Porous polymer monolithic column with surface-bound gold nanoparticles for the capture and separation of cysteine-containing peptides

A new porous polymer monolithic capillary column modified with gold nanoparticles that enables the selective capture of cysteine-containing peptides has been developed to reduce the complexity of peptide mixtures generated in bottom-up proteomic analysis. The column is prepared from a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith through reaction of some of its epoxide moieties with cysteamine to afford a monolith rich in surface thiol groups. In situ reduction of chloroauric acid within the column is then used to form gold nanoparticles attached to the surface of the pores of the monolith. This process preserves the excellent hydrodynamic properties of the monolithic column while providing a means to selectively retain cysteine-containing peptides from an analyte due to their high affinity for gold. Release of the retained peptides is subsequently achieved with an excess of 2-mercaptoethanol. The loading capacity determined for l-cysteine using frontal elution is 2.58 ?mol/m. Since the gold?thiol link is less stable at elevated temperatures, the adsorption capacity is recovered by washing the column at 80 C for 2 h. While regeneration is easy, the multiplicity of bonds between the monolithic support and the gold nanoparticles prevents their elution even under harsh conditions such as treatment with pure 2-mercaptoethanol or treatment with boiling water for 5 h. Application of the gold modified monolith in tandem with a packed C18 capillary column is demonstrated with baseline separation of a peptide mixture achieved in a two step process. The first involves retention of cysteine-containing peptides in monolith with reversed phase separation of all other peptides, while the retained peptides are released from monolith and separated in the second step.