Effect of sodium dodecyl sulfate on protein separation by hollow fiber flow field-flow fractionation.
Effects of protein denaturation and formation of protein-sodium dodecyl sulfate (SDS) complexes on protein separation and identification were investigated using hollow fiber flow field-flow fractionation (HF5) and nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). Denaturation and formation of protein-SDS complexes prior to HF5 separation resulted an increase in the retention of few protein standards due to unfolding of the protein structures and complexation, yielding ~30% increase in hydrodynamic diameter. In addition, low molecular weight proteins which could be lost from the HF membrane due to the pore size limitation showed an increase of peak recovery about 2-6 folds for cytochrome C and carbonic anhydrase. In the case of proteins composed of a number of subunits, denaturation resulted in a decrease in retention due to dissociation of protein subunits. A serum proteome sample, denatured with dithiothreitol and SDS, was fractionated by HF5, and the eluting protein fractions after tryptic digestion were analyzed for protein identification using nLC-ESI-MS-MS. The resulting pools of identified proteins were found to depend on whether the serum sample was treated with or without denaturation prior to the HF5 run due to differences in the aqueous solubility of the proteins. The enhancement of protein solubility by SDS also increased the number of identified membrane proteins (54 vs. 31).
Department of Chemistry, Yonsei University, Seoul, 120-749, South Korea.
Ki Hun
KH
Kim
Ju Yong
JY
Lee
Myeong Hee
MH
Moon
D000818:Animals; D004229:Dithiothreitol / Q000737:chemistry; D031521:Fractionation, Field Flow / Q000379:methods*; D006801:Humans; D011489:Protein Denaturation; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D044967:Serum / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D004229:Dithiothreitol