Continuous free-flow electrophoresis separation of cytosolic proteins from the 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 insolubility of certain classes of proteins (e.g. hydrophobic membrane proteins), as well as the amount of protein that can be processed. Here, we describe a simple procedure for resolving complex mixtures of proteins that involves a combination of free flow electrophoresis (FFE), a liquid-based isoelectric focussing (IEF) method, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were identified by peptide fragment sequencing using capillary column reversed-phase high performance liquid chromatography (RP-HPLC)/mass spectrometry (MS). An initial demonstration of the method was performed using digitonin/ethylenediaminetetraacetic acid EDTA extracted cytosolic proteins from the human colon carcinoma cell line, LIM 1215. Cytosolic proteins were separated by liquid-based IEF (pH range 3-10) into 96 fractions, and each FFE fraction was further fractionated by SDS-PAGE. Selected protein bands were excised from the SDS-PAGE gel, digested in situ with trypsin, and subsequently identified by on-line RP-HPLC/electrospray-ionization ion trap MS. Our results indicate that FFE is: (i) an extremely 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 powerful tool for cell-mapping proteomics). An up-to-date list of cytosolic proteins from the human colorectal carcinoma cell line LIM 1215 can be found in the Joint Protein Structure Laboratory (JPSL) proteome database. This information will provide an invaluable resource for future proteomics-based biological studies of colon cancer. The JPSL proteome database can be accessed through the World Wide Web (WWW) network (http://www.ludwig.edu.au/jpsl/jpslhome.html).
Hoffmann
P
P
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
Ji
H
H
Moritz
R L
RL
Connolly
L M
LM
Frecklington
D F
DF
Layton
M J
MJ
Eddes
J S
JS
Simpson
R J
RJ
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003110:Colonic Neoplasms / Q000737:chemistry* / Q000235:genetics; D003600:Cytosol / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D009363:Neoplasm Proteins / Q000235:genetics / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D020543:Proteome / Q000235:genetics / Q000302:isolation & purification*; D014357:Trypsin; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D010446:Peptide Fragments; D020543:Proteome; D014357:Trypsin