Development of a Multilane Channel System for Nongel-Based Two-Dimensional Protein Separations Using Isoelectric Focusing and Asymmetrical Flow Field-Flow Fractionation

摘要

A dual purpose multilane channel system to carry out isoelectric focusing (IEF) and asymmetrical flow field-flow fractionation (IEF-AF1FFF 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 approx3 h with the previously developed capillary isoelectric focusing-hollow fiber F1FFF (CIEF-HFF1FFF or CIEF-HF5) (Kang, D.; Moon, M. H. Anal. Chem. 2006, 78, 5789-5798) or approx36 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.