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.

Acquiring Structural Information on Virus Particles with Charge Detection Mass Spectrometry

“…In our earlier work on P22 [45], WT ‘PCs’ were PC-like particles (PLPs) that consisted only of 420 copies of 46.6-kDa coat protein and a distribution of 33.6-kDa scaffolding protein, as those were the only proteins expressed from the plasmid. Here, since the PCs were generated from a phage infection, they should contain 415 copies of coat protein, a distribution of scaffolding protein, 12 copies of 82.6-kDa portal protein (which occupies one of the 12 vertices of the icosahedral lattice), and the ejection proteins gp7, gp16, and gp20.…”

“…CDMS is particularly well-suited to the analysis of high-mass, heterogeneous analytes [45–47]. Here, we use CDMS to elucidate the assembly products that result from coat protein substitutions (A285T and A285Y).…”

Charge Detection Mass Spectrometry for Single Ions with an Uncertainty in the Charge Measurement of 0.65 e

“…By increasing the trapping time to 129 ms, we were able to improve the accuracy of the charge measurements to 1.3 e, though only 1% of the ions were trapped for this long [2]. This configuration has now been used to probe higher order multimers of pyruvate kinase [2], the mass distribution of 24-MDa bacteriophage P22 procapsids [30] late intermediates in the assembly of hepatitis B virus capsids [31] and woodchuck hepatitis virus [32].…”

Ion Mobility Spectrometry-Hydrogen Deuterium Exchange Mass Spectrometry of Anions: Part 3. Estimating Surface Area Exposure by Deuterium Uptake

“…Studies in this area have proceeded rapidly over the last two decades allowing the determination of protein complex masses and stoichiometries [18, 19]. It is recognized that the size of ions that can be interrogated by the approach is nearly limitless extending to the tens of megadaltons [20–22]. Additionally, collisional activation can be used to provide some information regarding individual protein subunits [23].…”