|Thannhauser, Theodore - Ted|
Submitted to: Journal of Biomolecular Techniques
Publication Type: Abstract Only
Publication Acceptance Date: 5/30/2006
Publication Date: 7/14/2006
Citation: Thannhauser, T.W., Yang, Y., Zhang, S. 2006. Comparison of protein identification by maldi-tof/tof and lc electrospray ionization tandem ms for 2d gel separated cauliflower samples. Journal of Biomolecular Techniques. 17:48.
Technical Abstract: In 2D-gel mapping, it is well-known that essentially identical proteins migrate to different positions in the gel (due to PTMs etc.) while some, seemingly, well resolved spots consist of multiple proteins. Clearly, this observation can have dire consequences for the validity of gel-based comparative studies. The goal of this work is to compare the results of protein identification using both the MALDI-TOF/TOF and LC-ESI/MS/MS approaches for 2D-gel separated cauliflower proteins with a view towards better understanding how these different identification tools can be used to help us properly interpret the 2-D electropherograms. Proteins were extracted from two types of cauliflowers: white colored homozygous wild-type (or/or) and orange colored heterozygote (Or/or). After 2D electrophoresis and image analysis, a total of 46 spots of interest were identified (> 2-fold up/down regulation). These were digested in situ and analyzed by mass spectrometry. Using the MALDL-TOF/TOF we were able to identify the 46 proteins from 43 of the 46 spots. Thirty-seven of these were unique. With the LC-ESI/MS/MS approach, we were able to identify 95 proteins with 80 unique Ids. Comparisons between MALDI and ESI/MS/MS results reveal that 36 out of 43 spots identified by MALDI match the top-hit identified by ESI/MS/MS. The remaining 7 spots identified by MALDI matched the second best hit found by LC/MS/MS. Since the highest scoring protein usually has a significantly lower ion score and less sequence coverage (due to its lower abundance) we conclude that the gel-based quantitation analysis is still reliable.