Mass spectrometry

TIBS - June 1976

N 136 all appear to consist of tandem repeating units within the genome with the basic unit containing the transcribed gene sequences and also a length of non-transcribed ‘spacer’ (NTS) DNA. The studies of D. Brown and others at Baltimore have been aimed at an understanding of the length heterogeneity in the NTS regions of the Xenopus Levis ribosomal RNA gene set. NTS regions of different lengths have been cloned and heteroduplexes between different length regions prepared and analyzed in the electron microscope. The length heterogeneity has thus been shown to result largely from the presence within the NTS region of an internally repetitive section containing a variable number of repeats of a basic 50 base pair unit. Preliminary analysis of the oocyte 5s RNA gene set of Xenopus has shown that here too, length heterogeneity results from variation in the number of copies of an NTS region repetitive sequence -- in this case 15 base pairs long. In speculating as to the function of these reiterated regions Brown suggests that they are probably generated by crossing-over events and may even serve as the recognition sites for homologous chromosome pairing and subsequent recombination. D. Clover (London) is engaged in a study of cloned ribosomal RNA repeat units from the Drosophila melanogaster genome. Two ribosomal RNA units of very different lengths are present in the Drosophila genome and these studies have shown that the longer unit is due to a completely novel DNA structural feature - the insertion of a stretch of DNA (5000 base pairs long and of unknown function) into the middle of a single coding region (the 2% ribosomal RNA gene). That is to say, in this ribosomal RNA unit two regions of DNA which should give rise to a single RNA molecule are non-continuous within the genome. It remains to be shown that this unit is actually used to give rise to functional 28s RNA at some stage in Drosophila development, but if this proves to be the case the implications for transcription are far-reaching indeed. S. Clarkson (Zurich) described the cloning and subsequent analysis of the initiator transfer RNA genes of Xenopus laevis, which are present at 300 copies per haploid genome. A repeat unit of 3 100 base pairs is generated by all the restriction enzymes tried to date and some of these have been cloned using E, as a vector. The transfer RNA coding sequences have been assigned to a 1900 base pair region of the cloned Hind 111 fragment but it is not clear if only one or multiple copies of the transfer RNA gene are present in this region. KATHY

BECKENHAM

SMITH

r

EMERGING TECHNIQUES

Mass spectrometry Howard

R. Morris

An important part of our understanding of how a biological system works is a knowledge of the structures (primary and higher) of the component parts of that system. Having determined structure one can begin to answer questions about interactions, mechanisms and the biochemistry of the system, and also use this better understanding in an applied way in medicine, via synthesis for example. Before this stage we are confronted with a structural problem on the molecular level, and its magnitude in the field of biochemistry is often greater than in the average chemical problem where a surfeit (milligrams or even grams) of pure or crystalline compound is available! In biochemistry one is more likely to encounter a few nanograms or micrograms of an impure material which has nevertheless elicited an enormous response to some bioassay or other. Clearly, traditional chemical or

Total Ion Current

spectroscopic methods of structure elucidation are of little value here. One field of study which is becoming increasingly important in this situation is mass spectrometry, some aspects of which are beginning to realize their potential in biochemistry and medicine. But what are these methods, why do they have such potential to the biochemist and what advantages does mass spectrometry offer over other probes? We can only begin to answer these questions in a short article but a large part of the answer lies in molecular weight. Picture a man who has isolated a few micrograms of an active component of some biological system; he has an Rr(or several) from chromatographic procedure, perhaps knows that it stains with a certain reagent, may even know that it contains particular functional groups, acids, alcohols, etc. These data, no matter how extensive, could tit almost an infinity of compounds. But suppose he can find the molecular weight of that compound, not just to the nearest whole number (although he would be grateful enough for that!) but to three or four decimal places, thus enabling an utomic composition to be calculated. The infinity is then reduced to a few possibilities. This information and much more can be available today by a combination of sophisticated microscale chemistry and mass spectrometry. Methods are improving daily and encompass developments in ionization technology such as chemical ionization and field desorption. The latter in particu-

Mass Spectrum No. Fig. 1. Temperature profile of u typical mixture indicating dl$eering compositions of components in consecutive spectra.

TIBS - June 1976

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lar can facilitate the study of quite involatile compounds (volatility is normally a prerequisite for mass spectrometry) such as free peptides, carbohydrates, drug conjugates and so on. A further feature of the mass spectrometer which sets it apart from most analytical instrumentation is the ability to analyze mixtures and yet obtain ‘clean’ data on each component. This is based upon the statistical improbability that the compounds present will volatilize at just the same temperature, and will last for the same amount of time (Fig. 1). By scanning the mass spectrum at different times and subtracting spectra one can obtain data on each component, even if unresolved. This leads to the important conclusion that a compound need not be ‘pure’ prior to analysis: an invaluable asset since many compounds of biochemical origin are exceedingly difficult to obtain pure in the chemical sense. An illustration of mixture analysis applied to protein chemistry is given in Fig. 2. An example of the use of mass spectrometry in structural work is given by a recent study of an endogenous opiate isolated from brain ~ Enkephalin [I]. The compound was thought to be a peptide of molecular weight about 1000. and one may well ask why bother with sophisticated methods when whole proteins can be adequately analyzed by the relatively simple dansyl-Edman sequencing procedure. Well, the compound was indeed studied by this method, but a definitive structure assignment (needed prior to synthesis) proved impossible because of several factors: the sequence obtained appeared to be too small for the amino acid analysis and molecular weight data, and there was also a suggestion of a new or novel amino acid based upon a fluorescence measurement. The latter possibility in particular demanded a different method of attack. Curiously enough (after my earlier statements) the mass spectrum of a derivative of Enkephalin showed no molecular ion (no molecular weight). This can often happen in practice, but does not necessarily spell disaster since the structure of a compound can be worked out from the ‘fragmentation pattern’ (the other signals of lower mass) in the mass spectrum. Using this approach it proved possible to deduce

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TIBS - June 1976

N 138 first that no novel amino acid was present, and second that the Enkephalin studied was composed of two closely related pentapeptide structures. Finally, it is worth stressing that the applications of mass spectrometry do not stop at structural elucidation. Far from it; methods for identifying the presence or absence of micro amounts of substance (perhaps a drug or its metabolite) in very complex biological extracts now often rely upon the continuous mass spectrometric screening of gas chromatographic column effluents with a direct gas chromatolink-up. spectrometry graphy-mass Furthermore, it is possible by techniques of ‘multiple-ion monitoring’ to quantitate components of a mixture relative to coldlabelled internal standards (perhaps deuterated). In competent hands drugs have been detected and measured down to 1O- I4 to 10 - is g level, and these methods already offer a viable and sometimes more specific alternative to radioimmunoassay [2].

Where have all the mutagens gone? It is an unfortunate fact that many actions taken by government or industry have an unintentional effect on some innocent third party. One such recent ‘innocent’ is the mutagenesis research worker who, due to the gradual tightening up in the chemical industry who find it unprofitable to produce substances which are required in small (but essential) quantities, as well as legal restrictions being imposed on the manufacture of certain chemical reagents, suddenly finds himself in a position where certain mutagens fundamental to his research are no longer commercially available. This state of affairs recently came to a head when supplies of triethylenenelamine (TEM) suddenly dried up. This particularly noxious substance is no longer commercially produced and private stocks are therefore dwindling. If TEM had no scientific importance then this matter would have passed unnoticed but it has already been the centre of some significant genetic research and certain lines of research still need the chemical. This whole question came to light when a group of geneticists were unable to

To those familiar with the wide areas of research interest now covered by mass spectrometry I have left a lot unsaid. Others one can only hope to have stimulated sufficiently to explore the subject further; it may be a valuable aid to your own biochemisty or in some cases it may offer the only solution to the problem in front of you. References I Hughes,

J.S., Smith, T.W., Kosterlitz, H. W., Fothergill, L. A., Morgan, B.A. and Morris, J. R. ( 1975) Nature 258, 577 2 Costa, D. and Holmstedt, B., eds (1973) Gas .C%romutography-Mars logy, Raven

Spec,trometrJ:

in Neurohio-

Press, New York

Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 4 edited by T.S. WORK, National institute

for Medical Research, Mill Hill, London, and E. WORK, Imperial College, London. 1976. viii+492 pages. US $51.95/Dfl. 130.00. ISBN 0-7204-4215-X Clothbound Part I Chemical Modification of Proteins Selected Methods and Analytical Procedures by A.N. GLAZER, R.J. DELANGE and D.S. SIGMAN, Department of Biological Chemistry, U.C.L.A. School of Medicine, Los Angeles, California, U.S.A.

Howard R. Morris is a Lecturer in the Biochemistry Department, Imperial College of‘&ience and Technology, Unbersity of‘London, U.K.

1975. ii+206 pages. US $11.25/Dfl. 28.00. ISBN o-7204-421 l-7 Paperback. Part II

Separation Methods for Nucleic Acids and Ollgonucleotldes

obtain any TEM from any of their own contacts or commercial sources. The environmental mutagenesis information centre at Oak Ridge, who run a computerized abstract service in environmental mutagenesis, were able to trace other research teams using the chemical and led this initial group of research workers to a small source. Not surprisingly, the owner was not too keen to be identified! However, it is obvious that TEM cannot be regarded as a long term research material. Worried by the disappearance of one of their compounds, the American Society of Environmental Mutagenesis have begun a search whereby they hope to pinpoint other mutagens which might be removed from commercial sale and also obtain the whereabouts of private stocks which might be distributed among the researchers to keep at least some lines of work running. If any reader feels he might be able to help in this search, contact Fred de Serres, President of the American Environmental Mutagenesis Society, NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709, U.S.A.

by HANNAH GOULD, Department of Biophysics, King’s College, London, UK and H.R. MATTHEWS, Department of Biophysics, Portsmouth Polytechnic, Portsmouth, U.K. 1976. iv+284 pages. US $15.25/Dfl. 38.00. ISBN o-7204-4213-3 Paperback. This volume in the widely acclaimed series Laboratory Techniques in Biochemistry and Molecular Biology is divided into two parts. The first part, Chemical Modification of Proteins, describes the theoretical and practical aspects of many experimental procedures in protein chemistry. The most valuable and useful chemical modification methods are presented critically and in their entirety. Major emphasis is placed on the methods necessary for quantitating amino acid derivatives generated by in vivo post-transcriptional and in vitro chemical modification. A special feature of this part is a summary of the experimental aspects of affinity and photoaffinity labelling. The second part, Separation Methods for Nucleic Acids and Oligonucleotidgs, discusses the choice of methodfor a given oligonucleotide or nucleic acid fractionation problem where the amount of material, approximate chain lengths and complexity can be estimated. The principles and application of paper chromatography, paper electrophoresis, equivalent thin layer techniques, ion exchange column chromatography and acrylamide gel electrophoresis are covered, and the necessary equipment and techniques are described.

North-Holland Publishing Company P.O. Box 211, Amsterdam, The Netherlands. Distributed in the U.S.A. and Canada by American Elsevler Publishing Company, INC. 52 Vanderbilt Ave., New York, N.Y. 10617 The Dutch guifder price is detinitlve. US $ prices srs subjsct to exchangs rats fluctuations. -.