Katharina Root1, Raphael Frey1, Donald Hilvert1, Renato Zenobi1
- Department of Chemistry and Applied Biosciences, ETH Zürich, CH-8093 Zürich, Switzerland
Chemical cross-linking combined with MALDI-MS was applied to structural analysis of a protein nanocontainer. Specifically, an engineered variant of lumazine synthase from Aquifex aeolicus (AaLS-13) was investigated that self-assembles into a capsid-like structure and is known to encapsulate other proteins by Coulombic attraction. Two complementary soft ionization techniques, MALDI-MS and native ESI-MS, were utilized to map the subunit stoichiometry of the high molecular weight capsid. In accordance with the previously reported cryo-electron microscopy structure of this protein container, only pentameric subunits were detected. This study highlights the possibility to map subunit stoichiometry via chemical cross-linking with glutaraldehyde followed by MALDI-MS. The same approach was used to study protein-protein interactions during encapsulation of GFP(+36) by the AaLS-13 capsid. Heterocomplexes between GFP(+36) and AaLS-13 multimers were not observed when mixed at maximal loading capacity (AalS-13 monomer:GFP(+36) 4:1). This is in agreement with the known fast encapsulation of GFP(+36) by the protein capsid, which essentially removes any free GFP(+36) from the solution. Exceeding the maximal loading capacity by addition of excess GFP(+36) results in aggregation.
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In-situ crosslinking with GA and then analysis by MALDI mass spectrometry led to the mapping of the subunit composition of the engineered AaLS-13 capsid. Protein samples were mixed with the sinapic acid matrix in a 1/1 ratio. 1 μl of the solution was placed on a stainless steel MALDI plate and dried in ambient conditions until crystallization occurred. The plate was analyzed via a MALDI TOF mass spectrometer modified with the CovalX HM2 detection system.