bc2001769_si_001.pdf (322.63 kB)
Mass Spectrometric Detection of Targeting Peptide Bioconjugation to Red clover necrotic mosaic virus
journal contribution
posted on 2011-10-19, 00:00 authored by Ruqi Wang, Dustin
M. Lockney, Michael B. Goshe, Stefan FranzenPlant virus nanoparticle (PVN) formulations constructed from Red clover necrotic mosaic virus by drug infusion and targeting
peptide conjugation can be employed as drug delivery tools. In this
investigation, we studied the cross-linked structures formed by application
of sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(sSMCC) and succinimidyl-[(N-maleimidopropionamido)-hexaethylene
glycol] ester (SMPEG) as heterobifunctional linkers in the bioconjugation
process. The plant virus formulations using several targeting peptides
cross-linked to the plant virus capsid were characterized by LC/MSE analysis, which produced at least 69% sequence coverage using
trypsin and chymotrypsin digestion. The results showed evidence for
several types of modification located in three domains of the capsid
protein. Extensive linker modifications on lysines or cysteines were
detected in all the domains, including both intended peptide–capsid
cross-links and unintended intracapsid cross-links. Surprisingly,
the most extensive peptide modification was observed in the R domain,
which is thought to be quite inaccessible to peptides and cross-linking
reagents in solution, since it is on the interior of the virus. These
results show that heterobifunctional linkers may not be the most efficient
method for attachment of peptides to plant virus capsids. As an alternative
conjugation strategy, maleimide peptides were used to conjugate with
the virus in a one-step reaction. Analysis by LC/MSE showed
that these one-step maleimide coupling reactions were more specific,
such as modifications of C154 and to a lesser extent C267, and provide
a means for achieving more effective PVN formulations.