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Brazilian Journal of Physics, vol. 29, no. 3, Septemb er, 1999 415 Mass Sp ectrometry of Biomolecules: From PDMS to MALDI Ronald D. Macfarlane Department of Chemistry Texas A&M University Col lege Station, Texas, USA May, 1999 Mother Nature the Ultimate Chemist: South America holds some of the richest rain forests in the world where Nature, through the abundance of the essentials of life has pro duced a rich sp ectrum of biological molecules. The early inhabitants of these regions, over thousands of years, have, through exp erimentation, identi ed plants and animals that contain comp onents that they have found to b e b ene cial for their health or so toxic that they can b e used as a weap on. Poisonous South American tree frogs Do anyofyou knowany examples of drugs or toxins that haveevolved to com- mercial pro ducts that have had their ori- and mass sp ectrometry gin from the exp erience of pre-mo dern history South Americans? When a plant or animal is identi ed as having pan of its chemical make-up, a sp ecies that has imp ortant physiological prop erties, that sp ecies is rst isolated and puri ed. The 3-dimensional chemical structure is While the knowledge of these imp ortant biological elucidated utilizing a variety of sp ectroscopic to ols. The comp ounds was passed on through generations from most information rich of these metho ds are NMR, IR, stories and the practice of medicine by learned mem- and x-ray di raction of pieces of the molecule and the b ers of these native communities, until recently, these whole molecule. A structure is prop osed that ts all of stories and \home remedies" were not recognized by the this information and a molecular mass determination is science communityashaving muchvalidity. Long b e- made that is a direct measure of the inventory of the fore p enicillin was discovered, the Native p eople knew atoms comprising the molecule. If the measured mass that a bad wound would heal more quickly if moldy do es not t the prop osed structure, them something bread were layered over the wound. is wrong with the prop osed structure and it must b e mo di ed. The molecular mass determination is made Although wehave reached a high level of sophis- by converting molecules of the sp ecies into gas phase tication in synthesizing biological comp ounds, Nature, molecular ions and the mass is measured by the inter- through the exp erience of millions of years, has devel- action of these ions with magnetic or electric elds or op ed synthesis pathways that are marvels in their com- a combination of these. plexity and ingenuity. Consequently, many scientists are not only isolating and identifying the active ingre- The sp eci c example that illustrates this scenario dients of a plant but also elucidating the pathways that is given by the Panamanian tree frog whose skin con- Nature uses to pro duce that sp ecies. The countries of tains a highly toxic molecule called tetro dotoxin. It's South America have b een a rich natural lab oratory for prop osed structure based on sp ectroscopic evidence is these activities. given b elow: 416 Ronald D. Macfarlane b ecause no gas phase molecular ions could b e pro duced in the mass sp ectrometer. In fact, a large fraction of Nature's biomolecules could not b e studied by mass sp ectrometry in that era b ecause of this fundamental problem. The reason for this can b e understo o d if the tetro dotoxin structure is examined in more detail. The Figure 1. Prop osed Structure of Tetro dotoxin. highly p olar groups on the structure result in a very strong dip ole/dip ole interaction b etween neighb oring molecules in a sample of tetro dotoxin which means that This molecule interferes with the electrical circuitry it is dicult to pull them apart by thermal agitation. of the heart and induces cardiac arrest. It is used in When the sample is heated to increase the intermolec- heart research to induced heart attacks in lab oratory ular excitation, the thermal energy excites internal vi- animals and to study the physiological action of this brations of the molecule and it disso ciates. This pro cess event. Attempts to obtain the molecular mass of this is depicted b elow. molecule by mass sp ectrometry in the early 1970's failed Figure 2. Thermal excitation of a tetro dotoxin matrix leading to decomp osition. A Chance Observation in a Nuclear Karl Wien and Hartmut Jungclas from Germany. This Physics Study Leads to a Solution to the phenomenon was so unexp ected and interesting that it Problem b ecame the topic of our future studies and the nuclear physics interests gradually dwindled to obscurity.We In 1972, our group at Texas A&M Universitywas b egan to coat the surface with a variety of di erent ma- studying the emission of radioactive recoil ions from terials including those with biological signi cance and surfaces using time-of- ight mass sp ectrometry. In ad- in all cases, we obtained molecular ions of whatever was dition to the exp ected ions, molecular ions of impuri- put on the surface. My nuclear physics colleagues who ties on the surface also app eared in the mass sp ectrum were working on problems in the same nuclear physics whichwere p ostulated to b e due to the ionization of eld also b ecame interested in this new adventure, and molecules on the surface of the sample by the nuclear since they all had the same nuclear physics technology 252 radiation. To test this hyp othesis, a Cf-source was were able to do the same thing in their lab. Manyof placed b ehind the sample and ssion fragments from these individuals, nuclear physicists turned mass sp ec- the sp ontaneous of this nuclide irradiated the sample. troscopists are also participating in this week's activ- Molecular ions of surface molecules were indeed pro- ities and have made ma jor contributions to the eld duced in high abundance and detected in the mass sp ec- over the past 2 decades. In addition to Wien and Jung- trum verifying the origin of the molecular ions in the clas, they include Yvon Le Beyec, Bo Sundqvist, Ken nuclear physics exp eriment. Key individuals partici- Standing, Helmut Voit, and Brian Chait. pating in this study in 1972 in our lab are participat- ing in the Desorption '98 meeting this week. They are Brazilian Journal of Physics, vol. 29, no. 3, Septemb er, 1999 417 Back to the Poisonous Frog and the this structure by MW determination b ecause of the in- Molecular Decomp osition Problem volatility/decomp osition problem. He had tried for sev- eral years using every new mass sp ectrometric metho d In 1973-4, when our studies on the use of ssion that had evolved but with no success. At the urging 252 fragments of Cf to generate mass sp ectra was b egin- of his new student, he sent us a sample to run and we ning to evolve, one of the students in my group b egan got a go o d result. At that time, we didn't knowitwas his graduate studies at Stanford Universityworking for a tough problem and we also didn't know ab out the Harry Mosher, a renowned natural pro ducts chemist. problem mass sp ectrometry was having with involatile Professor Mosher hid b een studying the tetro dotoxin biomolecules. The sp ectrum we got by sho oting ssion structure problem for several years and prop osed the fragments through a sample of this toxin is shown b e- structure shown ab ove. He was not able to verify low. Figure 3. First Mass Sp ectrum of tetro dotoxin obtained by PDMS very large biomolecules can survive the hostile environ- Naming the Metho d ment of the ssion track and b ecome intact gas phase Professor Mosher was very excited ab out this result. molecular ions. He suggested that we forget ab out this nuclear physics stu and really develop the metho d into a technology PDMS Research, 1974-1984 for natural pro duct studies. He also insisted that we come up with a name for it, something organic chemists During this era, our lab oratory at Texas A&M was could pronounce and rememb er and, after several at- 252 o o ded with requests to \run samples", primarily to de- tempts, came up with Cf PDMS or PDMS Plasma termine the MW of molecules of biological imp ortance. Desorption Mass Sp ectrometry. For the most part, these were pharmaceuticals b eing develop ed by drug companies and the MW determina- So how do Fission Fragments solve the tion was a requirement for acceptance as a drug for the Problem? public. Many of these comp ounds were known only by A ssion fragment is a high energy, highly-charged a co de. We got some nice lo oking sp ectra but had no ion that is a highly destructive sp ecies. The surprise idea what it all meant. Wemust have b een quite suc- was that a comp onent of its interaction with solids is cessful b ecause the sample came o o ding in. This was a gentle, resulting in the desorption and ionization of confusing time for us b ecause wewere still more nuclear fragile molecules without decomp osing them. Later scientist than mass sp ectroscopist, and in fact, we knew studies showed that the combination of high energy very little ab out the eld.

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