Desalting electroeluted proteins with hydrophilic interaction chromatography.

We describe a chromatographic procedure for the removal of sodium dodecyl sulfate (SDS) from proteins isolated by electroelution. It involves chromatography of electroeluates on poly(2-hydroxyethyl-aspartamide) silica, a support initially developed for hydrophilic interaction chromatography. The electroeluate, dialyzed against ammonium bicarbonate-SDS buffer, is directly injected onto the column, which is equilibrated in an n-propanol concentration greater than 60%. Bound proteins are eluted with a gradient of decreasing n-propanol. This procedure removes essentially all of the Coomassie blue-related contaminants and separates SDS from the protein. Due to the use of volatile buffer systems, the proteins are recovered in completely salt-free form, which facilitates further protein manipulation. After removal of the organic solvent from the chromatographic desalting step, the recovered proteins are directly amenable to N-terminal protein sequencing and, after evaporation of the organic phase, to enzymatic digestions and subsequent separation of fragments by reverse-phase HPLC.