3a. Proteins from the separation of HCV proteinase using pre-cast NuPAGE gel for the second dimension. Proteins were stained using colloidal coomassie blue.

One of the advantages of using silver staining over the coomassie brilliant blue staining is that due to the lower levels of protein that can be loaded, more crude samples can be loaded onto the gel. This would then allow whole cell extracts to be loaded without excessive treatment that can result in the loss of certain proteins. This was found when proteins from the fermentation of HCV proteinase were precipitated using tetrachloroacetic acid and acetone. Comparison of protein extracts with and without this precipitation procedure show that some proteins are lost this when procedure is used (Figure 3.5.1.3b).

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Figure 3.5.1.3b. Comparison of proteins from the fermentation of E.coli expressing HCV proteinase by 2-DE. Proteins from crude whole cells (A), and precipitated proteins from whole cells (B) were were separated by lEF for 40kVhr. Gels were silver stained.

3.5.1.4 Conclusions

In conclusion, the running conditions for lEF were found to give the best separation

of E.coli whole cell extracts when IPG pH 3-10 strips were run for a total of 40kVHr.

This was achieved using step gradients of the following: 360kVHr at 30V, 250kVHr at 500V, 500kVHr at 1000V, 38890kVHr at 5000V. More clarified protein samples that had been desalted using centrifugal filters followed the same protocol apart from the last step that was run at 8000kVHr at 5000V. A total of 5pg of protein was loaded onto each gel. Following lEF, and second dimension SDS-PAGE, protein spots were visualized using silver stain. It should be noted that protein spot samples that underwent mass spectrometry analysis (see chapter 2, section 2.10.5) were stained using coomassie brilliant blue, and therefore the protein loaded onto the gel was increased by approximately 50 fold.