Effect of strong detergents and chaotropes on the detection of proteins intwo-dimensional gels

The solubilization of a particular protein is mandatory for its subsequent resolution and detection in two-dimensional gels. However, the extraction s olutions, that are compatible with the first-dimensional separation step, s uch as urea and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate ( CHAPS), do not solubilize all proteins in a sample. We studied the effect o f various common, strong detergents and chaotropes, widely used as solubili zing agents, such as sodium dodecyl sulfate, lithium dodecyl sulfate and gu anidine hydrochloride, on the solubilization of the total and membrane prot eins of the bacterium Haemophilus influenzae. The proteins solubilized with each system were analyzed by two-dimensional electrophoresis and these of interest were identified by matrix assisted laser desorption/ionization-mas s spectrometry (MALDIMS). Use of sodium dodecyl sulfate, lithium dodecyl su lfate or guanidine hydrochloride for the solubilization of total proteins o f the microorganism resulted in the detection of several additional spots, representing mainly outer membrane proteins, in comparison with those detec ted in the soluble protein fraction. Solubilization of the proteins of the cell envelope fraction with sodium dodecyl sulfate did not result in a more efficient protein detection when compared to the extraction with the urea/ CHAPS system. When the dry immobilized pH gradient strips were rehydrated i n a solution containing the proteins of the membrane fraction solubilized w ith sodium dodecyl sulfate or lithium dodecyl sulfate, a larger number of p rotein spots were detected in comparison with strips that were rehydrated i n the urea/CHAPS solution. However, no improvement was observed in comparis on with protein application in sample cups. The additional proteins detecte d with the use of strong detergents and chaotropes are in the majority diff icult to solubilize and less hydrophobic proteins.