Two-dimensional protein separation with advanced sample and buffer isolation using microfluidic valves

Methods are described to achieve more efficient multidimensional protein separation in a microfluidic channel. The new methods couple isoelectric focusing (IEF) with high ionic strength electrophoretic separations by active microvalve control in a microchip. Several experiments demonstrating independent 2D separation were performed, and critical parameters for optimal chip performance were identified, including channel passivation, electroosmosis control, and IEF linearity control. This strategy can be used for integration of different-heterogeneous separation techniques, such as IEF, capillary electrophoresis, and liquid chromatography. This new device can be ideal for preseparation and preconcentration of complex biomolecule samples for a streamlined biomolecule analysis using mass spectrometry.