Feature

Weighing Up Molecules

New techniques help chemists look inside biological substances.

Bill Henderson and Brian Nicholson

Mass spectrometry is an essential technique in the modern chemistry and biochemistry laboratory. It serves as a very accurate means of "weighing" molecules; much can be learned from this information and from studying how the molecules fragment.

For molecules to be observed by a mass spectrometer, there are two essential requirements which must be met -- they must have an electric charge and they need to be present in the gas phase.

For small organic molecules, such as hydrocarbons, vaporisation is accomplished by heating the sample under a very low pressure; bombarding the resultant gas-phase molecules with a beam of electrons or some other highly energetic chemical species generates ions.

Such methods have been the mainstay of mass spectrometry for many years, but they have limitations. Many molecules will simply not withstand the harsh heating and electron bombardment regime of "classical" mass spectrometry. It is akin, in some respects, to hitting the molecules with a chemical sledgehammer! While softer ionisation techniques such as fast atom bombardment (using heavy atoms such as caesium or xenon) have extended the range of the technique, the ionisation is still rather harsh -- more like a foam-covered sledgehammer!

The introduction of a new, soft ionisation technique has revolutionised mass spectrometry by making it an appropriate technique for new ranges of compounds to investigate. This process has been named electrospray mass spectrometry or ESMS.

For this method, the sample is introduced into the mass spectrometer as a solution -- usually in a solvent containing a source of protons, such as water or methanol. The sample solution is simply sprayed into the source of the spectrometer and the solvent removed under a stream of gas. The spray is subjected to a high voltage, the effect of which is to form charged droplets, which then lose their solvent, thus producing intact ions in the gas phase. The exact mechanism of formation of gas phase ions remains an area of contention, but it is the outcome which is of interest -- it allows the production of gas-phase ions from a wide range of compounds, many of which were previously inaccessible to mass spectrometry.

These range from ionic inorganic compounds to high molecular mass proteins. Charged species do not need any additional ionisation, whereas uncharged molecules will typically join with a H⁺ or Na⁺ ion from the solution. Very non-polar species such as simple hydrocarbons are typically inaccessible to the electrospray technique, since they are uncharged, inert and extremely difficult to protonate, but these are the types of compounds which behave well under traditional, electron-impact mass spectrometry. The two methods are complementary weapons in the chemist's armoury.

Electrospray mass spectra can be tuned to give strong parent ions, but further information on molecular structure can be obtained. By increasing the voltage applied to the "windows", termed skimmer cones, through which the ions pass, increased fragmentation of the ions can be achieved. This allows the structure of the molecule to be probed in greater depth, by determining which groups are relatively easily lost from the main species.

Biochemists were quick to realise the potential of the electrospray technique, and it is now routinely possible to obtain electrospray mass spectra of biological molecules such as proteins. In such cases, the high molecular weight is actually well outside the range of modern detectors and cannot be measured directly, but the fact that mass detectors register the mass-to-charge ratio means that adding an appropriate number of charges to the protein gives several peaks within the range of the detector. From this it is possible to calculate the original molecular weight.

While electrospray has been rapidly developed for biochemical materials, applications to mainstream chemistry have been relatively few, particularly in the area of inorganic chemistry. Since many inorganic compounds are charged or highly polar, the electrospray technique is perfectly suited for their mass spectrometric analysis.

One interesting project at the University of Waikato, carried out in conjuction with Dr Chris Miles of AgResearch Ltd at Ruakura, has been the identification of complexes formed between the fungal toxin sporidesmin A (the primary cause of facial eczema in livestock) and zinc ions (which are administered to give protection against the ailment).

An anionic complex, [Zn(sporidesmin A)₂]^2-, can be detected by ESMS from mixtures of sproidesmin A, zinc ions, and a reducing agent, and it appears to be very stable. Whether or not such a complex is the primary source of protection in the animals remains to be seen -- more experiments are needed.

It is certainly feasible that the zinc strongly binds the sporidesmin A molecule, forming a stable, less toxic form, making it "unavailable" to cause the damaging effects of facial eczema. The electrospray technique is allowing us to investigate complexes formed between sporidesmin A and a range of metal ions, particularly transition metals having relatively low toxicities which are already present in biological systems, such as iron, copper and molybdenum.

The effect of other chemical species bonded to the zinc can also be easily and rapidly investigated -- just one of the projects currently underway on the electrospray mass spectrometer at Waikato.

Dr Bill Henderson is a lecturers in inorganic chemistry at the University of Waikato.
Professor Brian Nicholson is an inorganic chemist at the University of Waikato