Proteomics is the study of all expressed proteins. A major goal of proteomics is a complete description of the protein interaction networks underlying cell physiology. Before we discuss protein computational tools and methods, we will give a brief background of current proteomic technologies used in cancer diagnosis. For cancer diagnosis, both surface-enhanced laser desorption ionization (SELDI) and two-dimensional gel electrophoresis (2DE) approaches have been used.1,2 Recently protein-based microarrays have been developed that show great promise for analyzing the small amount of samples and yielding the maximum data on the cell’s microenvironment.3-5
The recent upsurge in proteomics research has been facilitated largely by streamlining of 2DE technology and parallel developments in MS for analysis of peptides and proteins. Two-dimensional gel electrophoresis is used to separate proteins based on charge and mass and can be used to identify posttranslationally modified proteins. A major limitation of this technology in proteomics is that membrane proteins contain a considerable number of hydrophobic amino acids, causing them to precipitate during the isoelectric focusing of standard 2DE.6 In addition, information regarding protein-protein interactions is lost during 2DE due to the denaturing conditions used in both gel dimensions. To overcome these limitations, two-dimensional blue-native gel electrophoresis has been used to resolve membrane proteins. In this process, membrane protein complexes are solubilized and resolved in the native forms in the first dimension. The separation in the second dimension is performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which denatures the complexes and resolves them into their separate subunits. Protein spots are digested with trypsin and analyzed by matrix-assisted laser ionization desorption time-of-flight mass spectrometry (MALDI-TOF MS). The 2DE blue-native gel electrophoresis is suitable for small biological samples and can detect posttranslational modifications (PTMs) in proteins. Common PTMs include phosphorylation, oxidation and nitrosation, fucosylation and galactosylation, reaction with lipid-derived aldehydes, and tyrosine nitration. Improvements are needed to resolve low-molecular-mass proteins, especially those with isoelectric points below pH 3 and above pH 10. This technique has low throughput (at the most 30 samples can be run simultaneously), and most of the steps are manual. Automatic spot-picking also needs improvement.