A pI-based protein fractionation method using solid-state buffers.
The analysis of very complex proteomes is dependent on efficient fractionation methods with low level of carry over from fraction to fraction. Among various possibilities the separation by ranges of isoelectric points for further analysis appears as attractive, but current methods involving an electrically driven migration in the presence of ampholyte carriers are not exempt of technical complications. In the present work a new separation concept is described involving the use of so-called solid-state buffers, in association with ion exchangers, to separate protein categories of different pI ranges with a low level of protein overlapping. Resin blends packed in separated columns are used under a cascade configuration of increasing or decreasing pH and, once proteins of different pI are adsorbed by individual resin blends, the columns are dissociated. From each column protein mixtures corresponding to a given pI range are collected by competitive desorption with salts so as to be ready for proteomic analysis. The process is rapid and does not involve electrical fields nor addition of carrier ampholyte material. The presence of potassium chloride during the separation prevents protein precipitation at the vicinity of their isoelectric points. The fractions thus obtained can be used for two dimensional electrophoresis and mass spectrometry analysis after the removal of salts.
Fortis
Frederic
F
Bio-Rad Laboratories, C/o CEA-Saclay, Gif-sur-Yvette, France.
Guerrier
Luc
L
Girot
Pierre
P
Fasoli
Elisa
E
Righetti
Pier Giorgio
PG
Boschetti
Egisto
E
D000327:Adsorption; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry; D002021:Buffers; D002627:Chemistry, Physical / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D013058:Mass Spectrometry / Q000379:methods*; D010843:Pichia / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*
D001798:Blood Proteins; D002021:Buffers; D011189:Potassium Chloride