Optimization of poly(GMA-co-EDMA) monolithic support for trypsin nanoreactor fabrication.
Fabrication of poly(glycidyl methacrylate-co-ethylene dimethacrylate) [also referred to as poly(GMA-co-EDMA)] monoliths was optimized as supporting material for trypsin digestion nanoreactors. Reaction parameters, such as polymerization time, porogen concentration, and monomer to crosslinker ratios, were evaluated in respect to the permeability of the resulting monolith and their effect on digestion efficiency, estimated by mass spectrometric analysis of a model protein cytochrome C. The structural homogeneity of the resulting monolithic support was checked by scanning electron microscopy. The best nanoreactor performance, measured by the reduction of nanoreactor backpressure and increased sequence coverage of cytochrome C, was achieved with 8% 2-octanol (porogen) 20%/20% glycidyl methacrylate to ethylene glycol dimethacrylate ratio and 5 h of polymerization time. Digestion of as low as 3 microg of cytochrome C with 77% sequence coverage was obtained using the optimized trypsin nanoreactor.
Monzo
Alex
A
Horváth Laboratory of Bioseparation Sciences, University of Innsbruck, Austria.
Rejtar
Tomas
T
Guttman
András
A
D000595:Amino Acid Sequence; D045304:Cytochromes c / Q000737:chemistry; D013058:Mass Spectrometry; D008768:Methylmethacrylates / Q000737:chemistry*; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D036103:Nanotechnology; D014357:Trypsin / Q000737:chemistry*
D008768:Methylmethacrylates; C020347:GMA-EDMA; D045304:Cytochromes c; D014357:Trypsin