Submitted to: Electrophoresis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/30/2006
Publication Date: 1/8/2007
Citation: Yang, Y., Thannhauser, T.W., Li, L., Zhang, S. 2007. Development of an integrated approach for evaluation of 2-D gel image analysis: Impact of multiple proteins in single spots on comparative proteomics in conventional 2-D gel/MALDI workflow. Electrophoresis. 28:2080-2094.
Interpretive Summary: Two-dimensional (2-D) electrophoresis continues to be the most utilized front end separation method for high throughput protein analysis (proteomics) despite the growing number of chromatographic alternatives. While it has many advantages, 2-D electrophoresis has a number of well known disadvantages, the most important being that it is not possible to resolve all the proteins produced by an organism in a single analysis. 2-D electrophoresis can resolve approximately 3,000 distinct protein spots but many organisms produce more than one million chemically distinct proteins. Thus, it is highly likely that most gel-spots will contain more than a single protein. This observation poses a significant challenge for the proper interpretation of comparative experiments in which changes in protein abundance are inferred from changes in gel spot staining intensities. Using two formats of mass spectrometry we evaluated the impact of the presence of multiple proteins in individual gel spots on the interpretation of gel image analysis. We conclude that the effects are negligible, providing the fold change used to determine the significance of increases or decreases in expression level is high. Furthermore, we investigate the use of an empirically derived quantity know as the “exponentially modified protein abundance index” (emPAI) as a means to properly distribute the observed change in normalized spot intensities among the various protein components of the spot. We then propose a novel workflow combining 2-D gel electrophoresis and liquid chromatography-electrospray ionization tandem mass spectrometry (2-D GeLC-MS/MS) with the emPAI analysis as a complementary alternative to the traditional gel-based quantitative proteomics analysis. This new approach will greatly facilitate studies designed to identify proteins associated with the ability to accumulate beta-carotene in cauliflower and improve aluminum tolerance in corn.
Technical Abstract: With 2-D gel mapping, it is often observed that essentially identical proteins migrate to different positions in the gel, while some seemingly well-resolved protein spots consist of multiple proteins. These observations can undermine the validity of gel-based comparative proteomic studies. Through a comparison of protein identifications using direct MALDI-TOF/TOF and LC-ESI/MS/MS analyses of 2-D gel separated proteins from cauliflower florets, we have developed an integrated approach to improve the accuracy and reliability of comparative 2-D electrophoresis. From 46 spots of interest, we identified 51 proteins by MALDI-TOF/TOF analysis and 108 proteins by LC-ESI/MS/MS. The results indicate that 75% of the analyzed spots contained multiple proteins. A comparison of hit rank for protein identifications showed that 37 out of 43 spots identified by MALDI matched the top-ranked hit from the ESI/MS/MS. By using the exponentially modified protein abundance index (emPAI) to determine the abundance of the individual component proteins for the spots containing multiple proteins, we found that the top-hit proteins from 40 out of 43 spots identified by MALDI matched the most abundant proteins determined by LC-MS/MS. Furthermore, our 2-D-GeLC-MS/MS results show that the top-hit proteins in 44 identified spots contributed on average 81% of the spots’ staining intensity. This is the first quantitative measurement of the average rate of false assignment for direct MALDI analysis of 2-D gel spots using a new integrated workflow (2-D gel imaging, “2-DGeLC-MS/MS”, and emPAI analysis). Here, the new approach is proposed as an alternative to traditional gel-based quantitative proteomics studies.