Rapid protein digestion and identification using monolithic enzymatic microreactor coupled with nano-liquid chromatography-electrospray ionization mass spectrometry

A novel monolithic enzymatic microreactor was prepared in the fused-silica capillary by in situ polymerization of acrylamide (AA), N-acryloxysuccinimide (NAS) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, which could offer very low back pressure, enabling the fast digestion of proteins. The performance of the monolithic microreactor was demonstrated by digesting cytochrome c at high flow rate, and the comparisons between the in-solution digestion and on-column reaction were made by a nano-high performance liquid chromatography-mass spectrometry (nano-HPLC-MS) system. The performance of the monolithic microreactor was demonostrated with the digestion of cytochrome c at the fast flow rate of 1 μL/min, which afforded a residence time of 7 s, yielding a sequence coverage of 54.81% using strict multiple database searching thresholds. Furture more, a mixture of four standard proteins was digested and analyzed using the on-line digestion and nano-HPLC-MS system. The results showed the promising of such a system in the analysis of protein mixture.