A sequential protocol of multidimensional fractionation was optimised to enable the comparative profiling of fractions of proteomes from cultured human cells. Differential detergent fractionation was employed as a first step to obtain fractions enriched for cytosolic, membrane/organelle, nuclear, and cytoskeletal proteins. Following buffer exchange using gel-permeation chromatography, cytosolic proteins were further fractionated by 2-dimensional chromatography employing anion-exchange followed by reversed-phase steps. Chromatographic fractions were shown to be readily compatible with 1- and 2-dimensional gel electrophoresis or with direct analysis by mass spectrometry using linear-MALDI-TOF-MS. Precision of extraction was confirmed by reproducible SDS-PAGE profiles, MALDI-TOF-MS spectra, and quantitation of trypsinolytic peptides using LC-MS/MS (MRM) analyses. Solid phases were immobilised in disposable cartridges and mobile-phase flow was achieved using a combination of centrifugation and vacuum pumping. These approaches yielded parallel sample handling which was limited only by the capacities of the employed devices and which enabled both high-throughput and experimentally precise procedures, as demonstrated by the processing of experimental replicates. Protocols were employed at 10？mg scale of extracted cell protein, but these approaches would be directly applicable to both smaller and larger quantities merely by adjusting the employed solid- and mobile-phase volumes. Additional potential applications of the fractionation protocol are briefly described. 1. Introduction Protein identification and quantitation are major steps towards full characterization of a proteome. Many proteomic projects classically employ 2-dimensional gel electrophoresis (2DE) and are limited by both the precision of the technique and by well-documented limitations in pI and molecular size constraints [1]. Proteome fractionation is desirable in potentially yielding reduced complexity and increased dynamic range and there have been numerous approaches developed including affinity-depletion [2] and immune depletion of major components [3], liquid isoelectric focussing (IEF) [4], GelC-MS [5], and multidimensional column liquid chromatographic (MDLC) protocols [6]. Differential detergent fractionation (DDF) has long been proposed a suitably robust alternative to more challenging and costly differential ultracentrifugation approaches [7] and indeed its use was recently commercialised [8]. For several decades, liquid chromatography has been a powerful tool for separating proteins,
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