International Journal of Ma88 Specttometry and Ion Physics, 53 (1983) 5-20 5

Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

PARTICLE INDUCED DESDRPTIDN AND THE ANALYSIS OF LARGE MOLECULES*

CATHERINE FENSELAU, J. YERGEY AND D. HELLER

Middle Atlantic Mass Spectrometry Facility, Department of Pharmacology, Johns
Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205 (U.S.A.)

ABSTRACT

This opening lecture in the Texas Symposillm on Particle Induced Desorption

is intended to reflect on the state of the art, and to pose questions to the
instrumentalists, the theoreticians and the analytical/bioorganic chemists
participating in the three respective subdivisions of the smposiun. Recent
studies from our laboratory are discussed which investigate the information
which can be deduced from the molecular ion envelope of an unknown middle mass
or 1 arge molecule, evaluate the need for unit resolution above 5000 amu, and
illustrate the ability of particle induced desorption to provide stable
isotope analyses, absolute and relative quantitation in mixtures, as well as
molecular weights and structural information.

INTRODUCTION
The technology, principles and ideas which were transferred a decade ago
by MacFarlane and Torgerson from nuclear to analytical chemistry have evolved
into the set of diverse yet related desorption techniques represented in this
vol une. Some of these phenomena have been better described by theoreticians
than others. Some of these methods have enjoyed more analytical success than
others. As a whole. these techniques enjoy the privilege of concomitant
interest from nuclear physicists, chemists and biologists. Much of the credit
for this unusually wide breadth of interaction goes to Professor R. D.
MacFarl ane.
The scope and analytical potential of techniques which use particle
induced desorption are illustrated in the first figure. On the left is the
molecular ion profile of bovine insulin (termed “the phthalate of the 80’s” by

*Opening Remarks from the Texas Symposium on Particle Induced Desorption held
May 18, 1983, in honor of Professor R. D. MacFarlane on the occasion of his
50th birthday,

0020-7381/83/$03.00 0 1983 Elsevier Science Publishers B.V.

6

1271
MacFarl ane) measured in a time-of-f1 ight mass spectrometer ionized by
(+20 charge state) parti cl es accelerated to 90 MeV in the Uppsal a accelerator
(ref.1). On the right is the protonated molecular ion envelope of porcine
insulin analyzed with a double focussin,g (energy and momentum) i nstrusent
of Nei r Johnson geometry ionized by xenon atoms accelerated to 8 KeV (ref.2).
Both spectra were acquired by particle induced desorption and computer support
was critical in both cases. The spectra can be differentiated by the lag time
of approximately one year between their acquisition, by the cost of the
i nstrunental systems used, by the characteristics of the bombarding particles,
by presentation of the sample in a sol id matrix in one case and a liquid
matrix in the other, and by the differences in the resolution provided by the
two analyzers.

Bovine
Insul in PORCINE INSULIN
Molecular
Ion

1
57302 IO

Fig.1. Molecular ion envelopes of insulin obtained by particle

1271+20
induced desorption with and xenon atoms,
 7

Resolution and molecular weights

A number of important questions must be considered as we develop our
capabilities for analysis of heavy bioorganic compounds by mass spectrometry.
What could we deduce about an unknown peptide from either of the molecular ion
regions shown in Figure l? Traditionally organic compounds have been
characterized by their nominal mass (C=l2, H=l, etc.) or by their monoisotopic
masses (comprising the most abundant isotope of each atom, C=12.0000,
H=l.O078, etc .) . The theoretical distributions of molecular ions calculated
by computer (ref.3) for porcine insulin, for glucagon and a trimeric gl ucagon
formula are presented in Fig. 2 and 3. It can be seen that the nominal mass
falls far below any of the actual molecular ions from a compound only as heavy
as gl ucagon. Fig. 2 and 3 also suggest that the abundance of the monoisotopic
ions will continually diminis,h relative to abundances of the major ions in
increasingly heavy molecular ion sets. A comparison of Fig. 1 and 2 suggests
that the weak monoisotopic ion of insulin may be hard to distinguish above the
noise even when the spectrum is recorded at unit resolution.

MONOISOTOP I c MASS (MH)’

AVERAGE MASS OW+

I
-----r-- \ n. ..

Fig.2. Theoretical distribution of protonated molecular ions

(M+~I)+ for porcine insulin.
8

. . .
4469 i-99
m/z

Fig.3. Theoretical distributions of molecular ions for glucagon

(ref.4.) and a glucagon trimer (ref.5.).
 9

If we lose the ability to distinguish and report the monoisotopic ion, can
we report instead the mast abundant molecular,ion as characteristic of an
unknown sample? Examination of Fig. 2 and 3 suggests that as the compound
becomes heavier it will be difficult to readily distinguish any particular
peak as most abundant. The envelopes become more nearly symmetrical. We
suggest that the most characteristic molecular weights which can be deduced
from spectra of peptides weighing more than 5000 are the average molecular
weights determined with good precision.
The question has been asked if high resolution accurate mass measurements
will provide the additional information on elemental compositions of middle
and heavy molecules as they do for species below 2000. The information in
TABLE 1 suggests that this will not be the case. TA8LE 1 lists the thirteen
most abundant isobaric ions which contribute to the ion current in the most
abundant peak of the insulin cluster. In total , more than 20 isobars
contribute to thi s peak.

TABLE 1
Composition of the most abundant+peak in the molecular ion cluster of porcine
insulin C256H382N65076S6 = (M+H) .

FORMULA* MASS X COMPOSITION OF THE PEAK AT M/Z=5777

lSN 34 5777 -629 _ I.688

1 s1
13C 34 5777 -633 19.907
1 %
13C 15 33s 5777.633 0.856
1 Nl 1
13C 15 5777.633 2.148
1 N2
15N 18 5777.637 0.967
1 OI
13C 15 V. 5777.637 0.523
1 NI 1
13C 15 5777.637 25.634
2 N1
13C 18 5777.641 11.409
1 Ol
13C12H115Nl 5777.641 1.036

13C 33 5777.641 5.029

2 %
13C 17 5777.645 3.074
2 Ol
13C 5777.648 100.00
3
1 3C22HI 5777.648 6.086

*Remainder of the molecular formula is made up of the most abundant

isotope of each element, 12C , ‘H, %, I%), 32S.
10

If we assume a consensus, on the basis of the arguments just presented,

that the accurate average mass is the most meaningful value which can be
reported to characterize a middle mass or large molecule, then one can ask
whether unit resolution is required or not. Fig. 4 shows the molecular ion
envelope obtained for porcine insulin using the Kratos MS-50 with the slits
open (-1500 resolution). This spectrum was obtained under the same
experimental conditions as the unit resolution spectrum in Fig. 1, except for
resol uti on. The numbers on the vertical axis indicate that seven times m many
”i ens” were counted by the computer at 1 ow resolution as at unit resolution.
One advantage of using low resolution in a double focussing instrunent is the
greatly increased sensitivity reflected in the ion counts. In TABLE 2 we pre-
sent the accurate average mass of insulin, calculated from these two molecular
i on envelopes . The accuracy is comparable and the precision is better for the
low resolution measurement, presumably because of its superior ion statisti

b 10253

Fig.4. Molecular ion envelope from porcine insulin obtained on a Nier-

Johns double-focussing mass spectrometer at low resolution (ref.2.).
TABLE 2

Average mass of porcine insulin.

__~~~~~
EXPERIMENTAL THEORETICAL
Unit Resolution Low Resol uti on

5779.00 5778.59
A= + .41 Y8*- + g6.37
s=.53 s=.22
(8) (5)

As a last consideration of the relative merits of unit and low resolution

measurements for peptides of this size, turn again to Fig. 1 and 2, and
compare the protonated molecular ion group calculated theoretically with that
recorded at unit resolution using fast atom bombardment. The experimentally
determined ion envelope is spread to both higher and lower masses than the
theoretical envelope. The lower ions probably result from losses of hydrogen
characteristic of fast atom bombardment. Supporting evidence for this is
derived from comparison of spectra of the hydrocarbon and fluorocarbon
sulfonate cluster ions (ref.6) presented in Fig. 5, Although the masses are
in the same range, the molecular ion envelopes recorded look quite different.
The point to be made is that losses of one and two hydrogen atoms provide a
downside tail on the hydrocarbon envelope. Comparable bond cleavages in the
fluorocarbon lead to fragment ions removed by at least 19 mass units.
The extra peaks on the high mass side of-the experimentally acquired
insulin envelope (comparing Fig. 1 and 2) are thought to arise by reduction of
disulfide linkages in some of the insulin molecules (ref.7). It can be argued
that unit resolution will permit us to assess the occurence and extent of this
reduction more .accurately.

The Desorption Process.

A complete description of desorption mechanisms wi 11 address:
Mechanisms by which the energy is transferred to the sample molecule
Mechanisms by which ions can be formed
Factors which determine which ions will leave the surface
Determinants of gas phase ion stabilities
The theoreticians have addressed the first problem with some success, for
the sol id matrix, commencing with discussions of nuclear and electronic
stoppi ng power. The chemists have addressed the second problem, particularly
for the liquid matrix, where ionization can be effected by predictable
chemical reac’tions. Studies of doubly charged ions are beginning to address
12

SODlull HEPTYLSULFOlU4TEN-15

CESIU~ FERFLwWIEXYLSULFME N-7

_-
C

Fig.5. Partial spectra of the cluster ions Na(C7H15S03Na)15+ and

Cs(C6F13S03Cs)7+ obtained using fast atom bombardment (ref.6.).
 13

the third category, and techniques for studying gas phase ion stabilities are
under development to address the fourth problem. The first , third and fourth
categories‘are addressed by other authors in this volune.
In considering desorption mechanisms it is important to distinguish the
nature of the ions observed. Quantitative measurements support the following
order of ease of formation:
R,N+ > (M+Na)+ B (M+H)+ > Mf

Preexisting ions, of which quaternary ammonium ions are a major example, are
most readily desorbed by 1 aser, fast atom bombardment, SIMS and field
deso,rption (ref -8). odd electron ions are formed by removal of an electron
under fast atom bombardment only when the sample has a very low ionization
potent i al , as in quinones and quinonediimines which readily undergo one
electron oxidations.
MacFarlane has included four “variations” in his definition of particle
induced desorption (ref .9) :
- heavy ion induced desorption
- secondary ion mass spectromctry
- fast atom bombardment
- laser desorption.

1249

t
IIONOISOTOPIC
!I+ 8110.5

8110

Fig.6. Partial spectra of the cluster ion CS(C~F~~SO~CS)~~’ obtained

at unit resolution of 8100 using fast atom bombardment (ref.6.).
14

Most mechanistic considerations to date have addressed size, charge, and

energy of the bombarding particle, but not the effects of flux. Substantial
differences exist in fluxes of the various techniques. Typical values are
sunmarized in TABLE 3 for the Cf-252 technique, “static” SIMS and fast atom
bombardment. The high primary flux of FAB generates a high secondary ion
flux, which facilitates the use of scanning sector analyzers, accurate mass
and high resolution determinatSons, tandem mass spectrometry and analysis of
metastabl e decompositions. The high flux also permits use of conventional
computer systems to acquire and process FAB spectra in real time. A cluster
ion recorded at unit resolution at 8100 is shown in Fig. 6 as an example of
the kind of measurements made possible by the high secondary ion flux
(ref.6).

TABLE 3
Typical primary particle flux.

P DMS Static SIMS FAB

1O-g A/ cm* 1O-5 A/cm*

lo3 parti cl es/set 6.3X10’ particlfs/ 6.3X1013 particlFs/

sec”cm set ‘cm

The term fast atom bombardment actually obscures the real innovation of
the two UMIST groups, which is the presentation of the sample in a liquid
matrix (ref .lO,ll) . Bombardment with atoms accelerated between 1 and 10 Kev
was reported in the literature at least as early as 1962 (ref.12) and more
recent studies in several labs (e.g. ref.13-15) suggest that the charge state
is not critical in this energy regime. In this context the term FAB would
appear to be unnecessarily redundant with the term SIMS. However, the physics
and chemistry of the mobile matrix are sufficiently distinct from those of the
solid matrix as to merit distinctive terminology.
The various contributions of the liquid matrix are summarized in Fig. 7
(ref.16). Chemical equilibria exist, of course, by which samples may be
i oni zed, and active protons may be exchanged for sodium cations, etc.
Solvation separates ions in solution and lowers the energy required to remove
the ions from the matrix (ref.17). The mobility of the liquid matrix
continually brings new sample ions to the surface and carries away the
radiolysis-1 i ke products of impact damage (ref .18). The analytical
effectiveness of the liquid matrix has led to its rapid acceptance world-wide.
However, mechanistic and theoretical considerations have addressed the solid
 15

DESORPTION
SPUTTERING

-
.

GLYCEROL

Fig.7. Schematic representation of the liquid matrix in fast atom

bombardment.
16

phase almost exclusively. We recommend that more consideration be given to

this key aspect of the most widely used technique for particle desorption of
organic mol ecu1 es.

Analytical Requirements of the Biochemist.

The organic or biochemist can obtain several different kinds of
information from a mass spectrun:
- molecular weights
- structure information from fragmentation patterns
- stab1 e isotope analysis
- quantitation of components within a mixture
Particle desorption techniques are well established as methods to provide
molecular weights from unknown organic samples. MacFarl ane and various
colleagues have accumulated a number of such credits for the Californium
technique, including Q*-nucleoside (MW 571) (ref.191, adenomycin (MW 758)
(ref.20)) bleomycin (MW 1313) (ref.21) and palytoxin (MW 2681.1+0.35)
(ref.22). Fast atom bombardment with a 1 i quid matrix has al so been
successfully appl icd to a number of structure problems. It has proven useful
for assessing molecular weights of unknown organic compounds containing
mu1 t i pl e charges , such as the cross-linked dimer derived by reaction of the
anticancer agent phosphoramide mustard with guanosine monophosphate (ref.23).
This compound contains two quaternary ammonium centers, however only
monocationic species are recorded (Fig. 8). Apparently an anionic site is
developed at one of the phosphate groups to provide three charged centers and
a net molecular charge of +l. [Desorpion of dicationic species has
independently been shown to be unfavorable in liquid FAB experiments
(ref.24)1. Organometal 1 i c compl exes compose an.other cl ass of compounds which
are readily analyzed by FAB in a liquid matrix. An example is shown in Fig.
9 of a novel mixed metal porphyrin dimer.
Thus far liquid fast atom bombardment has been used more for isotope
analysis than the other particle desorption techniques, In an early paper, a
FAB isotope analysis was found to compare satisfactorily with those made on
the same sample by field desorption and electron impact (ref.26). In another
instance isotope analysis by fast atom bombardment has been used to quantitate
dipalmitoylphosphatidylcholine in amniotic fluid as a method of assessing
fetal 1 ung maturity and risk (ref.27). Di palmi to.yl phosphati dyl chol i ne
carrying nine deuteriun atoms was used as internal standard in the assay. A
standard curve for this linear regression was constructed which had a
coefficient = 0.9994.
 17

NH2
1
O=P-OH 80
I
1

377

Hb OH Hd 6H

M = 874
363

(MH-79)+

796
+
(M+ Nal
I
a97

i I . II, 1
0 790 800 820 840 860 8430 So0 920

Fig-a. Fast atom bombardment spectrum of the dimeric adduct of

guanosine monophosphate cross-l inked by phosphoramide mustard.
Reprinted with permission of the American Chemical Society (ref.23.),
18

The challenge of mixture analysis correlation has been less satisfactorily

met by the particle bombardment techniques. A recent quantitative study of
standard mixtures of ionic and neutral surfactants showed that relative
intensities of organic cation peaks, present in FAB spectra did not
necessarily correspond to the relative molar amounts of compounds in the
sampl e (ref .28). On the other hand, careful studi es of glycopeptides which
have identical core structures and exhibit homologous (biological)
heterogeneity at both the sialic acid termini and the peptide end suggest that
quantitative assessments of the relative abundances within these mixtures can
be made (ref.29). It is important that the glycopeptide mixture be free of
contaminants such as sodium cations. One difference between these two
experiments may be the surface activity of the samples in glycerol.
To the four categories of information just discussed, we can add the
possibility of studying dynamic processes in the liquid matrix. Capriol i for
example has demonstrated that reactions may be followed which are catalyzed by
glycerol viable enzymes (ref.30).
In concluding, I want to summarize some of the questions a biochemist
might ask about particle desorption:
- How much sample do I need to prepare?
- How pure must it be?
- How heavy a molecule can we weigh?
- Can we identify components in a homologous mixture?
- Can we estimate relative proportions?
of a 1% component? of a 0.1% component?
- Can we detect an extra oxygen or methyl group?
reduction of a disul fide bond?
a deuterillm 1 abel?
Mass spectrometrists have some experience with the first four issues. The
fifth question,that of dynamic range,has not been discussed as much by
practicioneers of the various particle desorption techniques. The last
question comes back to resolution, and the requi rements debated at the
beginning of this article. Whether monisotopic masses or accurate average
masses are reported, it is possible and of real value to the biochemist to be
able to distinguish the presence or absence of a methyl group or an oxygen
atom introduced post-translationally into a peptide or protein. These
examples require the distinction of molecular weights, 14 or 16 atomic mass
units apart. It is also important to distinguish (M+H)+ ions from (M+Na)+ -
ions, a difference of 22 amu. In the molecular weight range of insulin the
distinction of post translational methylation would require resolution of
 19

about 400. In the mass range of interferon (20,000) 1 part in about 1400
would need to be resolved. The ability to distinguish even smaller molecular
weight changes would permit recognition of disulfide reduction, stable isotope
labels, cationization with lithium ions, etc. Although the analytical chemist
must mediate wttat is technologically and economically feasible, the
sophisticated biochemist can fully utilize the best sensitivity, dynamic
range, mass range and resolution we can provide.

POSITIVE ION FAE SPECTRUM

Fig.9. Partial fast atom bombardment spectrum of a mixed metal

face-to-face porphyrin dimer, CoAg(FTF4) (ref.25.).

ACKNOWLEDGEMENTS
The work at Hopkins was supported by a grant from the National Science
Foundation, PCM 8209954.
20

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