Gas-phase electrophoretic molecular mobility analysis of size and stoichiometry of complexes of a common cold virus with antibody and soluble receptor molecules.
Attachment of a nonaggregating monoclonal antibody and of a soluble recombinant receptor molecule to the icosahedral nonenveloped human rhinovirus serotype 2 was studied with a nanoelectrospray ionization gas-phase electrophoretic molecular mobility analyzer (nESI-GEMMA). The virus mass, as determined via nESI-GEMMA, was within instrument accuracy (+/-6%) close to the theoretical value (8 x 10(6) Da) calculated from the sum of all constituents of one virus particle (60 copies of each of the four viral capsid proteins, the RNA genome, and one copy of the RNA-linked protein VpG). The formation of virus-antibody complexes of different stoichiometries (up to a mass 12.5 x 10(6) Da corresponding to 30 attached antibodies) and virus-receptor complexes (up to a mass 8.8 x 10(6) Da corresponding to 12 attached receptor molecules) was monitored. Via the volume derived from the electrophoretic mobility diameter (EMD), the stoichiometry of the HRV complexes was calculated. The accuracy of the EMD was within +/-0.5 nm, which corresponds to an accuracy of +/-4 antibodies and +/-5 receptor molecules in the respective complexes. For the first time, we here demonstrate the use of nESI-GEMMA for the analysis of the size and stoichiometry of biomolecules in high-order complexes in real time under normal pressure conditions.
Laschober
Christian
C
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria.
Wruss
Juergen
J
Blaas
Dieter
D
Szymanski
Wladyslaw W
WW
Allmaier
Günter
G
D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D011991:Receptors, Virus / Q000737:chemistry*; D012229:Rhinovirus / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D012995:Solubility
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D011991:Receptors, Virus