A New Generation of Weights and Measures

Michael Peel
C&I Essay Competition

If someone told you that we are finding new ways to combat some of the gravest threats to human health by using technology which has existed since the First World War, you would probably give them some funny looks. If they went on to inform you that the machinery enabling this ground-breaking research was essentially a weighing machine, it might well provoke a search for hair on the palms of their hands. Yet in this apparent madness there is method - the method is electrospray mass spectrometry, even now giving us crucial information on life-threatening conditions as diverse as skin cancer and AIDS.

Mass spectrometry has traditionally been the blunderbuss in the organic chemist's battery of instrumentation. Whereas other methods give information on the molecules of life by observing their interaction with electromagnetic radiation and are governed by elegant theories of symmetry and quantum mechanics, the sheer physicality of mass spectrometry - literally weighing molecules by bombarding them with high-energy electrons until one or more of the sample's electrons 'drop off' - seems almost antediluvian.

This was the major stumbling block when it came to applying mass spectrometry to medical research. The species which underpin fundamental biological processes, particularly proteins, are invariably large, fragile and sensitive to temperature - features which do not co-exist easily with the high temperatures and high-energy electrons found in the average mass spectrometer.

The solution, developed in the late 1980s by a group led by the American John Fenn, was as most notable in its ingenuity as for its apparent simplicity. They reversed the process - instead of vaporisation followed by ionisation, the sample was instead dissolved, ionised in solution and then evaporated by application of an electric field to give gaseous molecules of sample encased in a protective shell of residual solvent. After removal of the shell by exposure to an inert drying gas, the sample can be analysed in the usual way.

The research has provided the basis for a quantum leap in our understanding of pathogens and how to combat them. The most dangerous invaders of the human body - notably viruses - are protein-based. To synthesise effective drugs we first need precise information on the make-up and structure of the proteins they will be acting against. This analysis has traditionally been done by chemically breaking off constituent amino acids one by one and sequencing the entire protein in this way. Since the relative mass of a protein may be many hundreds of thousands and the mass of an individual amino acid as low as 75, this represents a rather painstaking way of going about things. In an electrospray mass spectrometer, a protein can be fragmented by a gas stream resulting in a spectrum which shows a series of peaks, each one differing by the mass of one amino acid, allowing the whole protein to be sequenced in a single experiment, with a high degree of accuracy. A protein involved in malignant melanoma, the skin cancer caused by the ultraviolet radiation in sunlight, has been identified in this way.

The other major advantage of electrospray sequencing is that it is possible to analyse mixtures of proteins in a single experiment. Traditional chemistry-based methods can be used only with pure proteins. This advance has again had far-reaching consequences, this time in the analysis of proteins of the feline immunodeficiency virus, which is very similar to HIV, the human immunodeficiency virus, where seven of the nine proteins of the health-endangering 'active' genes of the virus were identified in a single electrospray experiment. The FIV work exemplifies the high sensitivity of the technique, typically to within 0.01% - comparable to a 5g uncertainty in the weight of a 50kg human being. Moreover, electrospray analysis of a protein of molecular weight 100,000 might require as little as one ten thousandth of a milligramme of sample. This is of crucial importance in an area of study where biologically active products may only be isolable in minute quantities.

Important though these uses of the new technique are, they are essentially updated versions of the earliest experiments to determine molecular weights. There is one feature of electrospray which is truly revolutionary. Electrospray mass spectrometers are the first weighing machines which can give us information on the shape of molecules - specifically, on protein conformation.

Information on the conformation of our enzymes - biological proteins - is the key to understanding the molecular basis of our day-to-day existence. Each enzyme is specific to a particular chemical transformation necessary for our continued well-being. The food we eat is acted on by enzymes to break it down into a form our bodies can use. The specificity is achieved through the shape of the enzyme's 'active site' being the same as that of the host species to be acted on. Only a species of the correct shape will interact successfully - this is the 'lock and key' theory of enzyme action.

Experiments in this area are founded on the fact that certain hydrogen ions in proteins will slowly change places with the hydrogen ions of water. Furthermore, the rate of exchange of a particular hydrogen ion is related to the structure of the protein at that point. 'Heavy' water contains two atoms of deuterium instead of two atoms of hydrogen. Deuterium is almost identical to hydrogen in all respects except that the ion D+ has a relative mass of 2, one more than H+. After an arbitrary time in D2O, some of the exchangeable hydrogen ions in the protein will have been exchanged for deuterium while others will be unchanged. A series of electrospray experiments, each corresponding to a different period of time of dissolution in D2O, therefore gives a conformational map of the protein and with it the possibility of forming theories relating protein structure to their functions.

Even today, a great deal of medicine and pharmacology is based on the empirical rather than the theoretical - we know from experience that certain drugs help with certain conditions, but we are either hazy on or ignorant of the detail on a molecular scale. Electrospray mass spectrometry could become as fundamental to medical science as the X-ray. Great ingenuity has been exercised in devising the technique; we now need to show comparable cunning in its application.

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