Enhanced protein digestion through the confinement of nanozeolite-assembled microchip reactors.

An on-chip microreactor was proposed toward the acceleration of protein digestion through the construction of a nanozeolite-assembled network. The nanozeolite microstructure was assembled using a layer-by-layer technique based on poly(diallyldimethylammonium chloride) and zeolite nanocrystals. The adsorption of trypsin in the nanozeolite network was theoretically studied based on the Langmuir adsorption isotherm model. It was found that the controlled trypsin-containing nanozeolite networks assembled within a microchannel could act as a stationary phase with a large surface-to-volume ratio for the highly efficient proteolysis of both proteins at low levels and with complex extracts. The maximum proteolytic rate of the adsorbed trypsin was measured to be 350 mM min-1 microg-1, much faster than that in solution. Moreover, due the large surface-to-volume ratio and biocompatible microenvironment provided by the nanozeolite-assembled films as well as the microfluidic confinement effect, the low-level proteins down to 16 fmol per analysis were confidently identified using the as-prepared microreactor within a very short residence time coupled to matrix-assisted laser desorption-time-of-flight mass spectrometry. The on-chip approach was further demonstrated in the identification of the complex extracts from mouse macrophages integrated with two-dimensional liquid chromatography-electrospray ionization-tandem mass spectrometry. This microchip reactor is promising for the development of a facile means for protein identification.