Distinct Dimerization Mechanisms in Silkworm and Spider Silk Proteins Revealed by Mass Photometry

We apply mass photometry to measure the mass distributions of individual protein assemblies in native silk dopes from <i>Bombyx mori</i> silkworms and <i>Latrodectus hesperus</i> spiders. In both systems, we find that protein dimers dominate, but with distinct stabilization mechanisms. In silkworm dope, ∼700 kDa protein particles are sensitive to treatment with dithiothreitol (DTT), forming a new ∼350 kDa species. These results are consistent with the reduction of disulfide-linked heavy-chain homodimers. In spider major ampullate (Ma) dope, ∼550 kDa protein particles dominate, consistent with MaSp1 homodimers and MaSp1-MaSp2 heterodimers. These MaSp dimers are resistant to DTT, indicating they are stabilized primarily through noncovalent interactions. Together, these results reveal fundamental differences in the molecular organization of prespun silk proteins between these systems, with potential implications for the spinning process and the mechanical properties of fibers. More broadly, our findings demonstrate that mass photometry is a powerful tool for characterizing silk protein multimerization and provide direction for future studies of higher-order assemblies under biologically relevant spinning conditions.