Hydrophobic interaction capillary electrochromatography of protein mutants. Use of lipid-based liquid crystalline nanoparticles as pseudostationary phase.
Nanoparticle-based hydrophobic interaction-capillary electrochromatography was utilized for separation of proteins with similar mass-to-charge ratio at neutral pH without organic modifier. Lipid-based liquid crystalline nanoparticles were prepared and used as pseudostationary phase,benefiting from their high biocompatibility, ease of preparation,and suspension stability at high concentrations.Use of laser-induced fluorescence enabled detection at high nanoparticle concentrations. Green fluorescent protein(GFP) and mutants of GFP harboring single or double amino acid substitutions with the same charge were separated in the described system but not in conventional capillary electrophoresis. Separation was achieved by increasing the salt concentration to promote hydrophobic interactions by shielding of the repulsive electrostatic interactions. In addition, the method was adapted to a capillary with an effective length of 6.7 cm, enabling fast separations and future applications on chip.
Nilsson
Christian
C
Pure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden.
Becker
Kristian
K
Harwigsson
Ian
I
Bülow
Leif
L
Birnbaum
Staffan
S
Nilsson
Staffan
S
D019943:Amino Acid Substitution; D053801:Capillary Electrochromatography / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008055:Lipids / Q000737:chemistry*; D050866:Liquid Crystals / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D053758:Nanoparticles / Q000737:chemistry*; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification
D008055:Lipids; D020033:Protein Isoforms; D049452:Green Fluorescent Proteins