ABSTRACT
As a step toward a fully automated front-end microfluidic chip for MS proteomics, we propose a system capable of performing online tryptic digest and ESI-MS, using a replaceable on-chip digestion microcolumn based on self-assembled magnetic particles.
Subject(s)
Microfluidic Analytical Techniques/instrumentation , Spectrometry, Mass, Electrospray Ionization/instrumentation , Trypsin/chemistry , Dimethylpolysiloxanes/chemistry , Magnetics , Microfluidic Analytical Techniques/methods , Proteins/analysis , Proteomics/methods , Spectrometry, Mass, Electrospray Ionization/methodsABSTRACT
A microreactor for proteinase K (PK)-mediated protein digestion was developed as a step towards the elaboration of a fully integrated microdevice for the detection of pathological prion protein (PrP). PK-grafted magnetic beads were immobilized inside a polydimethylsiloxane (PDMS) microchannel using a longitudinal magnetic field parallel to the flow direction and a magnetic field gradient, thereby forming a matrix for enzymatic digestion. This self-organization provided uniform pore sizes, a low flow resistance and a strong reaction efficiency due to a very thin diffusion layer. The microreactor's performance was first evaluated using a model substrate, succinyl-ala-ala-ala-paranitroanilide (SAAAP). Reaction kinetics were typically accelerated a hundred-fold as compared to conventional batch reactions. Reproducibility was around 98% for on-chip experiments. This microsystem was then applied to the digestion of prion protein from brain tissues. Controlled proteolysis could be obtained by varying the on-chip flow rate, while a complete proteolysis of normal protein was achieved in only three minutes. Extracts from normal and pathological brain homogenates were finally compared and strong discrimination between normal and pathological samples was demonstrated.
