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Electrospray mass spectrometry (EMS) is used to study large molecules without fragmentation. A pure macromolecular sample is dissolved in a solvent system, injected into a high voltage electric field, and studied. EMS coupled with time-of-flight analysis and collision cooling reduces fragmentation. Isotopes can affect EMS determinations, making monoisotopic samples preferable.
A mass spectrometer (MS) is an electronic instrument used to identify chemical structure. In most mass spectrometry procedures, molecules are electrically bombarded, resulting in ionization with fragmentation. The fragments are then magnetically accelerated towards sensing and recording devices, resulting in specific peaks and intensities that researchers can study as a kind of “molecular fingerprint”. The electrospray mass spectrometer (EMS) works differently, without causing fragmentation. This makes it valuable in studying large species or macromolecules.
If it is sufficient to determine the simple chemical bond, the use of an electrospray mass spectrometer will probably not be necessary. For larger molecules such as peptides, however, molecular shape and molecular folding, even molecular interaction with surrounding molecules, are just as important. In such cases, it is essential that the molecule remain unfragmented. The necessary delicacy dictates the use of an electrospray mass spectrometer, which does not require the use of either high temperatures or a vacuum.
When using an electrospray mass spectrometer, a pure macromolecular sample is first dissolved in a solvent system, which is then injected via a narrow bore needle into a high voltage electric field. The solvent rather than the solute gets the brunt of the bombardment. When the liquid reaches a critical charge level, the solution violently breaks into aerosol-sized droplets, whose charge causes them to individually repel each other. Soon the droplets evaporate, depositing their multiple charges on the still intact molecules, which lengthen due to intermolecular repulsion. In this state their structure, even at high levels of complexity, can be studied and determined.
The first successful intact protein spectrum was produced in 1989 by researchers at Yale University in Connecticut. Progress in the EMS technique was rapid, and in 1996 chemist Carol Robinson detected spectral peaks that could be associated not just with a single structure, but with a coenzyme-protein complex. A major improvement since then is the coupling of the electrospray mass spectrometer with time-of-flight (TOF) analysis. Collision cooling takes this improvement a step further, reducing the fragmentation of immense structures produced by heat.
One difficulty encountered in electrospray mass spectrometer determinations is that introduced by elementary isotopes. This is because the peaks depend on the mass-to-charge ratio. The mass of a fragment or molecule, divided by the number of discrete charges it carries, determines its location. Different elemental isotopes contribute different masses, perhaps the most critical variance being that between carbon-12 and carbon-13. For this reason, samples of complex molecules should be monoisotopic whenever possible.
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