Isoelectric beads for proteome pre-fractionation. II: experimental evaluation in a multicompartment electrolyzer.
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isoelectric, buffering membranes, delimiting each compartment and acting, in pairs, as isoelectric traps, have been replaced by isoelectric buffering beads, operating on the same principle, but allowing unhindered migration of proteins (lack of sieving properties, contrary to typical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.
Fortis
Frédéric
F
Ciphergen Biosystems-Biosepra, Cergy-Pontoise, France.
Girot
Pierre
P
Brieau
Olivier
O
Castagna
Annalisa
A
Righetti
Pier Giorgio
PG
Boschetti
Egisto
E
D001798:Blood Proteins / Q000737:chemistry; D002021:Buffers; D045424:Complex Mixtures / Q000302:isolation & purification; D045304:Cytochromes c / Q000737:chemistry; D004572:Electrolysis; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry; D006801:Humans; D007475:Ion Exchange Resins; D007525:Isoelectric Focusing; D008863:Microspheres; D020543:Proteome / Q000302:isolation & purification*; D012260:Ribonucleases / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001798:Blood Proteins; D002021:Buffers; D045424:Complex Mixtures; D029968:Escherichia coli Proteins; D007475:Ion Exchange Resins; D020543:Proteome; D045304:Cytochromes c; D012260:Ribonucleases