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

摘要

Sequential adsorption of poly(styrene sulfonate) and trypsin in nylon membranes provides a simple, inexpensive method to create stable, microporous reactors for fast protein digestion. The high local trypsin concentration and short radial diffusion distances in membrane pores facilitate proteolysis in residence times of a few seconds, and the minimal pressure drop across the thin membranes allows their use in syringe filters. Membrane digestion and subsequent MS analysis of bovine serum albumin provide 84percent sequence coverage, which is higher than the 71percent coverage obtained with in-solution digestion for 16 h or the <50percent sequence coverages of other methods that employ immobilized trypsin. Moreover, trypsin-modified membranes digest protein in the presence of 0.05 wt percent sodium dodecyl sulfate (SDS), whereas in-solution digestion under similar conditions yields no peptide signals in mass spectra even after removal of SDS. These membrane reactors, which can be easily prepared in any laboratory, have a shelf life of several months and continuously digest protein for at least 33 h without significant loss of activity.