Facile Trypsin Immobilization in Polymeric Membranes for Rapid, Efficient Protein Digestion

摘要

Sequential adsorption of poly(styrene sulfonate) andntrypsin in nylon membranes provides a simple, inexpensivenmethod to create stable, microporous reactors for fastnprotein digestion. The high local trypsin concentration andnshort radial diffusion distances in membrane pores facilitatenproteolysis in residence times of a few seconds,nand the minimal pressure drop across the thin membranesnallows their use in syringe filters. Membranendigestion and subsequent MS analysis of bovine serumnalbumin provide 84% sequence coverage, which is highernthan the 71% coverage obtained with in-solution digestionnfor 16 h or the <50% sequence coverages of othernmethods that employ immobilized trypsin. Moreover,ntrypsin-modified membranes digest protein in the presencenof 0.05 wt % sodium dodecyl sulfate (SDS), whereasnin-solution digestion under similar conditions yields nonpeptide signals in mass spectra even after removal of SDS.nThese membrane reactors, which can be easily preparednin any laboratory, have a shelf life of several months andncontinuously digest protein for at least 33 h withoutnsignificant loss of activity.