Comparative Proteomics

If complex mixtures are analyzed, a lot of information can be obtained by a proper sample preparation and subsequent electrophoretic and mass spectrometric analysis. In Figure 1, difference between calcium binding proteins in the plasma membranes of rat liver (left) and fast growing Morris hepatoma 7777 is shown. These proteins have been identified as annexins. Note the increased levels of annexins with lower molecular weights (between 30 and 35 kDa) in the extract from Morris hepatoma 7777 plasma membranes.

Selective solubilization by use of different reagents, combined with electrophoretic analysis and nano LC-ESI MS/MS is a powerful tool for the analysis of membrane proteins from normal and malignant tissues such as rat liver and hepatocellular carcinomas Morris hepatomas. As shown in figure 2, a reliable detection of differences between isolated plasma membrane fractions from normal and malignant tissue is possible. It is the first step on the long and complicated way towards detection of reliable biomarkers for hepatocellular carcinomas.

complex mixtures and subsequent SDS-PAGE

Figure 1

SDS-PAGE of rat liver plasma membranes. Membrane proteins were solubilized with one percent CHAPS in the absence of (left) or presence of EDTA (right). Arrows - proteins which are extracted after calcium complexing.

Left – Buffer with 2 mM CaCl 2.

Right – Buffer with 10 mM EDTA.

Figure 2

TX100

Comparison of proteins identified in Triton X100 (TX100) solubilizates from liver (NL) and Morris hepatoma 7777 (M Hep) plasma membranes. The total number of ProteinPilot identifications, using the entire NCBI nr database, but after filtering out all non-rat proteins (as annoted in nr), at three different confidence levels is shown in the table in the upper left. The Venn diagram illustrates the overlap between 95% confidence proteins. The log–log plot of protein scores reveals the correlation between experimental samples. ProteinPilot scores (S) are related to confidence (C) by the formula: C = 100 × (1 − 10−S)%. Therefore, the lines at S = 1.3 and 2.0 demark the 95% and 99% confidence thresholds. Because no peptide assignment can contribute more than two to a protein score, those protein identifications above 2.0 (>99% in the table) must have two or more peptides contributing to the identification. Note: To place the unique proteins on the log-scaled graph, their protein scores were arbitrarily set to 1.

Figure 3

SDS-PAGE of calcium binding proteins (annexins) from plasma membranes of rat liver (left) and Morris hepatoma 7777 (right).

Other proteins have been removed by previous extraction with one percent Triton X100. (see Fig 2)

However, 2-D electrophoresis can yield much more information. As shown in Figure 3, the two lower bands from the SDS-PAGE in Figure 2, contain more than 10 polypeptides with very similar molecular weights that can be distinguished by differences in pI.

complex mixtures and subsequent SDS-PAGE
Figure 4

Silver stained 2-D electrophoresis of the Morris hepatoma 7777 plasma membrane proteins extracted with 25 mM EGTA from the Triton X100 insoluble pellet. Proteins identified by SELDI-TOF mass spectrometry in the labeled spots/groups of spots are listed in Table 1 and Table 2.

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