Charge detection mass spectrometry of bacteriophage P22 procapsid distributions above 20 MDa

Abstract

Rationale: Charge state resolution is required to determine the masses of ions in electrospray mass spectrometry, a feat which becomes increasingly difficult as the mass increases. Charge detection mass spectrometry (CDMS) circumvents this limitation by simultaneously measuring the charge and the m/z of individual ions. In this work, we have used electrospray CDMS to determine the number of scaffolding proteins associated with bacteriophage P22 procapsids.

Methods: P22 procapsids containing a native cargo of scaffolding protein were assembled in E. coli and purified via differential centrifugation. Electrospray CDMS was used to measure their mass distribution.

Results: The procapsid peak was centered at 23.60 MDa, which indicates that they contain an average of ~112 scaffolding proteins. The distribution is relatively narrow, less than 31 scaffolding proteins wide. In addition, a peak at 19.84 MDa with a relative abundance of ~15% is attributed to empty capsids. Despite having the same sizes in solution, the empty capsid and the procapsid have significantly different average charges.

Conclusions: The detection of empty capsids is unexpected and the process that leads to them is unknown. The average charge on the empty capsids is significantly lower than expected from the charge residue model, which probably indicates that the empty capsids have contracted in the gas phase. The scaffolding protein presumably limits the contraction of the procapsids. This work shows that electrospray CDMS can provide valuable information for masses greater than 20 MDa.

Figure 1

Negative-stain TEM micrograph of P22 procapsids in 25 mM ammonium acetate. Scale bar is 100 nm.

Figure 2

Mass spectrum of P22 procapsids. The measured masses are binned into a histogram with bin size 167825 Da (the mass of five scaffolding proteins). The procapsid peak is centered at 23.60 MDa. In addition there is a smaller peak centered at 19.84 MDa which is attributed to empty capsids.

Figure 3

Charge versus mass scatter plot for all the ions in Fig. 2. The charge is in units of elementary charge (e). The empty capsids’ charge distribution is centered at 325 charges and the procapsids’ charge distribution is centered at 427 charges.