High throughput tryptic digestion via poly (acrylamide-co-methylenebisacrylamide) monolith based immobilized enzyme reactor

High throughput tryptic digestion via poly (acrylamide-co-methylenebisacrylamide) monolith based immobilized enzyme reactorA poly (acrylamide-co-methylenebisacrylamide) (poly (AAm-co-MBA)) monolith was prepared by thermal polymerization in the 100 or 250 mu m i.d. capillary. The monolithic support was activated by ethylenediamine followed by glutaraldehyde. Trypsin was then introduced to form an immobilized enzyme reactor (IMER). The prepared IMER showed a reliable mechanical stability and permeability (permeability constant K = 2.65 x 10(-13) m(2)). With BSA as the model protein, efficient digestion was completed within 20 s, yielding the sequence coverage of 57%, better than that obtained from the traditional in-solution digestion (42%), which took about 12 h. Moreover. BSA down to femtomole was efficiently digested by the IMER and positively identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). To test the applicability of IMER for complex sample profiling, proteins extracted from Escherichia coli were digested by the IMER and further analyzed by nanoreversed phase liquid chromatography-electrospray ionization-mass spectrometry (nanoRPLC-ESI-MS/MS). In comparison to in-solution digestion, despite slightly fewer proteins were positively identified at a false discovery rate (FDR) of similar to 1% (333 vs 411), the digestion time used was largely shortened (20 s vs 24 h), implying superior digestion performance for the high throughput analysis of complex samples. (C) 2011 Elsevier B.V. All rights reserved