Measurement of the accurate mass of a 50 MDa infectious virus

Abstract

RATIONALE Bacteriophage P22 is believed to contain a total of 521 copies of 9 different proteins and a 41,724 base pair genome. Despite its enormous size and complexity, phage P22 can be electrosprayed, and it remains intact in ultra-high vacuum where its molar mass distribution has been measured. METHODS Phage P22 virions were generated by complementation in Salmonella enterica and purified. They were transferred into 100 mM ammonium acetate and then electrosprayed. The masses of individual virions were determined using charge detection mass spectrometry. RESULTS The stoichiometry of the protein components of phage P22 is sufficiently well-known that the theoretical molar mass can be determined to within a narrow range. The measured average molar mass of phage P22, 52,180±59 kDa, is consistent with the theoretical molar mass and supports the proposed stoichiometry of the components. The intrinsic width of the phage P22 mass distribution can be entirely accounted for by the distribution of DNA packaged by the headful mechanism. CONCLUSIONS At over 50 MDa, phage P22 is the largest object with a well-defined molar mass to be analyzed by mass spectrometry. The narrow measured mass distribution indicates that the virions survive the transition into the gas phase intact.

Integrative structure and functional anatomy of a nuclear pore complex

“…2A): (1) use of synthetic concatemers of tryptic peptides or QconCATs 51 to define the relative stoichiometry of each component by quantitative MS in affinity-captured NPCs; (2) In vivo calibrated imaging analysis of GFP-tagged Nups 52 , to quantify the absolute copy number per NPC of Nups selected to represent each major module of the NPC; and (3) Charge Detection MS to measure the total mass of affinity-captured NPCs 53 . For the calculation of the integrative NPC structure, the final copy numbers were rounded to fit the known NPC C 8 -symmetry and these values are indicated in Supplementary Table 2A.…”

“…The CDMS instrument is described in detail elsewhere 53,65 . Briefly, the measurements are made by trapping single ions in a linear electrostatic ion trap.…”

Neutral mass spectrometry of virus capsids above 100 megadaltons with nanomechanical resonators

“…More recently, MS research has devoted its efforts to expanding the mass range accessible with conventional techniques, up to the 10-MDa range (4). Alternative MS techniques, such as Charge Detection Mass Spectrometry (CDMS) (5,6) can analyze single ions with masses up to a GDa. Nevertheless, to date, no commerciallyavailable MS instruments can measure masses above a MDa.…”

Native Mass Spectrometry from Common Buffers with Salts That Mimic the Extracellular Environment

“…Sodium is ubiquitous in nature but is generally believed to be incompatible with ESI mass spectrometry when present above low mm concentrations.The use of submicrometer ESI tips is the only method to produce protein and protein complex ions directly from av ariety of buffers that contain nonvolatile salts at concentrations that mimic the extracellular environment (135-150 mm Na + )and does not require any additional preparation of the sample,such as buffer exchanging into ammonium salt solutions.T his demonstration of the applicability of this method to av ariety of different buffers with different properties (all with high ionic strengths of NaCl and all of which are commonly used in protein chemistry laboratories) eliminates concerns that aqueous ammonium salts will alter the native forms and functions of proteins.This method makes native MS more compatible with other traditional biophysical techniques.T hese results suggest that this method could be promising for monitoring extracellular fluid directly with MS without sample preparation or clean up. Any potential issues with emitter tip clogging could be reduced using filters or by laser vaporization of contaminants at the emitter tip.V iruses or other biological molecules in extracellular fluid could be ionized and mass analyzed directly by new MS techniques [25] using this method which may ultimately be useful in applications such as early detection of infections.…”