Protein Concentration by Hydrophilic Interaction Chromatography Combined with Solid Phase Extraction

Summary

Hydrophilic interaction chromatography (HILIC) is a variant of normal phase chromatography, in which analyte molecules attach to a solid support (e.g., poly [2-hydroxyethyl] aspartamide silica) by the action of a mobile phase containing a high amount of organic modifier such as acetonitrile or propanol. Elution of analyte molecules is achieved, when the resin is washed with a solution devoid of organic solvent. The method and its basic principles have been extensively described by Alpert et al. (1). Applications of HILIC include the isolation of membrane proteins, electroeluted from SDS-PAGE gels (2), glycopeptides (3), and post-translationally modified protein variants (4).

Here, an extended application of hydrophilic interaction chromatography is described, which allows nonselective enrichment of proteins from various dilute sources before two-dimensional gel electrophoresis (2-D PAGE). The use of this approach is demonstrated by processing protein containing samples from high resolution, preparative isoelectric focusing (IEF) separations achieved by carrier free electrophoresis (free flow electrophoresis [FFE]), described in (5). Furthermore the concept of compatible recovery is shown which allows buffer exchange, concentration and recovery of bound proteins in one step directly into a sample buffer required for 2-D PAGE.