Sodium dodecyl sulfate-capillary gel electrophoresis analysis of rotavirus-like particles.
This work describes the application of a sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) method for the analysis of triple 2/6/7 and double-layered 2/6 rotavirus-like particles (RLPs), candidate vaccines against rotavirus infection. SDS-CGE analysis of RLPs resulted in peaks that could be attributed to the viral proteins (VP2, VP6 and VP7) according to their apparent molecular mass (MWapp). Samples containing the glycoprotein VP7 were analysed after deglycosylation with PNGase F. Upon deglycosylation, VP7 MWapp decreased 4-7kDa consistent with a degree of glycosylation of approximately 12-21%. VP2 was eventually detected in the form of more than one related proteins, despite the small areas due to the relative low mass proportion of this protein in the particle (16%). The effect of analytical parameters such as capillary temperature on method performance was evaluated. MWapp values estimated by SDS-CGE were compared with values obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The method described in this work proved to be fast, consistent and reproducible, representing a feasible alternative to the laborious conventional electrophoresis for the characterization of RLPs.
Mellado
Maria Candida M
MC
ITQB-UNL/IBET, Apartado 12, P-2781-901 Oeiras, Portugal.
Franco
Catarina
C
Coelho
Ana
A
Alves
Paula M
PM
Simplício
Ana Luísa
AL
D000956:Antigens, Viral / Q000032:analysis; D015153:Blotting, Western; D036022:Capsid Proteins / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D046529:Microscopy, Electron, Transmission; D008970:Molecular Weight; D012401:Rotavirus / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000956:Antigens, Viral; D036022:Capsid Proteins; C442545:VP2 protein, Rotavirus; C061332:VP6 protein, Rotavirus; C050779:VP7 protein, Rotavirus; D012967:Sodium Dodecyl Sulfate