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United States Department of Agriculture

Agricultural Research Service

Title: Analysis of Detergent Extraction Methods and 1d Sds-Page for Enrichment of Plant Membrane Proteins and Subsequent Detection by Ms/ms

Authors
item Lee, Joohyun
item Garrett, Wesley
item Feng, Jian - JOHNS HOPKINS UNIVERSITY
item Naiman, Daniel - JOHNS HOPKING UNIVERSITY
item Cooper, Bret

Submitted to: BARC Poster Day
Publication Type: Abstract Only
Publication Acceptance Date: May 1, 2006
Publication Date: May 1, 2006
Citation: Lee, J., Garrett, W.M., Feng, J., Naiman, D.Q., 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]. BARC Poster Day. p. 31.

Technical Abstract: 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 coupled with mass spectrometry, is able to overcome some of the deficiencies of 2D-PAGE. Still MudPIT is not readily compatible with common 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 detergents and detergent clean-up methods that may be compatible with mass spectrometry. In comparison to our standard MudPIT methods, 1D SDS-PAGE separation followed by sequential MS/MS analysis of peptides eluted from the gel resolved 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 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. Finally, we examined docecyl beta-maltoside (DBM), an acid-labile detergent that is compatible with mass spectrometry. DBM increased protein solubilization over our standard procedures and allowed us to detect more proteins. We conclude that combined DBM/MudPIT and 1D SDS-PAGE analyses are beneficial for sampling a large number of proteins. Several replicates of each analysis will help resolve a variety of proteins, which can result in increased coverage of membrane-spanning proteins by virtue of increased sampling.

Last Modified: 4/18/2014
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