Two dimensional liquid phase separations of proteins using online fractionation and concentration between chromatographic dimensions

Multi-dimensional liquid chromatography is often presented as an alternative to two-dimensional (2-D) gel electrophoresis for separating complex protein mixtures. The vast majority of analytical-scale 2-D LC systems have employed either off-line fractionation or stepped gradients in the first dimension separation. The latter severely restrict flexibility in setting up the first dimension gradient. We propose a novel two-dimensional LC system that employs online fractionation of proteins into a series of small reversed phase trapping columns. These traps effectively decouple the two separation dimensions and avoid problems associated with off-line fraction collection. Flexibility in determining the gradient programs for the two separations is thus enhanced. The reduced diameter of the trapping columns concentrates analyte between chromatographic dimensions. The apparatus is coupled with online electrospray time-of-flight mass spectrometry to characterize ribosomal proteins of Caulobacter crescentus.