Staying Alive: Measuring Intact Viable Microbes with Electrospray Ionization Mass Spectrometry
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Mass spectrometry has traditionally been the technology of choice for small molecule analysis, making significant inroads into metabolism, clinical diagnostics, and pharmacodynamics since the 1960s. In the mid-1980s, with the discovery of electrospray ionization (ESI) for biomolecule analysis, a new door opened for applications beyond small molecules. Initially, proteins were widely examined, followed by oligonucleotides and other nonvolatile molecules. Then in 1991, three intriguing studies reported using mass spectrometry to examine noncovalent protein complexes, results that have been expanded on for the last 25 years. Those experiments also raised the questions: How soft is ESI, and can it be used to examine even more complex interactions? Our lab addressed these questions with the analyses of viruses, which were initially tested for viability following electrospray ionization and their passage through a quadrupole mass analyzer by placing them on an active medium that would allow them to propagate. This observation has been replicated on multiple different systems, including experiments on an even bigger microbe, a spore. The question of analysis was also addressed in the early 2000s with charge detection mass spectrometry. This unique technology could simultaneously measure mass-to-charge and charge, allowing for the direct determination of the mass of a virus. More recent experiments on spores and enveloped viruses have given us insight into the range of mass spectrometry’s capabilities (reaching 100 trillion Da), beginning to answer fundamental questions regarding the complexity of these organisms beyond proteins and genes, and how small molecules are integral to these supramolecular living structures.
KeywordsMass spectrometry Intact Virus Bacteria Viability Charge detection Electrophoretic mobility Noncovalent interactions
These breakthroughs were compelling for both chemists and biologists, and for our lab they begged a bigger question: Can whole organisms be examined with mass spectrometry? By virtue of their homogeneity and relatively small size (megadaltons), the logical organism to test to answer this question was a virus. An intact virus, or virion, consists of genetic material in the form of RNA or DNA surrounded by a protein shell known as a capsid and may be helical or icosahedral in nature. Some viruses are further encased by a lipid membrane called a viral envelope, which helps in evading the immune system. While advances in viral research have been largely associated with the development of physical techniques such as X-ray crystallography [11, 12] and electron microscopy [13, 14], the development of mass spectrometry to mass measure viral ions was recognized as having the potential to further facilitate their characterization. Another motivation for analyzing viruses was that while convincing evidence existed regarding the observation of noncovalent interactions with mass spectrometry [1, 2, 3, 4, 5, 6, 7, 8], a common question was (and still is) whether native conformations are preserved throughout the vaporization, ionization, and mass analysis within the vacuum of the mass spectrometer . A third question, reminiscent of the Manhattan Project where Calutron mass spectrometers were used to separate uranium isotopes , is whether this technology can be used as a viable separation and collection device for biomolecules. Our work on viruses attempted to address these issues with the analysis of the 40 MDa tobacco mosaic virus.
Instrumentation for Viral Analysis
The first breakthrough in accurately measuring the mass of a complete virus particle required the development of a novel detector capable of simultaneously measuring charge, m/z, and velocity of an ion. Dr. Henry Benner created this technology by modifying an ESI-TOF mass spectrometer with an additional metal flight tube attached to a charge-sensitive preamplifier [2, 17, 18]. The charge-sensitive preamplifier creates an image current for single ions as they pass through this device and have the added benefit of a signal with a magnitude proportional to the number of charges they are carrying. To demonstrate the capability of this charge detector design, an entire intact genome was initially analyzed . This represented, at that time, the single largest molecule to be successfully mass analyzed. Charge detection mass analysis circumvents the problem of detecting large ions by making a simultaneous measurement of charge (z) and m/z ratio for individual ions; therefore it enables the direct measurement of mass (as opposed to m/z). Strictly speaking, one could say that this was the first true “mass spectrometer.”
Although the initial observations of a single virus ion may seem trivial given today’s mass spectrometry technology, back in the 1990s in the Lawrence Livermore lab, monitoring the data being generated by the instrument as each single ion passed through the detector was thrilling. At the time, this was a completely unique mass spectrometer at the forefront of sensitivity and mass measurement capability. These instrument alterations were highly influential in studying supramolecular living structures by mass spectrometry. Since the 1990s, even larger biomolecules have been analyzed using charge detection devices with improvements made by the Jarrold lab on the additional flight tube with optmized dimensions, alignment, and programming parameters. In this system, peaks can be rejected if they have baseline fluctuations, signals from more than one droplet, or when signficant differences between the entrance and exit peaks are observed . This improved setup results in a tighter distribution and detectable mass limits reaching 1014 Da (100 trillion daltons).
Another interesting offshoot of ESI-MS has been the coupling of ESI to ion mobility spectrometry (IMS) and differential mobility analysis, better known as gas-phase electrophoretic molecular mobility analyzers (GEMMA) . These devices provide confident measure of nanometer-sized biomolecules and noncovalent complexes with sub-nanometer resolution, and additional validation that these complexes survive the analysis process. IMS allows for the analysis of the cross-sectional area of an ion to be measured and has been widely applied to intact protein complexes [21, 25, 26, 27, 28, 29]. This technique has been used to detect intact bacteriophage MS2 at 24 ± 2 nm , and human rhinovirus with a diameter of 29.8 ± 0.3 nm . Interestingly, since the measurements are based on the electrophoretic mobility (EM) measurements that are independent of known virus features, these results provide strong evidence that no large-scale disruption of tertiary or quaternary structure of the capsid occurred during ESI desolvation and ionization .
Although the ability to detect megadalton-sized particles is at the heart of investigating microbe behavior, it is not only size that matters. In native microbe analysis, the resolving power, sensitivity, and sampling rate are crucial for deciphering relevant and specific information about the intact particles and how they interact with modulators of function. Consequently, these aspects have been continually improved in both CDMS and GEMMA over the years. Today, even conventional mass spectrometers can be harnessed to obtain high-resolution mass spectra of megadalton-sized complexes. QTOFs are a particularly good choice for simple modifications to aid in the transmission of large ions. For example, analysis of an 18 MDa intact capsid from bacteriophage HK97 was performed on a QTOF with xenon collision gas at higher pressures and voltages. The instrument is capable of resolving masses up to 40 GDa, but the practical limitation lies within the ability to desolvate the ions, thus reducing the limit to ~20 MDa . An Orbitrap mass analyzer has also been configured to accommodate the analysis of megadalton-sized capsids. With an additional gas flow to the HCD cell, optimized radio frequency (rf) voltages, and the removal of analogue filters, an upper mass limit of ~5 MDa was achieved, but restricted by the low pressures required in the C-trap . The benefit of using a modified QTOF or Orbitrap mass analyzer to measure megadalton particles is the capability of generating high-resolution spectra without the need for a completely different system.
Virus and Spore Viability
There have since been other reports of testing viability after the electrospray ionization of a virus. Hogan et al. used EM to analyze bacteriophage MS2, λ, T2, and T4 with a highly monodisperse electrospray source operated in cone jet mode to produce a near steady state diffusion charge distribution . An airborne sampler was teed off from the electrospray source to collect and test the viability of the aerosolized virus particles. Bacteriophage MS2, a small, single-unit virus, was found to be viable, whereas the λ bacteriophage had low amounts of viability and the large, multi-subunit viruses T2 and T4 bacteriophages had no viability. The length of bacteriophages T2 and T4 exceeded the mean diameter of the droplets (171 nm), yet both T2 and T4 had mean diameters of 87.03 and 88.32 nm, respectively. It was surmised that the T2 and T4 bacteriophages were not the whole viruses, but only the protein capsid heads that remained intact through the electrospray process. Viruses with sizes exceeding the droplet diameter would be exposed to more mechanical and electrical stress, leaving them more susceptible to noncovalent bond disruption.
Another interesting study involving much larger bacterial cells was recently reported by Pratt and Austin . Two different species, E. coli and B. subtilis, were electrosprayed, ensuring complete deagglomeration and desolvation, electrostatically deflected, and collected to assess whether individual cells in various charge states could survive. Bacterial particles with higher charge or lower mass were deflected more strongly as shown in Figure 3. This device, called a “Bug Trap,” consists of two parallel stainless steel plates with an applied electric field acting as a particle deflector, and a slotted collection plate to capture the charged particles. After electrospraying a bacteria sample, the collector channels were washed with sterile water, transferred to petri dishes, and incubated overnight at 39 °C. The E. coli did not survive complete desolvation and charging under the applied electric field, whereas B. subtilis did survive. The major difference between these two strains is the ability for B. subtilis to form spores, whereas E. coli does not. This protective spore coat helped this bacterium survive the electrospray process in both atmospheric (~20% recovery) and vacuum (<0.1% recovery) conditions, albeit at low charge states. To keep microorganisms viable as they traverse the harsh conditions of electrospraying and acceleration through electric fields, the size of the microorganism with respect to the droplet diameter becomes important as does the strength of the noncovalent interactions maintaining the native structure. Reducing mechanical and electrical stress by controlling droplet size can be balanced with the ability to completely desolvate the particle to effectively separate it based on electrophoretic mobility and m/z, which is necessary to give accurate and well-resolved measurements. Taken together, these results show that the viability of the microorganism was strain-dependent, as determined by their size and structural stability.
Conclusion: Revolution to Evolution
The mass spectrometer has seen dramatic improvements in the past three decades and the revolution of ESI has generated an evolution in the entire mass spectrometer from front to back. ESI has clearly been the method of choice for examining noncovalent interactions in the gas phase, especially intact viruses, yet it is interesting that desorption/ionization techniques (e.g., MALDI) have not been used extensively, despite both of these methods also being relatively soft. This may be because analyzing macromolecular complexes by MALDI is confounded by the need for a denaturing sample preparation involving matrix application, laser optimization, and reduced detector sensitivity in the ultrahigh m/z range . However, gentle ablation methods, such as DESI  or nanostructure-based desorption and ionization , coupled with CDMS or modified TOF-MS, may provide possible alternatives for soft desorption/ionization. For the ESI itself, nebulization techniques could also be improved to increase the viability of the microorganisms. As discussed above, previous studies observed that bacteria or viruses with a more spherical shape and smaller diameters were more likely to survive the journey through the MS. Further investigation on the size of nebulizer droplets, desolvation methods, and temperature/voltage during ion transfer would be useful in keeping larger microorganisms alive through the MS process.
Some would say that mass spectrometry is now a mature technology; however, a truly comprehensive understanding of a biological system requires the ability to examine all of its components: the genetic material, proteins, macromolecular structures, and small molecules. While there have been many advances in MS instrumentation for analyzing these systems, a complete solution remains tantalizingly out of reach. What are the next mass spectrometry technologies that will allow us to simultaneously observe genes, proteins, metabolites, and their interactions within the complexity that is a living biological entity? Perhaps we need to turn to our students for the next revolution.
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