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1.  Limited Proteolysis Via Millisecond Digestions in Protease-Modified Membranes 
Analytical chemistry  2012;84(19):8357-8363.
Sequential adsorption of poly(styrene sulfonate) (PSS) and proteases in porous nylon yields enzymatic membrane reactors for limited protein digestion. Although a high local enzyme density (~30 mg/cm3) and small pore diameters in the membrane lead to digestion in < 1 s, the low membrane thickness (170 μm) affords control over residence times at the ms level to limit digestion. Apomyoglobin digestion demonstrates that peptide lengths increase as the residence time in the membrane decreases. Moreover, electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) on a large myoglobin proteolytic peptide (8 kD) provides a resolution of 1–2 amino acids. Under denaturing conditions, limited membrane digestion of bovine serum albumin (BSA) and subsequent ESI-Orbitrap MS analysis reveal large peptides (3 kD–10 kD) that increase the sequence coverage from 53 % (2-s digestion) to 82 % (0.05-s digestion). With this approach we also performed membrane-based limited proteolysis of a large Arabidopsis GTPase, ROOT HAIR DEFECTIVE 3 (RHD3), and showed suitable probing for labile regions near the C-terminus to suggest what protein reconstruction might make RHD3 more suitable for crystallization.
PMCID: PMC3683959  PMID: 22950601
2.  Facile Trypsin Immobilization in Polymeric Membranes for Rapid, Efficient Protein Digestion 
Analytical chemistry  2010;82(24):10045-10051.
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.
PMCID: PMC3052767  PMID: 21087034

Results 1-2 (2)