A Capillary Monolithic Trypsin Reactor for Efficient Protein Digestion in Online and Offline Coupling to ESI and MALDI Mass Spectrometry

摘要

We describe the preparation of a capillary trypsin immobilizednmonolithic enzyme reactor (IMER) for a rapidnand efficient digestion of proteins down to the femtomolenlevel. Trypsin was immobilized on a poly(glycidyl methacrylate-nco-acrylamide-co-ethylene glycol dimethycrylate)nmonolith using the glutaraldehyde technique. Digestionnefficiencies of the IMER were evaluated using modelnproteins and protein mixtures as well as chemically crosslinkednlysozyme regarding the addition of denaturants andnincreasing digestion temperature. The trypsin IMERndescribed herein is applicable for the digestion of proteinnmixtures. Even at a 1000-fold molar excess of onenprotein, low-abundance proteins are readily identified, inncombination with MS/MS analysis. An online setup of thenIMER with reversed phase nano-HPLC separation andnnano-ESI-MS/MS analysis was established. The greatnpotential of the trypsin IMER for proteomics applicationsncomprise short digestion times in the range of secondsnto minutes, in addition to improved digestion efficiencies,ncompared to in-solution digestion.