DIGE Analysis Software and Protein Identification Approaches.

Abstract

Two-dimensional difference gel electrophoresis (2D-DIGE) is a high-resolution protein separation technique, with the excellent dynamic range obtained by fluorescent tag labeling of protein samples. Scanned images of 2D-DIGE gels show thousands of protein spots, each spot representing a single or a group of protein isoforms. By using commercially available software, each protein spot is defined by an outline, which is digitized and correlated with the quantity of proteins present in each spot. Software packages include DeCyder, SameSpots, and Dymension 3. In addition, proteins of interest can be excised from post-stained gels and identified with conventional mass spectrometric techniques. High-throughput mass spectrometry is performed using sophisticated instrumentation, including matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), MALDI-TOF/TOF, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Tandem MS (MALDI-TOF/TOF or LC-MS/MS) analyzes fragmented peptides, resulting in amino acid sequence information, which is especially useful when protein spots are low abundant or where a mixture of proteins is present.