Given the complexity of 2D gels, which typically display hundreds of proteins on a single gel, image analysis systems are required to detect and quantify the spots. I am currently using the Phoretix 2D software (developed by Nonlinear Dynamics) to analyse 2D gels scanned at 600 dpi and saved as TIFF files. This software follows the typical general steps for analysis of gels: spot detection, spot editing, background correction, matching of the gels to a reference gel, normalization, isoelectric point and molecular weight calibration.
Parameters for automatic spot detection can be entered by using the set-up wizard. Once the optimal automated detection parameters are established, they can be applied to all of the other gels in the experiment or saved for later applications. Spot detection often needs to be manually reviewed by adding, deleting or editing spots that were not detected by the automated spot detection algorithm. Although an extensive set of tools is available for manual editing, this work can sometimes be time consuming. The software provides three automatic methods of background subtraction (mode of non spot, lowest on boundary, and average on boundary); in addition, a manual option is available. The software automatically determines a number of parameters of each spot in each gel (i.e., spot volume, area, optical density).
The most critical step in the analysis is the gel matching. The gels can be matched automatically and the process can be improved by adding user-defined seeds. However, in my experience, the results are rarely satisfactory and, in general, manual work is required. When automatic matching is used, it is necessary to confirm manually that each spot had been identified correctly by the software. Vectors connecting the matched spots can be visualized; I found this function particularly useful because it is easy to understand that the matching is correct when the vectors have similar lengths and directions. During matching, each spot is automatically named with the number of the corresponding spot in the reference gel. Importantly, when a reference gel is created, it is possible to share it between experiments. In addition, it is easy to add (automatically or manually) additional spots to the reference gel. Several options are available for spot normalization (i.e., normalize to single value, to total spot volume, to total volume ratio). Isoelectric point and molecular weight of every spot on each gel can be estimated by assigning known values to a few spots.
Once the analysis is complete, there is an extensive range of data visualisation tools to identify changes in protein expression (i.e., creating histograms). Although these tools can quickly give an idea of protein expression, I found them of little value from a scientific point of view. I prefer using statistical program for the analysis, therefore, I usually include in tables the data of interest (very easy to do) and export them to Microsoft Excel.
The software can support without problems the simultaneous analysis of a large number of gels (I analyzed over 100 gels simultaneously). However, in relation to the processor used, the software may work very slowly and therefore it is advisable to separately analyze a limited number of gels. A lot of free hard disk space, good graphics card and large screen may simplify the analysis. A large screen is particularly relevant because manual work often requires the use of enlarged images of the gels.
Obviously, the development of 2-D electrophoresis gel image analysis is a continuously ongoing process. I have used Phoretix 2D since 2000 and I have found it to be a very useful tool in the analysis of my 2D gels. However, manual spot editing and matching are still the most critical and time-consuming steps. Depending on the expertise of the operator and the complexity of the pattern, spot editing can take up to several hours per gel. The software is quite easy to use and new users can quickly gain familiarity with it.
Simone Ferrero, M.D.
Ph.D. Student
Reproductive Physiology Laboratory
St Bartholomew’s Hospital, London