title: Facile trypsin immobilization in polymeric membranes for rapid, efficient protein digestion.
abstract: 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 84% sequence coverage, which is higher than the 71% coverage obtained with in-solution digestion for 16 h or the <50% sequence coverages of other methods that employ immobilized trypsin. Moreover, trypsin-modified membranes digest protein in the presence of 0.05 wt % 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.
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, United States.
authors.forename: Fei
authors.initials: F
authors.lastname: Xu
authors.forename: Wei-Han
authors.initials: WH
authors.lastname: Wang
authors.forename: Yu-Jing
authors.initials: YJ
authors.lastname: Tan
authors.forename: Merlin L
authors.initials: ML
authors.lastname: Bruening
mesh_terms: D000818:Animals; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D008722:Methods; D011108:Polymers; D011506:Proteins / Q000378:metabolism*; D012710:Serum Albumin, Bovine; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D011108:Polymers; D011506:Proteins; D012710:Serum Albumin, Bovine; D014357:Trypsin