This chapter describes the development of the two-dimensional separation methods using flow field-flow fractionation (F4) and isoelectric focusing for proteomics utility. The methods described here are the rapid, non-gel-based, on-line, two-dimensional separation methods in which proteins are separated by isoelectric focusing (IEF) in the first dimension according to differences in isoelectric point (pI) followed by size based separation using F4 (either hollow fiber F4 or multilane asymmetrical F4 channels) in an orthogonal direction. In this chapter, the capillary IEF-HF5 and IEF-AF4 methods are described with the demonstration of system performances using protein standards. Also described are the applications to human urinary proteome samples in which proteome fractions are collected and tryptic digested for the proteomic analysis using nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS-MS).
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