Defining the stoichiometry and cargo load of viral and bacterial nanoparticles by Orbitrap mass spectrometry

Abstract

Accurate mass analysis can provide useful information on the stoichiometry and composition of protein-based particles, such as virus-like assemblies. For applications in nanotechnology and medicine, such nanoparticles are loaded with foreign cargos, making accurate mass information essential to define the cargo load. Here, we describe modifications to an Orbitrap mass spectrometer that enable high mass analysis of several virus-like nanoparticles up to 4.5 MDa in mass. This allows the accurate determination of the composition of virus-like particles. The modified instrument is utilized to determine the cargo load of bacterial encapsulin nanoparticles that were engineered to encapsulate foreign cargo proteins. We find that encapsulin packages from 8 up to 12 cargo proteins, thereby quantifying cargo load but also showing the ensemble spread. In addition, we determined the previously unknown stoichiometry of the three different splice variants of the capsid protein in adeno-associated virus (AAV) capsids, showing that symmetry is broken and assembly is heterogeneous and stochastic. These results demonstrate the potential of high-resolution mass analysis of protein-based nanoparticles, with widespread applications in chemical biology and nanotechnology.

Figure 1

Schematic of the Exactive Plus instrument.

Figure 2

Native MS of virus-like particles on the modified Exactive Plus. Shown are encapsulin (top), adenovirus dodecahedron (top-middle), adeno-associated virus serotype 1 (bottom-middle) and cowpea chlorotic mottle virus (bottom). Corresponding structures are also shown on the graphs. The quoted masses represent the average ± standard deviation over all charge states in the spectrum. For detailed peak assignments, see Supporting Information Table S2.

Figure 3

Identifying and quantifying foreign cargo encapsulation (Teal Fluorescent Protein, TFP) in bacterial encapsulin. (a) Native MS spectrum of encapsulin at high collision energy, showing dissociation of up to two encapsulin monomeric subunits. (b) Zoom-in of the peaks corresponding to the first (top) and second (bottom) dissociation products; colors according to number of encapsulated cargo molecules. (c) Total intensity of all identified encapsulin-cargo stoichiometries. For detailed peak assignments, see Supporting Information Table S3.

Figure 4

Determining the VP1/VP2/VP3 stoichiometry in AAV1. (a) Experimental spectrum of AAV1 capsids recorded at 32 ms transient time. The AAV1 capsid structure is shown with scattered copies of VP1/VP2. (b) Simulated spectra of AAV1 capsids with different VP1/VP2/VP3 stoichiometries. The simulated spectra illustrate how there is partial overlap between peaks of capsids with different VP1 and VP2 copy numbers.