Protein Expression Mapping

Nearly all cellular functions are determined by the activity of proteins. Proteins act as catalysts, receptors or structural components that are required for the life of a cell. Many cellular processes are performed by complexes of several different proteins. It is essential that the protein components of these complexes be expressed at the same time and in the same place for the cell to function efficiently. Therefore, an understanding of cellular function at the molecular level requires knowledge of the patterns of expression of all of the component proteins. 

1 Protein expression mapping by 2D gel electrophoresis and mass spectrometry 

The use of 2D gel electrophoresis and mass spectrometry to identify proteins was discussed in Chapter 2. Protein expression mapping involves the use of these methodologies to compare expression patterns in different cell types or in the same cell type that has been exposed to different 

The utility of protein expression mapping using 2D gel electrophoresis and mass spectrometry has been demonstrated for several experimental systems. One application has been to assess the differences in protein expression between normal and cancerous cells. For example, expression mapping has been used to identify protein markers for bladder cancer (Ostergaard et al., 1999). This was accomplished by identifying proteins released into the urine of patients with and without bladder cancer using 2D electrophoresis and mass spectrometry. 

The Gmuender (2001) study also illustrates the limitations in terms of the quantitative reproducibility of protein expression mapping using 2D gels. They found that 32% of the spots on the 2D gels exhibited greater than 

---

D) polyacrylamide gels. These types of experiments have been performed for more than twenty years to build databases of proteins expressed from certain cell or tissue types (Anderson and Anderson, 1996 O Farrell, 1975). Although this remains an important component of proteomics research, the field has expanded due to the development of additional technologies. Proteomics can be broadly divided into two areas of research (i) protein expression mapping, and (ii) protein interaction mapping. 

Figure 3.1. Protein expression mapping using 2-D electrophoresis and mass spectrometry. The purpose is to compare protein expression patterns between cell types or in the same cell type under different growth conditions. Proteins are extracted from the different cell types and separated by 2D gel electrophoresis. Image analysis programs are used to compare the spot intensities between gels and identify proteins that are differentially expressed. The protein of interest is excised from the gel and its identity is determined by mass spectrometry. The power of the method increases greatly if the identity of a large number of proteins on the gel is known and present in a database because information can then be obtained without further mass spectrometry.

The difficulty with protein arrays is that proteins do not behave as uniformly as nucleic acid. Protein function is dependent on a precise, and fragile, three-dimensional structure that may be difficult to maintain in an array format. In addition, the strength and stability of interactions between proteins are not nearly as standardized as nucleic acid hybridization. Each protein-protein interaction is unique and could assume a wide range of affinities. Currently, protein expression mapping is performed almost exclusively by two-dimensional electrophoresis and mass spectrometry. The development of protein arrays, however, could provide another powerful... 

Figure 7.1. Protein expression mapping using an antibody array. The antibody array consists of monoclonal antibodies specific for a set of proteins in the organism of interest gridded onto a filter. To determine if a protein is expressed under the conditions being tested, a crude lysate is obtained and the proteins within the lysate are labeled with a fluorescent tag. The lysate is applied to the filter and the proteins are allowed to bind to the relevant antibody. Bound proteins are visualized via the fluorescent tag.

FIGURE 1.2 2D liquid protein expression map of the HCT-116 human colon adenocarcinomacell line. The x-axis is in p/units from 4.0 to 7.0 in 0.2 increments. They-axis is percent B of the RP-HPLC gradient. The gray scale of the bands represents the relative intensity of each band by UV detection at 214 nm. From Yan et al. (2003) with permission of the American Chemical Society. (See color plate.)... 

FIGURE 1.2 2D liquid protein expression map of the HCT-116 human colon adenocarcinoma cell line. (See text for full caption.)... 

Krapfenbauer K, Fountoulakis M, Lubec G. A rat brain protein expression map including cytosolic and enriched mitochondrial and microsomal fractions. Electrophoresis 2003 24 1847-1870. 

Krapfenbauer, K., Fountoulakis, M. and Lubec, G. (2003) A rat brain protein expression map including cytosolic and enriched mitochondrial and microsomal fractions. Electrophoresis 24, 1847-1870. Langen, H., Bemdt, P., Roder, D., Caims, N., Lubec, G. and Fountoulakis, M. (1999) Two-dimensional map of human brain proteins. Electrophoresis 20, 907-916. 

S.P. Zheng, K.A. Schneider, T.J. Barder, and D.M. Lubman, Two-dimensional liquid chromatography protein expression mapping for differential proteomic analysis of normal and O157 H7 Escherichia coli. Biotechniques, 35, 1202-1212 (2003). 

Currently, protein expression mapping is often performed by coupled 2DE or HPLC and MS-MS technologies. However, the development of protein microarrays (protein chips) may provide another powerful method to explore protein expression (protein profiling) and function on a genome-wide scale (Fung, 2004 LaBaer and Ramachandran, 2005 Hultschig et al, 2006). Figure 16.8 shows some potential applications for protein microarrays. 

---