|Feng, Jian - JOHNS HOPKINS UNIVERSITY|
|Naiman, Daniel - JOHNS HOPKINS UNIVERSITY|
Submitted to: American Society for Mass Spectrometry
Publication Type: Abstract Only
Publication Acceptance Date: March 1, 2006
Publication Date: March 1, 2006
Citation: Lee, J., Garrett, W.M., Feng, J., Naiman, D., Cooper, B. 2006. Analysis of detergent extraction methods and 1d sds-page for enrichment of plant membrane proteins and subsequent detection by ms/ms [abstract. American Society for Mass Spectrometry. Technical Abstract: Novel Aspect: Evaluation of commercial membrane protein extraction kits; establishing protocols to analyze small amounts of plant samples by 1D SDS-PAGE MS/MS. Introduction: Although 2D-PAGE is a major technique used in the plant proteomics field, the lack of resolution for hydrophobic-membrane proteins, low abundant proteins, and basic proteins is a real obstacle to profiling whole plant proteomes with this separation method. On the other hand, MudPIT, a LC-based peptide separation technique, is able to overcome some of the deficiencies of 2D-PAGE. Still MudPIT is not readily compatible with many detergents that are useful for extracting hydrophobic proteins from a sample. With this in mind, we have attempted to enrich for membrane proteins using various detergent-containing commercial kits, separated the proteins by 1D SDS-PAGE, evaluated trypsin digested peptides by MS/MS and have compared these findings to those produced using our routine MudPIT methodology. Methods: A Membrane Extraction Kit (Sigma) and a Signal Protein Extraction Kit (Bio-Rad) were used to isolate proteins from Arabidopsis leaves. Plant samples were also extracted with home-made SDS-Tris buffer or with acetone/TCA for comparison. Proteins were separated in 4-12 % gradient Novex Bis-Tris gels. Each lane was sliced into 32 fractions, proteins were digested in the gel and peptides extracted. Peptides were analyzed by LC-MS/MS. MS/MS spectra were searched using Mascot, and the identified proteins were evaluated by Protein Panorama, our custom software that assembles a probability-based and parsimonious set of non-redundant proteins. Preliminary Data: We are evaluating different extraction procedures and shotgun proteomics analyses to 1) increase the number of proteins we are able to detect in a single analysis and 2) increase the number of membrane-spanning proteins that we can detect. In comparison to a MudPIT analysis, 1D SDS-PAGE separation followed by sequential MS/MS analysis of peptides eluted from the gel resolves several hundred more proteins. Alone, the 1D SDS-PAGE gel method is simple and robust, and is compatible with ion trap mass spectrometers coupled with an autosampler, HPLC pump, switching valve and microspray source. This method should be of interest to plant biologists who are looking beyond 2D-PAGE MS/MS to identify plant proteins but do not have MudPIT capabilities. We also show that the 1D SDS-PAGE separation process is compatible with detergents that are part of commercial membrane protein enrichment protocols, which are not compatible with routine MudPIT protocols. However, these commercial kits do not necessarily enrich for an overwhelming amount of membrane-spanning proteins as determined by ARAMEMNON, a public portal useful for identifying proteins with membrane spanning domains. We conclude that combined MudPIT and 1D SDS-PAGE analyses are beneficial for sampling a larger number of proteins and propose several replicates of each analysis to achieve maximum protein sample, which can result in increased coverage of membrane-spanning proteins by virtue of increased sampling. We are currently evaluating other detergents, commercial kits and are evaluating sequential extractions of insoluble plant materials that may help enrich for membrane-spanning proteins.