Capillary size-exclusion chromatography as a gel-free strategy in plasma proteomics

Abstract

Although 2D-PAGE has been widely used as the primary method for protein separation, difficulties in displaying proteins with an extreme values of isoelectric point (pl), molecular size and hydrophobicity limit the technique(1). In addition, time consuming steps involving protein transfer and extraction from the gel-pieces can result in sample loss. Here, we describe a novel protein separation technique with capillary size-exclusion chromatography (CSEC) for rapid protein identification from human plasma. The method includes protein fractionation along with molecular size followed by in-solution tryptic digestion and peptide analysis through reversed phase liquid chromatography (RPLC) coupled to nanoflow electrospray-tandem mass spectrometry (ESI-MS/MS)(2-4). Tryptic peptides are applied on a 100 mu m i.d. x 10 mm length pre-column and then separated on a 75 mu m x 200 mm analytical column at -100 nL/min flow rate. Proteins were identified over the wide ranges of pi (3.7-12.3) when this technique was applied to the analysis of 1-2 mu L of human plasma. This gel-free system provides fast fractionation and may be considered a complementary technique to SIDS-PAGE in proteomics.