Overview of Experimental Procedure 311 Proteomic Analysis of Cancer Tissues

To analyze the differences of protein expression profiles between cancer tissues and corresponding non-cancerous tissues, the proteomic differential display method was used. In this method, 2-DE and MS were used to identify the proteins. We first separated proteins from cancer tissues and corresponding non-cancerous tissues by 2-DE. Then the protein spots of the samples from cancer tissues were compared to the spots of the samples from corresponding non-cancerous tissues by using software for proteomic differential display.

Particular protein spots for which expression was different between cancerous and non-cancerous tissues were cut out from the gels, and were identified by means of MS. Figure 1 shows a workflow of these methods. The proteomic differential display method is a common method to analyze the profiling of protein expression. Usually 2-DE and MS have been used.

Recently, however, other new developments in protein detection technologies such as fluorescence two-dimensional difference gel electrophoresis (2-D-DIGE), isotope-coded affinity tags (ICAT), and isobaric tag for relative and absolute quantitation (iTRAQ) have been used. The 2-DE makes it possible to separate proteins according to both their charge in isoelectoric focusing (IEF) gels and their molecular weight in sodium dodecyl sulfate (SDS)gels (10,11).

The 2-DE technique has advantageous characteristics to compare the expression of a huge number of proteins simultaneously and to examine post-translational modifications of the protein spots. The technique of 2-D-DIGE makes it possible to run two or three differently labeled protein samples on the same gel simultaneously. It can exclude variability existing among gels to run two or three protein samples on the same gel (12,13,14,15). Isotope-coded affinity tag (ICAT) labeling, a new quantitative method, was recently performed (16). ICAT makes it possible to comprehensively analyze two comparable samples immediately. After labeling protein samples with isotope tags of 12C or 13C, they are separated by HPLC and identified by MS.

iTRAQ is a recently developed LC-based protein quantitation technique that utilizes four isobaric amine-specific tags. The principal advantages of iTRAQ are that

Fig. 1. Workflow of proteomic differential display methods.

four samples can be analyzed simultaneously, thereby reducing the amount of MS time needed for analysis, and it is more sensitive than other techniques (17).

3.1.2. Proteomic Analysis of Auto-antibodies in Patients

To detect auto-antibodies expected as cancer biomarkers for HCC, we analyzed serum auto-antibodies immunoreacting to proteins in cancer tissue obtained from patients with HCC. Tissue proteins were separated by 2-DE, transferred onto PVDF membranes, and immunoblotted with autologous sera. By comparing each immunoblot pattern, we identified immunoreactive spots with stronger staining intensity in cancer tissues than in corresponding non-cancerous tissues. Matched proteins on 2-DE gels were identified by MS. Figure 1 shows a workflow of these methods.

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