title: Charge reduced electrospray size spectrometry of mega- and gigadalton complexes: whole viruses and virus fragments.
abstract: The ability to analyze and identify large macromolecular complexes whose molecular weight is beyond the analyzable range of mass spectrometry is of great interest. The size of such complexes makes them suitable for analysis via mobility size spectrometry. In this work, charge reduced electrospray size spectrometry was used for the analysis of bacteriophage viruses with total molecular masses ranging from 3.6 MDa up to the gigadalton range. The electrospray source used was operated in "cone jet" mode with a mean droplet diameter of 170.56 nm. Bacteriophage MS2 was found to have a mobility diameter of 24.13 +/- 0.06 nm and remain highly viable after the electrospray process. Larger bacteriophages T2 and T4 have lengths greater than the diameter of the electrospray jet and droplets; thus, they could not be completely enclosed and were found to fragment at the virus capsid head-tail noncovalent interface during either the jet formation or jet breakup process. No viable T2 or T4 virions were detectable after being electrosprayed. While the exact mechanism of fragmentation could not be determined, it is proposed here that macromolecular fragmentation at noncovalent interfaces occurs due to mechanically and electrically induced stresses during jet formation and jet breakup. Bacteriophage T4 capsid heads were found to be statistically significantly larger than bacteriophage T2 capsid heads, with a mean peak diameter of 88.32 +/- 1.02 nm for T4 and 87.03 +/- 0.18 nm for T2. While capsid head fragments were detectable, tail and tail-fiber fragments could not be detected by size spectrometric analysis. This is attributed to the fact that the contractile tails of bacteriophage T2 and T4 virions mechanically deform to a varying degree while confined within the smaller jet and droplets. Further evidence of contractile tail deformation during the electrospray process was found by measuring the size spectrum of bacteriophage lambda, which has a noncontractile tail. Bacteriophage lambda had two distinct peaks in its size spectrum, one corresponding to the capsid head and the other corresponding to the tail fragment. Size spectrometry was also used for rapid quantification of virus concentrations, thus demonstrating its full capabilities in the analysis of large macromolecular complexes.
authors.affiliation: Aerosol and Air Quality Research Laboratory, Environmental Engineering Science Program, Washington University in St. Louis, Missouri 63130, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Hogan
authors.forename: Eric M
authors.initials: EM
authors.lastname: Kettleson
authors.forename: Bala
authors.initials: B
authors.lastname: Ramaswami
authors.forename: Da-Ren
authors.initials: DR
authors.lastname: Chen
authors.forename: Pratim
authors.initials: P
authors.lastname: Biswas
mesh_terms: D001435:Bacteriophages / Q000737:chemistry*; D008970:Molecular Weight; D010316:Particle Size; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation* / Q000379:methods*