Premium
Isoelectric beads for proteome pre‐fractionation. II: Experimental evaluation in a multicompartment electrolyzer
Author(s) -
Fortis Frédéric,
Girot Pierre,
Brieau Olivier,
Castagna Annalisa,
Righetti Pier Giorgio,
Boschetti Egisto
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401112
Subject(s) - isoelectric focusing , chromatography , chemistry , isoelectric point , fractionation , electrophoresis , proteome , membrane , sodium dodecyl sulfate , mass spectrometry , polyacrylamide gel electrophoresis , desorption , gel electrophoresis , adsorption , biochemistry , organic chemistry , enzyme
Proteome pre‐fractionation in multicompartment electrolyzers is proposed here, with substantial modifications as compared to the standard technique. First of all, the classical isoelectric, buffering membranes, delimiting each compartment and acting, in pairs, as isoelectric traps, have been replaced by isoelectric buffering beads, operating on the same principle, but allowing unhindered migration of proteins (lack of sieving properties, contrary to typical continuous membrane barriers). Secondly, the isoelectric beads are not made in the conventional manner, with ionic acrylamide derivative monomers throughout their central core, but are composed of a hard, ceramic core, coated with an amphoteric buffering polymer. This minimizes mass transfer resistance of proteins that are transiently adsorbed onto the beads. As a result, significantly reduced separation times, of the order of ca. 3 h, are required for developing steady‐state patterns, as compared to the lengthy times (overnight and much longer) in conventional multicompartment electrolyzers operating with isoelectric membranes. Examples of separation of standard marker proteins, as well as entire Escherichia coli lysates and human serum proteins, are given. The obtained fractions are analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, two‐dimensional gel electrophoresis and by surface enhanced laser desorption/ionization mass spectrometry.