ARS | Publication request: Reproducibility of an Integrated Quantitation Method Coupling 2D GeLC-MS/MS with the emPAI for Comparative Proteomics

Submitted to: Journal of Biomolecular Techniques
Publication Type: Abstract Only
Publication Acceptance Date: January 25, 2008
Publication Date: February 16, 2008
Citation: Yang, Y., Zhang, S., Thannhauser, T.W. 2008. Reproducibility of an Integrated Quantitation Method Coupling 2D GeLC-MS/MS with the emPAI for Comparative Proteomics. Journal of Biomolecular Techniques. Vol. 19:52.

Technical Abstract: In 2D gel mapping, most protein spots consist of multiple proteins posing a significant challenge for the proper interpretation of gel-based comparative experiments. Previously we introduced an approach integrating 2-D difference gel electrophoresis and LC-MS/MS analysis with the exponentially modified protein abundance index to correctly distribute a spot’s volume to each of its protein constituents. However, the experimental variation associated with the procedure was not fully established. Here we evaluated the reliability of this method by analyzing replicate sets of samples. Protein was extracted from an aluminum tolerant cultivar of maize under Al free and treated conditions and separated by 2-D DIGE. After image analysis, spots of interest were excised, digested and analyzed by nLC-MS/MS. Protein identifications were made and associated emPAI values were generated for each spot by Mascot 2.2 through searching the maize database. 27 matched spot pairs from the control and treated states were analyzed in duplicate. The results show that all contain multiple proteins, averaging 10 proteins/spot. The volume of each spot was distributed among its constituent proteins on the basis of their mol fraction as determined by the emPAI. The fractional spot volume for individual proteins was compared between control and treated samples. Many instances were found in which down regulated proteins were discovered in up-regulated spots and vice verse, demonstrating the importance of dissecting and distributing the change in spot volume. Approximately 90% of the protein identifications obtained from replicate spots matched and their mol fractions were found to be nearly identical. Unmatched proteins and instances where the mol fractions of replicates deviated greatly were limited to the lowest abundance proteins and do not impact the overall results significantly, suggesting the approach is reliable.