Analysis of Megadalton-Sized DNA by Charge Detection Mass Spectrometry: Entropic Trapping and Shearing in Nanoelectrospray

Theanalysis of nucleic acids by conventional mass spectrometryis complicated by counter ions which cause mass heterogeneity andlimit the size of the DNA that can be analyzed. In this work, we overcomethis limitation using charge detection mass spectrometry to analyzemegadalton-sized DNA. Using positive mode electrospray, we find twodramatically different charge distributions for DNA plasmids. A lowcharge population that charges like compact DNA origami and a muchhigher charge population, with charges that extend over a broad range.For the high-charge population, the deviation between the measuredmass and mass expected from the DNA sequence is consistently around1%. For the low-charge population, the deviation is larger and morevariable. The high-charge population is attributed to the supercoiledplasmid in a random coil configuration, with the broad charge distributionresulting from the rich variety of geometries the random coil canadopt. High-resolution measurements show that the mass distributionshifts to slightly lower mass with increasing charge. The low-chargepopulation is attributed to a condensed form of the plasmid. We suggestthat the condensed form results from entropic trapping where the randomcoil must undergo a geometry change to squeeze through the Taylorcone and enter an electrospray droplet. For the larger plasmids, shearing(mechanical breakup) occurs during electrospray or in the electrosprayinterface. Shearing is reduced by lowering the salt concentration.

Automated summary

Conventional mass spectrometry of nucleic acids is complicated by counter ions, limiting the analysis of DNA size. This study overcomes this limitation using charge detection mass spectrometry to analyze megadalton-sized DNA. Different charge distributions are observed for DNA plasmids, with a low-charge population resembling compact DNA and a higher-charge population with a broad charge range. The high-charge population is attributed to supercoiled plasmids in random coil configuration, while the low-charge population is attributed to a condensed form of the plasmid.