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. 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, Franz Hillenkamp, 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 Michael Karas 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.