Continuous free-flow electrophoresis separation of cytosolic proteins fromthe human colon carcinoma cell line LIM 1215: A non two-dimensional gel electrophoresis-based proteome analysis strategy

The conventional approach for analyzing the protein complement of a genome involves the combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometric based protein identification technologies. While 2-DE is a powerful separation technique, it is severely limited by the insolubilit y of certain classes of proteins (e.g. hydrophobic membrane proteins), as w ell as the amount of protein that can be processed. Here, we describe a sim ple procedure for resolving complex mixtures of proteins that involves a co mbination of free flow electrophoresis (FFE), a liquid-based isoelectric fo cussing (IEF) method, and sodium doclecylsulfate-polyacrylamide gel electro phoresis (SDS-PAGE). Resolved proteins were identified by peptide fragment sequencing using capillary column reversed-phase high performance liquid ch romatography (RP-HPLC)/mass spectrometry (MS). An initial demonstration of the method was performed using digitonin/ethylenediaminetetraacetic acid ED TA extracted cytosolic proteins from the human colon carcinoma cell line, L IM 1215. Cytosolic proteins were separated by liquid-based IEF (pH range 3- 10) into 96 fractions, and each FFE fraction was further fractionated by SD S-PAGE. Selected protein bands were excised from the SDS-PAGE gel, digested in situ with trypsin, and subsequently identified by on-line RP-HPLC/elect rospray-ionization ion trap MS. Our results indicate that FIFE is: (i) an e xtremely powerful liquid-based IEF method for resolving proteins; (ii) not limited by the amount of sample that can be loaded onto the instrument; and (iii) capable of fractionating intact protein complexes (a potentially pow erful tool for cell-mapping proteomics). An up-to-date list of cytosolic pr oteins from the human colorectal carcinoma cell line LIM 1215 can be found in the Joint Protein Structure Laboratory (JPSL) proteome database. This in formation will provide an invaluable resource for future proteomics-based b iological studies of colon cancer. The JPSL proteome database can be access ed through the World Wide Web (WWW) network (http://www.ludwig.edu.au/ jpsl /jpslhome.html).