A dual purpose multilane channel system to carry out isoelectric focusing (IEF) and asymmetrical flow field-flow fractionation (IEF-AFlFFF or IEF-AF4) was developed for the high-speed fractionation of a proteome in two dimensions (2D): isoelectric point (pI) and hydrodynamic diameter (d(s)). Separation of proteins is initially achieved by differences in pI using IEF in an open thin segment, which is formed by interconnecting the beginning part of six parallel flow FFF channels in the lateral direction. After IEF, each protein pool of a different pI interval is simultaneously separated in an orthogonal direction by d(s) in six individual AF4 channels. The developed IEF-AF4 multilane channel system provides ultimate nongel, elution based, and 2D protein separation at an improved separation speed; the entire separation can be processed within 30 min, compared to approximately 3 h with the previously developed capillary isoelectric focusing-hollow fiber FlFFF (CIEF-HFFlFFF or CIEF-HF5) (Kang, D.; Moon, M. H. Anal. Chem. 2006, 78, 5789-5798) or approximately 36 h with 2D-polyacryamide gel electrophoresis (2D-PAGE). An initial evaluation of IEF-AF4 was performed to investigate the influence of ampholyte concentration and IEF voltage on the separation of standard protein mixtures.