Journal article
Limited proteolysis via millisecond digestions in protease-modified membranes
YJ Tan, WH Wang, Y Zheng, J Dong, G Stefano, F Brandizzi, RM Garavito, GE Reid, ML Bruening
Analytical Chemistry | AMER CHEMICAL SOC | Published : 2012
DOI: 10.1021/ac3019153
Abstract
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 millisecond 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 kDa) provides a resolution of 1-2 amino acids. Under denaturing conditions..
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Awarded by National Institute of General Medical Sciences
Funding Acknowledgements
We are grateful to the U.S. National Institutes of Health (Grant GM 080511 to M.L.B.), the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (Award Number DE-FG02-91ER20021 to F.B.), the National Science Foundation (MCB Grant 0948584 to F.B.), and the MSU-REF Center Program (Grant REF03-016, R.M.G) for funding portions of this work.