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

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 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 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

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. Wenow know that manyof

density and short-excitation time was they key feature the samples we studied have b ecome imp ortant drugs

of the interaction that makes PDMS work and that even currently in use. These include vancomycin, bleomycin,

418 Ronald D. Macfarlane

amphotericin, and thiostreptin. Although wewere suc- This metho d was well established b efore PDMS

cessfull with these molecules, the mass sp ectrometry came along. Low energy ions b ombard the surface of

community did not embrace our endeavors for several a sample pro ducing gas phase ions of the matrix. The

reasons: fear of radioactivity, use of time-of- ight and metho d was used extensively for characterizing the sur-

not a magnet or quadrup ole, low mass resolution, and faces of inorganic sp ecies b efore 1974 and continues to-

you had to wait for more than a minute to obtain the day. The mechanism involves collisions of the incident

mass sp ectrum. It was a time when editors of jour- ion with surface atoms that are ejected. Molecular ions

nals were more sympathetic to our manuscripts than were also present in the mass sp ectrum whichwere an

reviewers. \unwanted background". When PDMS was intro duced,

it was realized that a comp onent of the SIMS pro cess

also was similar to PDMS, the rapid dep osition of en-

Accelerator Studies, 1976-present

ergy, and molecular ions of involatile molecules could

also b e desorb ed by these low energy ions. From 1976

During the time when wewere actively involved in

to the present, SIMS has b een a memb er of the Desorp-

\running samples", mynuclear physics colleagues, who,

tion `xx family, led by Benninghoven at Munster and

like most physicists, generally distain chemistry, b egan

Standing in Winnip eg. A friendly rivalry emerged dur-

to tackle the problem of mechanism of PDMS using

ing this era where a series of pap ers were presented on

their nuclear accelerators. It was from these exp eri-

SIMS catching up to PDMS in terms of numb er of ions

ments, that the prop erties of the incident ion were elu-

desorb ed and the mass range. In terms of acceptance by

cidated and the interaction of high energy ions with

the mass sp ec. community, SIMS had the same problem

solids as a eld in its own b egan to emerge. These ac-

as PDMS b ecause it did not use sophisticated magnets

tivities were initiated and sustained byVoit at Erlan-

and have high mass resolution. This acceptance issue

gen, Sundqvist at Uppsala, Le Beyec at Orsay, Wien

comes up in several places here b ecause it was a real

at Darmstadt, da Silveira at Rio and Tombrello at

dilemma. Thousands of mass sp ec. p eople were es-

Caltech. Intense and enduring friendships with these

sentially ignoring the work of the PDMS/SIMS p eople

groups for the past 25 years have b een one of the rich-

despite the successes.

est comp onents of my professional and private life. We

have maintained this closeness through the series of

The Beginning of Being Accepted

Desorption `xx meetings that have taken place in won-

The rst mass sp ectrometry meeting where PDMS

derful and interesting sites that include Brazil as a two-

results were presented was in 1976. It was the last pa-

time favorite of this group. Over the years, the group

per of a week-long meeting and 4 p eople were in the

has expanded as the eld has grown and develop ed new

audience they were waiting for their plane. But as

ro ots and branches. It is this feature that I wish to ad-

it turned out, they were very imp ortant p eople and

dress next.

each b ecame part of the PDMS family. They were

highly resp ected in the mass sp ec. community p oli-

Secondary Ion Mass Sp ectrometry

tics and 3 of the 4 b ecame President of the American

SIMS

So ciety for Mass Sp ectrometry and the fourth is now

President of a large mass sp ectrometry company in the

US. It was through these individuals and their fore-

sight and unsel sh devotion to the go o d of the eld

that PDMS b egan to b e a part of this community.To

Frank Field, Hank Fales, Jim McCloskey, and Ian Jar-

dine, the Desorption `xx group can b e grateful to these

bridge builders. For Frank Field and Victor Tal'Rose,

their strong intellectual leadership b ecame the base for

acceptance by the mass sp ectrometry community.

Brazilian Journal of Physics, vol. 29, no. 3, Septemb er, 1999 419

The Comp etition Heats Up, Laser to an op erator who then went b ehind a curtain, pre-

Desorption is Intro duced pared the samples and inserted them into a mass sp ec-

trometer. Out came high quality sp ectra and the big

It was generally accepted by the mass sp ectrome-

news was that a \prop er" mass sp ectrometer was used

try community that there must b e a b etter way than

i. e. a magnetic sector instrument. The professor in

PDMS to obtain mass sp ectra of biomolecules but us-

charge was Michael Barb er and the metho d is known as

ing the same general idea. In the late 1970's, Meuzze-

fast atom b ombardment. The incident sp ecies is a high

lar in Holland b egan to obtain impressive mass sp ectra

energy neutral atom and the matrix is a liquid matrix.

using laser excitation. When I heard one of his rst

A high yield of ions, a steady current of these ions is

talks on his work, I had the feeling that the days of the

pro duced, and it is compatible with the technology cur-

monop oly that PDMS had on the eld were numb ered.

rently in use by the mass sp ectrometry communityat

Laser desorption had so much going for it in terms of

that time. High resolution, instant grati cation in the

control of the excitation. The pulsed laser was a nat-

time required to get the data, imp ortant criteria for ac-

ural for the TOF technique. However, the promise did

ceptance, was a part of the FAB technique. Within the

not convert to reality in the next few years. One of the

next year, I heard Mickey Barb er present his metho d

Desorption `xx participants, , to ok up

and results at a meeting in London, the last talk of the

the challenge of laser desorption at that time and for

meeting, and I knew then that PDMS was on its wayto

several meeting Hillenkamp kept us informed on the

b ecome part of the past history of mass sp ectrometry.

progress of the development of the metho d. The mass

Most of the imp ortant pharmaceutical problems could

sp ectrometry community and commercial mass sp ec-

now b e solved byFAB and the demand for PDMS mea-

trometer makers patiently waited for something more

surements was reduced to almost nothing. The mass

attractive.

sp ectrometry communityembraced FAB and there was

a public impression that now there was a metho d far

Fast Atom BombardmentFAB makes

analyzing involatile biomolecules. This p erception was

its debut

not challenged by the Desorption `xx community I'm

not sure why; intellectual maturity?, unsel sh devotion

to the science and not the publicity?, .....? and the

FAB researchers chose not to b e a part of the Desorp-

tion `xx communityeven though the mechanisms of the

pro cess were closely related. In later years, Mickey and

I had several go o d interactions and conversations. In

the last I didn't knowitwould b e the last, I got up

the courage to ask him a question I had p ondered since

I rst heard ab out FAB and the basis for the ques-

tion was the lack of the development of a link b etween

FAB and the other desorption/ionization metho ds. My

In the early 1980's, wewere working with Kenneth

question: \Mickey, did PDMS haveany in uence on the

Reinhart, a natural pro duct chemist at Illinois, on a

developmentofFAB? Mickey: \You damn right it did!

series of p otentially imp ortantantibiotics derived from

Wewere so angry that you were able to run all those

marine organisms. These were tough molecules to an-

samples on a TOF instrument that we put one of your

alyze. In the middle of the studies, Ken told us ab out

sp ectra on the wall of our lab as a daily reminder that

a metho d he heard was just develop ed in England that

wemust nd a way to run these samples on a prop er

was an imp ortant breakthrough for the analysis of in-

mass sp ectrometer." I cherish that moment.

volatile biomolecules. He was going to England imme-

diately with his samples and would rep ort back. When

he returned, he told the story of a clandestine op eration

literally clo cked in secrecy where he handed his samples

420 Ronald D. Macfarlane

The Post-FAB era and the birth of

MALDI

What kept PDMS going after FAB was intro duced

was the ability to study larger biomolecules. Largely

through the work of the Uppsala group, the mass range

limit was pushed up to close to 40, 000 for a protein.

But it was hard work. At the same time, biotechnology

and medicine was b ecoming more sophisticated and the

desire to obtain accurate and precise molecular weights

of biop olyimers was b ecoming more acute. It was ob-

vious from our studies that there was a limit to the

size of a protein that could b e desorb ed by a ssion

fragment. This was a bit of disapp ointment b ecause

our own interests were heading in a direction in medi-

cal research where wewanted to use PDMS to charac-

terize proteins involved in human health and disease.

It was clear that PDMS was not going to b e used in

Dateline: January 30, 1998, Scott & White

these studies. In the late 1980's, at an International

Hospital, Temple, Texas. A51year old white

Science meeting in Bordeaux, Franz Hillenkamp gave

female admitted to emergency ro om with chest

a talk, imb edded deep in the program on the work he

pain rated as 9 out of 10 with radiation to

and were doing with laser desorption; a

the upp er jaw and asso ciated with shortness of

breakthrough and a new acronym: MALDI. You will

breath, nausea, and diaphoresis. A new tech-

nique for monitoring the severity of the heart

hear more ab out this metho d from Franz in the next

damage is employed. MALDI sp ectra, recorded

dayortwo. This time, my resp onse to his talk was

over a p erio d of 3 days on the proteins presentin

absolute excitement! I could see my desire to havea

the patients HDL, shows an increase in the con-

metho d available to accomplish my long term goal: to

centration of the acute phase resp onse protein,

make a contribution to medicine, had a chance. I close

serum amyloid SAA, an indication of extensive

this long set of lecture notes with an excerpt of dream

damage to the inferior myo cardial wall. Attend-

ing physician, Catherine J. McNeal, MD, Ph.D. that has b ecome reality.

Brazilian Journal of Physics, vol. 29, no. 3, Septemb er, 1999 421

MALDI sp ectra of a heart patient recorded over a p erio d of 3 days. A sp ecial thanks to Dave Russell and his group in the

Texas A&M, Department of Chemistry, Lab oratory for Biological Mass Sp ectrometry for obtaining these data for us. It was

carried out on a very long Persp ective Biosystems Instrument.