%0 Journal Article
%T An Economical High-Throughput Protocol for Multidimensional Fractionation of Proteins
%A David John Tooth
%A Varun Gopala Krishna
%A Robert Layfield
%J International Journal of Proteomics
%D 2012
%I Hindawi Publishing Corporation
%R 10.1155/2012/735132
%X 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,
%U http://www.hindawi.com/journals/ijpro/2012/735132/