2D Electrophoresis: Isotachophoresis Followed by Isoelectric Focusing

The primary challenge to proteomic analysis of complex biological fluids is the vast diversity of proteins, >10⁵, and their broad dynamic range, >10⁸. These issues can be simultaneously addressed by developing separation platforms with high peak capacities and which can concentrate low-abundance proteins (1 mg/ml). Since no single separation technique can accomplish this task alone, it is necessary that several orthogonal "dimensions" of separation be multiplexed into a single platform.

This paper describes a 2D nonlinear electrophoresis system on a PDMS chip which combines isotachophoresis (ITP) followed by isoelectric focusing (IEF). ITP is used as the first dimension to preconcentrate low-abundance proteins in the sample. The concentrated sample zones are then fed into channel segments through which IEF is applied to separate them based on differences in their isoelectric points (ΔpI). A mixture of three fluorescent proteins is used to demonstrate that they are successfully resolved into three bands in the IEF dimension after ITP preconcentration. Further integration of a third dimension is then possible after IEF.