Image Analysis Workflow for 2-D Electrophoresis Gels Based on ImageJ

Software-based image analysis is a crucial step in the biological interpretation of two-dimensional gel electrophoresis experiments. Recent significant advances in image processing methods combined with powerful computing hardware have enabled the routine analysis of large experiments. We cover the process starting with the imaging of 2-D gels, quantitation of spots, creation of expression profiles to statistical expression analysis followed by the presentation of results. Challenges for analysis software as well as good practices are highlighted. We emphasize image warping and related methods that are able to overcome the difficulties that are due to varying migration positions of spots between gels. Spot detection, quantitation, normalization, and the creation of expression profiles are described in detail. The recent development of consensus spot patterns and complete expression profiles enables one to take full advantage of statistical methods for expression analysis that are well established for the analysis of DNA microarray experiments. We close with an overview of visualization and presentation methods (proteome maps) and current challenges in the field.

The aim of this study was to analyze modifications in the plasma protein map during an acute coronary syndrome (ACS) using proteomics. Proteomics is a new technology that allows the detection and identification of several proteins at a given time in a sample. Plasma from 19 patients, 11 with acute myocardial infarction (AMI) and 8 with unstable angina (UA), was investigated. The control group included nine age-matched volunteers. In two-dimensional electrophoresis using a pH range of 4 to 7, constant differences were found in at least four different areas within the plasma protein map. In area 1, we identified the presence of seven alpha(1)-antitrypsin (AAT) isoforms in plasma from control subjects. alpha(1)-antitrypsin isoform 1 was undetectable in plasma from UA and AMI patients. The AAT isoforms 5, 6, and 7 were reduced in plasma from AMI patients when compared with UA patients. Three fibrinogen gamma chain isoforms were identified in area 2. Fibrinogen gamma chain isoforms 1 and 2 were increased in AMI patients with respect to UA patients. Five apolipoprotein A-I isoforms were identified in area 3. All of them were reduced in plasma from AMI patients with respect to UA patients. In area 4, the gamma-immunoglobulin heavy chains were detected and were found increased in plasma from ACS patients. Plasma proteomic analysis makes it possible to develop a map of the protein isoforms that are expressed in plasma during an ACS.