Capillary isoelectric focusing of physiologically derived proteins with on-line desalting of isotonic salt concentrations.
Capillary isoelectric focusing within capillaries (cIEF) is a powerful and practical method for high-resolution separation of components within complex biological mixtures. However, a major problem has always existed; separation performance is usually degraded by the presence of salts within the sample. Normally this requires the removal of these components by some off-line sample cleanup method, prior to analyte separation by cIEF. In this study, we have shown it is possible to efficiently remove high salt levels from samples by on-line voltage ramping of the applied CE voltage. To allow this technique to be used effectively, a customized version of an existing method to internally coat a fused-silica capillary has been developed and examined for interexperimental reproducibility. We describe the systematic examination of the desalting process and its optimization through the use of model protein systems. Furthermore, we demonstrate the automated application of this on-line desalting cIEF scheme to studies of whole human blood and human cerebrospinal fluid which have undergone no manipulation or work up prior to cIEF analysis.
Clarke
N J
NJ
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA.
Tomlinson
A J
AJ
Schomburg
G
G
Naylor
S
S
D001798:Blood Proteins / Q000032:analysis*; D002556:Cerebrospinal Fluid Proteins / Q000032:analysis*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D011142:Polyvinyl Alcohol / Q000737:chemistry; D012492:Salts / Q000737:chemistry*; D014466:Ultraviolet Rays
D001798:Blood Proteins; D002556:Cerebrospinal Fluid Proteins; D012492:Salts; D011142:Polyvinyl Alcohol