Peptide separations by slab gel electrophoresis in pluronic F127 polymer liquid crystals.
Proteomics and peptidomics could benefit from simple methods for high-resolution separation of oligopeptides analogous to slab gel electrophoresis of proteins. Gels of Pluronic F127 copolymer surfactant were investigated as media for slab gel electrophoresis of oligopeptides using a trypsin digest of myoglobin. Concentrated solutions of Pluronic F127 are fluid at low temperatures (</= 5 degrees C), but become a gel-like micellar liquid crystal upon warming. Nucleic acids are well separated by electrophoresis in these gels as previously shown by Rill and Liu. Good separations of myoglobin tryptic peptides were accomplished by electrophoresis on slab gels of 24% Pluronic F127 or 15% polyacrylamide using the alkaline Laemmli buffer system (without sodium dodecyl sulfate). Labeling of peptides with the succinimidyl ester of Cascade Yellow (CY) prior to electrophoresis allowed sensitive detection with blacklight illumination at 365 nm. Labeled tryptic peptides were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. An inverse dependence of electrophoretic mobility on mass of peptides with charge Z = -1 was observed in both media. Two-dimensional (2-D) electrophoresis of myoglobin peptides on polyacrylamide, then on Pluronic media, at pH 8.3 indicated that the primary separation mechanism of most peptides was the same in both media. A few off-diagonal spots indicated that some peptides were preferentially retarded in Pluronic gels, perhaps due to hydrophobic effects. The ease of gel preparation and peptide recovery are advantages of Pluronic F127 gels for oligopeptide electrophoresis. The two media can be combined conveniently for 2-D electrophoresis, providing means to facilitate protein identification and peptidomics.
Rill
Randolph L
RL
Department of Chemistry and Biochemistry, College of Medicine, The Florida State University, Tallahassee, FL, USA. Randolph.Rill@med.fsu.edu
Al-Sayah
Mohammad A
MA
D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D004586:Electrophoresis; D015180:Electrophoresis, Gel, Two-Dimensional; D005456:Fluorescent Dyes / Q000737:chemistry; D005782:Gels / Q000737:chemistry; D009211:Myoglobin / Q000032:analysis* / Q000737:chemistry; D009842:Oligopeptides / Q000032:analysis* / Q000737:chemistry; D020442:Poloxamer / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013501:Surface-Active Agents / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism; D000099166:Polyacrylamides
D000180:Acrylic Resins; D005456:Fluorescent Dyes; D005782:Gels; D009211:Myoglobin; D009842:Oligopeptides; D020442:Poloxamer; D013501:Surface-Active Agents; D014357:Trypsin; D000099166:Polyacrylamides