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RADIATION RESEARCH
161, 346–358 (2004)
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Soft X-Ray-Induced Decomposition of Amino Acids: An XPS, Mass
Spectrometry, and NEXAFS Study
Yan Zubavichus,a,1 Oliver Fuchs,b Lothar Weinhardt,b Clemens Heske,b,2 Eberhard Umbach,b Jonathan D. Denlingerc and
Michael Grunzea
a
Angewandte Physikalische Chemie, Universität Heidelberg, INF 253, 69120 Heidelberg, Germany; b Experimentelle Physik II, Universität
Würzburg, Am Hubland, 97074 Würzburg, Germany; and c Advanced Light Source, 1 Cyclotron Road, Berkeley, California 94720
(8–10). Amino acids, which are the building blocks of proteins, are among the simplest organic molecules of biological relevance and thus serve as convenient model systems
in studies of radiation damage.
Radiation-induced chemical modifications in the solid
state can be monitored by X-ray photoelectron spectroscopy
(XPS), which is sensitive to changes in the overall surface
composition and to chemical transformations of functional
groups. In this approach, the X-ray beam both damages and
probes the sample (11, 12). XPS has often been applied to
study amino acids and oligopeptides (13–22), and it is commonly observed that these molecules decompose under prolonged or intense irradiation. However, the atomistic mechanism of these radiation-induced chemical processes has
not been the subject of a detailed investigation. To our
knowledge, only one paper (23) devoted to the XPS characterization of soft X-ray-induced damage to a single amino
acid, lysine, has been published so far. In this paper, Bozack
et al. point out three important tendencies in the XPS spectra: an attenuation of a component in the C 1s signal corresponding to the carboxylic carbon, strong changes in the
N 1s peak interpreted as a deprotonation of the initially
protonated amino group of the zwitterionic amino acid, and
a decrease in the amount of oxygen relative to that of carbon. The authors conclude that all the changes can be explained by a decarboxylation of the molecule, preceded by
a proton transfer from the amino group to the carboxyl
group (zwitterion → neutral molecule transition). However,
the authors did not analyze the variations in the overall
stoichiometry, in particular, changes involving the nitrogen
atoms. Survey XPS spectra shown in ref. (23) (Fig. 2a and
b) suggest that not only the oxygen:carbon ratio but also
the nitrogen:carbon ratio decreases, in contrast to the fact
that the nitrogen:carbon ratio should be increased if only
decarboxylation (loss of gaseous CO2) is involved.
Near-edge X-ray absorption spectroscopy (NEXAFS),
similar to XPS, gives detailed structural information with
both atomic and functional group specificity. Recently, this
method was applied to the characterization of various amino acids (24, 25), and an excellent paper reporting on detailed experimental and theoretical analyses of carbon K-
Zubavichus, Y., Fuchs, O., Weinhardt, L., Heske, C., Umbach, E., Denlinger, J. D. and Grunze, M. Soft X-Ray-Induced
Decomposition of Amino Acids: An XPS, Mass Spectrometry,
and NEXAFS Study. Radiat. Res. 161, 346–358 (2004).
Decomposition of five amino acids, alanine, serine, cysteine,
aspartic acid, and asparagine, under irradiation with soft X
rays (magnesium Ka X-ray source) in ultra-high vacuum was
studied by means of X-ray photoelectron spectrometry (XPS)
and mass spectrometry. A comparative analysis of changes in
XPS line shapes, stoichiometry and residual gas composition
indicates that the molecules decompose by several pathways.
Dehydration, decarboxylation, decarbonylation, deamination
and desulfurization of pristine molecules accompanied by desorption of H2, H2O, CO2, CO, NH3 and H2S are observed with
rates depending on the specific amino acid. NEXAFS spectra
of cysteine at the carbon, oxygen and nitrogen K-shell and
sulfur L2,3 edges complement the XPS and mass spectrometry
data and show that the exposure of the sample to an intense
soft X-ray synchrotron beam results in the formation of C-C
and C-N double and triple bonds. Qualitatively, the amino
acids studied can be arranged in the following ascending order of radiation stability: serine , alanine , aspartic acid ,
cysteine , asparagine. q 2004 by Radiation Research Society
INTRODUCTION
Biological macromolecules, such as proteins or DNA, are
known to be very sensitive to ionizing radiation. On one
hand, this problem has a fundamental importance in biology and medicine (e.g. mechanisms of mutagenesis and
methods of radiation protection), but on the other hand, it
hinders the investigation of biologically important systems
by physical methods using intense beams of photons or
charged particles. Radiation damage manifests itself in the
disturbance of a long-range crystalline or supramolecular
order (1–3) and in chemical modifications of the system
under study, e.g. free radical formation (4–7) or mass loss
Address for correspondence: Universität Heidelberg, INF 253, 69120
Heidelberg, Germany; e-mail: [email protected].
2
Address after April 1, 2004: Dept. of Chemistry, University of Nevada, Las Vegas, NV 89154-4003.
1
346
X-RAY DAMAGE OF AMINO ACIDS: XPS, MS AND NEXAFS
347
FIG. 1. Molecular formulas of the five amino acids studied (as neutral molecules).
shell edge NEXAFS spectra of all 20 major amino acids
has been published (26). Recently, NEXAFS spectroscopy
was used efficiently in studies of soft X-ray-induced chemical modifications of organic polymers (27).
Useful information on the fragmentation of organic molecules under ionizing radiation can also be obtained by
mass spectrometry. Electron impact or chemical ionization
of amino acids in the gas phase induces a highly specific
fragmentation pattern that is the basis of the wide use of
mass spectrometry in qualitative and quantitative analysis
of proteins and their mixtures (28–30). Fragmentation patterns of solid amino acids under bombardment with fast
Ar1 ions (secondary ion mass spectrometry, SIMS) have
been studied extensively by Benninghoven and coworkers
(31–35) and by other authors (36).
Gohlke and coworkers studied the fragmentation of glycine in the gas phase through a dissociative electron attachment mechanism by means of negative-ion mass spectrometry (37). They pointed out that at least eight different
resonant dissociation pathways can be detected at kinetic
energies of the incident electrons in the range of 0–15 eV.
Accordingly, a variety of low-molecular-mass anionic fragments such as O/NH2, OH, CN, H2CN, HCO2, H2C2NO,
H2C2O2/H4C2NO, and H4C2NO2 can be produced by that
process.
In this paper, we monitor the radiation-induced decomposition of five amino acids—L-alanine (Ala), L-serine
(Ser), L-cysteine (Cys), L-aspartic acid (Asp), and L-asparagine (Asn)—at room temperature using time-resolved XPS
and mass spectrometry. Formally, these amino acids are
derived from alanine by substituting one of the hydrogen
atoms at the b-carbon position by a specific functional
group, i.e. -OH (serine), -SH (cysteine), -COOH (aspartic
acid), or -CONH2 (asparagine) (Fig. 1). Hence this series
of amino acids allows a direct and systematic comparison
of the influence of these functional groups on the radiation
stability of the target molecules. To gain deeper insight into
the mechanism(s) of the radiation-induced chemical modifications and the chemical nature of the products formed, a
series of NEXAFS spectra at the K-shell absorption edges
of carbon, oxygen and nitrogen and at the L2,3 edges of
sulfur were measured using synchrotron radiation for one
of the above amino acids, cysteine.
MATERIALS AND METHODS
Commercially available polycrystalline powders of amino acids (Sigma-Aldrich Chemie GmbH, purity .98%) were finely ground in a mortar
and pressed into sputter-cleaned indium foil to form a uniform layer.
Excess powder, which did not adhere to the foil, was removed with a
brush. Samples were loaded into the ultra-high vacuum chamber, pumped
for 12–14 h, and then exposed to X rays.
X-ray photoelectron spectra were measured using a VG ESCAscope
spectrometer (38) equipped with a dual-anode X-ray source, a hemispherical energy analyzer, and a multi-channeltron detector. A magnesium Ka
X-ray source operated at 300 W (15 kV 3 20 mA) was positioned about
20 mm in front of the sample surface. XPS spectra were recorded in the
fixed analyzer transmission (FAT) mode with a pass energy of 40 eV. In
this regimen, the FWHM of the Ag 3d5/2 line was 1.3 eV. The linearity
of the energy scale was checked using Cu 2p3/2 (932.7 eV), Ag 3d5/2
(368.3 eV), and Au 4f7/2 (84.0 eV) core-level lines measured on freshly
sputtered metal foils. C 1s, O 1s, N 1s and S 2p (the latter only for
cysteine) detail spectra were taken for all five amino acids with energy
steps of 0.1 eV. The acquisition time was chosen as a compromise between the signal-to-noise ratio in the spectra and the rate of radiationinduced decomposition. On average, every scan took 5–7 min and thus
one measurement cycle (carbon, nitrogen, oxygen and sulfur regions)
required 15–20 min. To monitor changes, the spectra were taken repeatedly over 3–4 h. At least 10 cycles were recorded for each of the amino
acids.
All spectra were taken at room temperature. In several cases, the temperature at the sample surface was measured during irradiation with a
standard chromel-alumel thermocouple and showed that the radiation load
is not sufficient to raise the sample temperature by more than about 5 K.
Since amino acid powders are insulating, the positions of the spectral
features in XPS were affected by charging. A standard procedure for the
energy correction in XPS implies assigning a binding energy of 284.4
eV to the aliphatic carbon in C 1s spectra (39). However, this procedure
could not be applied in our case since short-chain organic molecules with
strongly electronegative functional groups were the objects of the study,
and the level of contamination was relatively low according to the quantitative analysis (see below). Therefore, to compensate for the observed
shift, the high binding energy features in the C 1s spectra (corresponding
to carbon atoms of the carboxyl groups) were assigned to a binding energy of 288.4 eV. A similar value of 288.2 eV was determined in an
extended XPS study of simple amino acids, dipeptides and polypeptides
by Clark et al. (13).
For the quantitative analysis, XPS spectra were background-subtracted
using polynomial functions, fitted using symmetrical Gaussian functions,
348
ZUBAVICHUS ET AL.
FIG. 2. Detailed C 1s, O 1s, and N 1s XPS spectra of pristine alanine (Ala), serine (Ser), cysteine (Cys), aspartic
acid (Asp), and asparagine (Asn): experimental data (solid lines) and fitting results (dotted).
and integrated. Due to considerable inhomogeneous broadening, a line fit
using only Gaussian line shapes and thus neglecting the small Lorentzian
contribution was sufficient. Intensities of the shake-up satellites were not
taken into account. Standard atomic sensitivity factors (40) were used,
which were empirically corrected for the analyzer transmission so as to
achieve the correct 1:2:1 Si:C:O stoichiometry for a polydimethylsiloxane
[Me2SiO]n reference sample. The overall accuracy of the applied quantitative analysis procedure is limited to about 10%.
Mass spectra were measured simultaneously with the XPS spectra using a Transpector residual gas analyzer attached directly to the main vacuum chamber of the ESCAscope. The analyzer was equipped with an
electron impact ion source (102 eV), a quadrupole mass filter, and an
electron multiplier detector. Before switching on the X-ray source, the
base pressure in the main UHV chamber was ;1 3 1029 mbar. According
to the mass spectra, hydrogen, water, nitrogen/CO and argon were the
dominant species in the residual vacuum. After the X-ray source was
switched on, the pressure in the UHV chamber started to increase gradually due to a radiation-induced outgassing of the amino acid powders
despite continuous pumping by a 230 liter/s Varian StarCell VacIon
pump. The rates of increase in pressure and qualitative changes in the
mass spectral patterns were characteristic for the specific amino acid under study. During exposure, mass spectra were measured repeatedly every
10–15 min for positively charged ions over a mass-to-charge ratio (M/z)
range from 0 to 200 amu with a scan step of 1 amu and 256 ms of
acquisition time per channel. The detection limit of the electron multiplier
detector is around 10213 Å.
The positively charged cationic species registered by the detector of
the mass spectrometer were produced in the ionizer, not necessarily reflecting the radiation-induced desorption products. Nevertheless, X-rayinduced desorption is the only mechanism of mass transfer from the solid
surface to the gas phase. Measurements were stopped when the pressure
in the main chamber exceeded ;5 3 1028 mbar to prevent damage of
high-voltage devices operating in the UHV chamber. No pressure increases were detected in control measurements with the same experimental
conditions but a blank sample holder instead of an amino acid sample.
The NEXAFS spectra of cysteine were measured at the undulator beam
line 8.0 of the Advanced Light Source (ALS, Lawrence Berkeley National
Laboratory, Berkeley, CA) in the total fluorescence yield mode. Photon
energy scanning was achieved by a coupled movement of the undulator
gap and the spherical grating monochromator (SGM). The intensity of
the emitted photon beam was measured by a channeltron detector and
then normalized to the reference current from a grid of freshly evaporated
gold placed in the excitation beam before the sample. To slow down the
radiation damage to cysteine, the X-ray beam flux was reduced by minimizing the entrance and exit slits of the monochromator. The total flux
on the sample is estimated to 1012 photons/s. A series of 10–15 consec-
utive scans were recorded for all regions with an energy step of 0.05–
0.2 eV and a dwell time of 0.5–2 s per point, resulting in an overall
acquisition time for each spectrum of 30–120 s for carbon, nitrogen and
oxygen K-shell edges and 600 s for sulfur L2,3 edges.
The photon flux density in the NEXAFS experiment was roughly three
orders of magnitude higher than in the laboratory XPS experiment. A
(desirable) quantification of the radiation doses actually applied (instead
of exposure times) appeared impossible due to the unknown microscopic
structure and morphology of the amino acid grains in the sample. Thus,
and also because of a lack of beam time and required equipment, we
abstained from a detailed calibration of the photon fluxes from the two
different radiation sources.
RESULTS
XPS Characterization of Pristine Amino Acids
Initial XPS spectra of the amino acids in the C 1s, N 1s,
and O 1s regions are depicted in Fig. 2. In most cases, the
spectra have complicated asymmetric shapes due to contributions of several functional groups and shake-up satellites.3 For the assignment of the spectral features, it must
be taken into account that the form of amino acids that is
most stable in the solid state is a zwitterion with a protonated amino group and a deprotonated carboxyl group.
The higher binding energy component in the C 1s spectra
evidently corresponds to carbon atoms in carboxyl or carbamido groups (the latter is present only in asparagine),
which are known to induce a strong chemical shift toward
higher binding energies (EB). The low EB parts of the spectra contain partially resolved contributions from other substituents at carbon such as -OH, -NH31, -SH or -CH3. The
two major components of the C 1s spectra are arbitrarily
3
The term shake-up satellites denotes lower-kinetic-energy satellites of
major XPS lines originated from two-electron processes, in which the
outgoing photoelectron apparently loses a part of its energy to induce an
electron transition within the valence shell. For organic substances, p-p*
shake-up satellites are most common. The energy shift typically amounts
to 5–10 eV, and the intensity does not exceed a few percent of the main
line’s intensity.
349
X-RAY DAMAGE OF AMINO ACIDS: XPS, MS AND NEXAFS
TABLE 1
Results of a Quantitative XPS Analysis (at. %) for Amino Acids: Nominal
Stoichiometry, Pristine State, and after Specified Durations of Exposure to Soft X Rays
Ala
Ser
Cys
Asp
Asn
Stoichiometry
Pristine
25 min
50 min
95 min
120 min
140 min
170 min
Stoichiometry
Pristine
20 min
40 min
80 min
110 min
150 min
Stoichiometry
Pristine
25 min
50 min
80 min
110 min
150 min
240 min
Stoichiometry
Pristine
20 min
50 min
90 min
150 min
210 min
260 min
Stoichiometry
Pristine
40 min
70 min
110 min
150 min
230 min
Calk
Ccarb
C
OC5O
OOH
O
NNH3
1
NNH2
N
S
33.3
30.7
30.2
30.4
35.1
37.8
42.0
45.0
28.6
31.4
31.7
33.4
37.5
42.9
46.2
28.6
34.7
34.4
34.0
35.7
37.8
39.7
43.5
22.2
22.2
23.4
24.6
28.4
34.3
44.4
48.5
22.2
22.5
23.8
23.9
25.6
26.3
29.8
16.7
14.8
15.0
14.3
13.0
11.8
11.6
10.8
14.3
12.5
12.9
12.8
11.9
11.4
9.9
14.3
14.1
14.2
13.8
12.9
13.0
12.1
10.7
22.2
20.8
21.1
19.6
18.8
17.0
14.7
13.8
22.2
21.2
21.2
21.1
21.0
20.5
19.4
50.0
45.5
45.2
44.7
48.1
49.6
53.6
55.8
42.9
43.9
44.6
46.2
49.4
54.3
56.1
42.9
48.8
48.6
47.9
48.6
50.8
51.9
54.2
44.4
43.0
44.5
44.2
47.2
51.3
59.1
62.3
44.4
43.7
45.0
45.0
46.6
46.8
49.2
33.3
34.6
34.7
35.1
32.0
29.0
26.5
24.7
28.6
26.7
26.5
29.7
25.3
18.9
16.5
28.6
27.5
26.0
26.3
23.5
21.3
19.3
15.6
33.3
33.7
32.7
33.9
31.9
28.1
22.7
19.6
33.3
33.7
32.5
32.1
30.2
29.5
26.8
0.0
1.8
2.2
2.5
2.8
4.5
3.1
3.6
14.3
14.4
13.8
9.7
10.0
11.5
10.8
0.0
0.0
0.9
1.4
3.0
3.1
3.4
3.5
11.1
12.1
11.5
10.3
8.9
8.7
6.8
6.5
0
0
0
0
0
0
0
33.3
36.4
36.9
37.6
34.8
33.5
29.6
28.3
42.9
41.1
40.3
39.4
35.3
30.4
27.3
28.6
27.5
26.9
27.7
26.5
24.4
22.8
19.1
44.4
45.8
44.2
44.2
40.8
36.8
29.5
26.1
33.3
33.7
32.5
32.1
30.2
29.5
26.8
16.7a
16.3
15.9
15.3
14.0
13.5
12.9
11.1
14.3
15.0
13.2
11.1
10.0
8.1
6.9
14.3
11.8
11.3
11.4
11.4
11.3
10.3
8.6
11.1
11.1
10.1
10.4
10.0
9.4
7.3
6.6
11.1
9.7
9.4
8.8
7.9
7.3
5.0
0.0
1.7
2.0
2.5
3.1
3.4
3.9
4.6
0.0
0.0
1.9
3.3
5.3
7.2
9.6
0.0
3.0
3.7
3.3
3.6
3.4
4.3
6.5
0.0
0.0
1.1
1.2
1.9
2.6
4.2
5.1
11.1
13.0
13.0
14.1
15.3
16.4
19.0
16.7
18.0
17.9
17.8
17.1
16.9
16.8
15.7
14.3
15.0
15.1
14.4
15.3
15.3
16.5
14.3
14.8
15.0
14.7
15.0
14.7
14.6
15.1
11.1
11.1
11.2
11.6
11.9
12.0
11.5
11.7
22.2
22.7
22.4
22.9
23.2
23.7
24.0
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
14.3
9.0
9.5
9.7
10.0
10.1
10.7
11.6
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Note. Notations of components used in the fitting are explained in the text and shown for pristine amino acids in
Fig. 2).
a
For calculation of stoichiometric NNH3 fractions, amino acids are assumed to be 100% zwitterionic.
1
denoted Ccarb (288.4 eV) and Calk (285–287 eV), respectively, as shown in the left panel of Fig. 2.
The oxygen spectra in the central panel of Fig. 2 also
exhibit at least two components. The dominating low EB
component observed at 531.4 6 0.2 eV is assigned to the
keto-oxygens (OC5O) of carboxyl or carbamido groups. In
the case of a deprotonated carboxyl group, both oxygen
atoms contribute to the signal at this binding energy. The
higher binding energy component at about 532.8 eV, clearly
distinguishable in the spectra of serine and aspartic acid, is
due to the hydroxyl oxygens (OOH) of either the hydroxyl
groups (serine) or the nondeprotonated COOH groups (aspartic acid).
The two components sufficient to describe the N 1s spectra (right panel of Fig. 2) correspond to protonated (401.4
6 0.2 eV) and unprotonated (399.7 6 0.2 eV) NH2 groups.
The latter is most pronounced in the spectrum of asparagine, which has an amido group (see Fig. 1).
The results of a quantitative analysis of the XPS spectra
for the pristine amino acids are shown in Table 1 in comparison with the nominal stoichiometry and the changes as
a function of the radiation exposure (to be discussed below). The intensity ratios between the components in the
O 1s and N 1s spectra confirm that the zwitterionic state is
found predominantly at the surface of the pristine amino
acid powder crystallites. The overall compositions of the
pristine amino acids are close to the expected nominal stoichiometries: i.e., the procedure used for sample preparation
avoided significant contamination of the surfaces.
Upon prolonged exposure to X rays, significant changes
350
ZUBAVICHUS ET AL.
FIG. 3. Time evolution of detailed C 1s, O 1s, and N 1s XPS spectra of alanine during continuous exposure to
X rays.
both in the XPS line shape and surface composition were
observed for all amino acids. The time evolution of the C
1s, O 1s, N 1s and S 2p (Cys) XPS spectra for alanine,
serine, cysteine, aspartic acid and asparagine is shown in
Figs. 3–7. Results of the quantitative analysis are summarized in Table 1. In the quantitative analysis, the same fitting components as shown for the pristine amino acids in
Fig. 2 were used. The following trends are observed:
6. Changes in the relative amount of nitrogen are not significant and are different among the samples studied: A
decrease was observed for alanine and an increase for
serine and aspartic acid, whereas the changes for cysteine and asparagine were less than the statistical error
of our quantitative analysis; meanwhile, nitrogen:carbon
ratios decreased for all amino acids as a result of the
irradiation.
1. The fraction of the carboxyl-type carbon (Ccarb) decreases gradually with irradiation time for all five amino acids.
2. The relative amount of carbon increases significantly as
a result of the X-ray exposure.
3. Only small variations in O 1s XPS peak shapes are observed, although the OOH component at high EB exhibits
a clear tendency to increase with respect to the OC5O
component.
4. The relative amount of oxygen decreases significantly.
5. The ratio of unprotonated to protonated amino groups
(NNH2/NNH31) shows a pronounced increase.
If we assume that the zwitterions → neutral molecule
transition and the decarboxylation are the two predominant
processes caused by X irradiation of amino acids (23), we
would expect that (a) the rate of protonation of -COO2 to
produce -COOH (which could be monitored by an appearance of the OOH component in the O 1s spectra) should be
equal to the rate of deprotonation of the -NH31 groups, and
(b) the rate of decrease in oxygen percentage should correlate with the decrease in the fraction of the carboxyl-type
carbon atoms.
The results of our quantitative analysis (see Table 1)
show that these effects are not observed: The protonation
FIG. 4. Time evolution of detailed C 1s, O 1s, and N 1s XPS spectra of serine during continuous exposure to X
rays.
X-RAY DAMAGE OF AMINO ACIDS: XPS, MS AND NEXAFS
351
FIG. 5. Time evolution of detailed C 1s, O 1s, N 1s, and S 2p XPS spectra of cysteine during continuous exposure to X rays.
of the carboxyl groups is not comparable to the deprotonation of the amino groups, and the loss of oxygen is higher
than expected from the decrease in the Ccarb fraction. This
implies that other mechanisms are involved in the observed
radiation-induced chemical modifications. For instance, the
two aforementioned discrepancies can be reconciled if we
assume that a dehydration through scission of the Ccarb-OH
bond occurs. It must be stressed that a thermally activated
dehydration of carboxylic acids and amino acids in particular is well known (35). Nevertheless, to achieve a qualitative agreement between all the observed changes, even
more decomposition routes need to be taken into account.
To gain more insight into such alternative decomposition
routes, we have performed a detailed mass spectrometry
study of the gas-phase environment, which will be discussed in the following section.
Compositional Changes of the Gas Phase as Monitored
by Mass Spectrometry
Exposure of the amino acids to X rays leads to substantial changes in both the quantitative and qualitative com-
position of the residual gas in the experimental chamber.
Mass spectral patterns of the five amino acids after prolonged exposures to X rays are depicted in Fig. 8. In this
figure, ion currents are plotted on a logarithmic scale to
emphasize the contributions of low-concentration species.
No significant peaks in the range of 80–140 amu, which
would correspond to the molecular ions of the respective
amino acids, were detected. This indicates that it is not
evaporation of amino acids that is responsible for the observed changes in the mass spectral patterns, but rather radiation-induced decomposition accompanied by the release
of respective low-molecular-mass gaseous species, as expected from the XPS results. The most pronounced changes
occur for the following masses (M/z): 2, 16, 18, 28 and 44
amu, as shown in Fig. 9 (ion currents as functions of exposure time). Although these peaks are always present in
the mass spectral patterns of the vacuum chamber (Fig. 8a),
their intensities increase noticeably after the start of X-ray
exposure. In some cases, new components emerge. For example, during exposure of cysteine, a set of three mass
peaks at 32, 33 and 34 amu with constant intensity ratios
FIG. 6. Time evolution of detailed C 1s, O 1s, and N 1s XPS spectra of aspartic acid during continuous exposure
to X rays.
352
ZUBAVICHUS ET AL.
FIG. 7. Time evolution of detailed C 1s, O 1s, and N 1s XPS spectra of asparagine during continuous exposure
to X rays.
appears in the spectra (Fig. 8d). These peaks can be attributed to S1, HS1 and H2S1, respectively. The peaks at 2,
18 and 44 amu correspond to H21, H2O1 and CO21, respectively. Mass 28 is attributed to molecular ions of nitrogen and carbon monoxide. We suggest that variations in the
intensity of this peak are mostly due to changes in the con-
centration of CO since no sources of molecular nitrogen are
present in the experimental setup. Furthermore, H2CN1
could also contribute to this peak. Earlier, H2CN2 was detected as a product of the electron impact-induced fragmentation of gaseous glycine (33). Moreover, HCN is one
of the major pyrolysis products of amino acids (41). The
FIG. 8. Mass-spectral patterns of the residual gas in the main UHV chamber. Panel a: typical pattern of the UHV chamber before switching on the
X-ray source; panel b: irradiation of alanine for 185 min; panel c: irradiation of serine for 120 min; panel d: irradiation of cysteine for 260 min; panel
e: irradiation of aspartic acid for 260 min; panel f: irradiation of asparagine for 240 min.
X-RAY DAMAGE OF AMINO ACIDS: XPS, MS AND NEXAFS
353
FIG. 9. Concentration changes of principal residual gas components during X-ray exposure. Panel a; alanine; panel b: serine; panel c: cysteine;
panel d: aspartic acid; panel e: asparagine. aIon currents of H21 (M/z 5 2) are divided by 10.
peak at 16 amu is usually ascribed to O1, which is frequently found in UHV environments since it can be produced by fragmentation of any oxygen-containing molecule
(primarily water, carbon dioxide, and carbon monoxide).
However, changes in the intensity of this peak during X
irradiation of amino acids did not correlate with changes in
the intensity of the peaks at 18, 28 and 44 amu, and in
some cases, this peak even became the dominant one (see,
for instance, Fig. 9a). Therefore, it is assumed that it is due
to a great extent to another chemical species, e.g., NH21.
This ion could be formed by a direct detachment of the
amino group from an amino acid molecule by a heterolytic
scission of the C-N bond or by a fragmentation of gaseous
ammonia, NH3, in the ionizer of the mass spectrometer. In
summary, therefore, X irradiation of amino acids leads to
a substantial enrichment of the residual vacuum with H2,
NH2, H2O, CO, CO2, H2S (the latter only for cysteine), and
possibly also H2CN.
A comprehensive quantitative interpretation of the data
is difficult due to the dynamic character of the experimental
process: The pumping speed provided by the ion pump
varies significantly from one gaseous species to another and
depends on the overall pressure in the UHV chamber. Furthermore, a desorption of weakly bound species (e.g. H2O)
from the walls of the UHV chamber can be stimulated by
the increase in the total pressure. Hence we limit ourselves
to a qualitative analysis of the mass spectra.
The time evolution of the mass spectral patterns for the
five amino acids during irradiation has several common features. For instance, molecular hydrogen (M/z peak at 2
amu) remains the dominant component in the residual gas
for all amino acids throughout the exposure time, prevailing
over the other components by approximately one order of
magnitude. The water peak (M/z 18 amu) shows a peculiar
behavior: It increases rapidly in the early stages of the irradiation but then, after roughly 2 h of exposure, reaches a
354
ZUBAVICHUS ET AL.
saturation level or even passes through a maximum. We
believe that these changes are due to a significant extent to
the radiation-induced chemical dehydration of amino acids
rather than to desorption of physisorbed water, which would
have been a logical suggestion taking into account the highly hygroscopic character of the amino acids. First, the amino acids were purchased as anhydrous powders, and second, quantitative XPS analysis showed no significant
amount of over-stoichiometric oxygen, which could have
been assigned to physisorbed water.
Despite the similarities, there are distinct differences in
the time-dependent mass spectral patterns of the five amino
acids studied, which could reflect peculiarities of their decomposition mechanisms. In particular, in the case of alanine (Fig. 9a), the NH2 peak shows the fastest growth in
the initial stages of the irradiation while CO (or, partly,
H2CN) becomes the dominant species in the late stages.
Except for the aforementioned low-molecular-mass components, the residual gas is enriched in this case with heavier species, giving rise to peaks at 73, 56 and 45 amu, as is
clearly seen in Fig. 8b. These three peaks can be attributed
to [M-NH2], [M-NH2-OH], and [M-CO2] ions, respectively.
Here the standard nomenclature of mass spectrometry is
used; M denotes the molecular ion of alanine (Mw 5 89
amu). In the case of serine (Fig. 9b), the water peak (M/z
5 18) shows a pronounced growth in the early stages of
irradiation, indicating a fast dehydration of serine. In the
case of cysteine, major gaseous decomposition products, as
detected by mass spectrometry, are NH2 and CO2, which
evolve in parallel as a function of exposure time (Fig. 9c).
After prolonged exposure of cysteine, the mass spectral pattern shows a set of distinct peaks at 98–104 amu (Fig. 8d).
The origin of these peaks is presently unclear. They are
probably due to derivatives of 2-methylthiazolidine (Mw 5
103 amu). As has been shown (42), 2-methylthiazolidine is
also formed upon the pyrolysis of cysteine. For aspartic
acid, the dominant evolving species is CO2 (Fig. 9d),
whereas for asparagine it is H2O (Fig. 9e).
NEXAFS Spectra of Cysteine
When comparing the decomposition effects of amino acids in our NEXAFS and XPS spectra, three major factors
must be taken into account:
1. Intense soft X-ray beams generated by an undulator
beam line at a third-generation synchrotron source were
used for NEXAFS data collection. In this case, the photon density on the exposed sample area is higher by
three to four orders of magnitude than in the case of a
laboratory X-ray source, and thus the radiation-induced
decomposition could proceed much faster.
2. For the measurements of XPS spectra, non-monochromatized radiation with peak photon intensity at the energy of the magnesium Ka, 1253.6 eV, and a wide
Bremsstrahlung tail was used, whereas for NEXAFS, the
photon energy of the monochromatic beam was varied
over a certain range in the proximity of the resonant
excitation threshold. Due to the resonant character of the
excitation, and especially because of the drastically
modified Auger and autoionization decay channels of
the primary core hole, both the kinetics and the mechanisms of the decomposition reactions could change significantly (43, 44).
3. For the current NEXAFS measurements, the fluorescence yield mode of detection was used. The probing
depth of this technique is of the order of a few 1000 Å,
i.e., about two orders of magnitude greater than that of
XPS.
Nevertheless, we will show (for the case of cysteine) that
some general conclusions from the NEXAFS data can be
used to clarify and supplement the suggested mechanisms
of the radiation-induced decomposition derived from the
XPS and mass spectrometry data.
NEXAFS spectra of cysteine taken at the carbon, oxygen
and nitrogen K-shell and sulfur L2,3 edges are shown in Fig.
10 (normalized to the heights of the edge jumps). Significant spectral changes are observed as a function of irradiation time, which reveal themselves through the appearance
of new peaks, a redistribution of intensities between the
spectral features, and pronounced variations in the overall
intensity of the signal. Noticeable changes were observed
even between first scans on pristine cysteine measured with
different parameters (curves labeled ‘‘pristine*’’ and ‘‘pristine’’ in the left panel of Fig. 10): ‘‘pristine*’’ was measured with smaller entrance and exit slits of the monochromator and higher scanning speed, resulting in a total dose
reduction by a factor of 10 compared to ‘‘pristine’’. This
indicates that the beam damage is very fast and the results
of the radiation-induced processes become significant after
exposure of a sample for tens of seconds.
For carbon K-shell edge NEXAFS, only the so-called
p*-resonance range was measured (284–292 eV). The most
representative spectrum of pristine cysteine (curve ‘‘pristinea’’ in the left panel of Fig. 10) reveals an intense peak
at ;289.0 eV and a low-energy shoulder at ;287.7 eV.
The shape of the spectrum is quite close to the carbon Kshell edge NEXAFS spectrum of cysteine reported by Kaznacheyev et al. (26). The peak at ;289 eV is the spectral
signature of the carboxyl group and is attributed to the C
1s → p* (C5O) transition (24–26). The low-energy shoulder is assigned to a C 1s → s* (C-S) transition on the basis
of theoretical calculations within the static exchange
(STEX) approximation (26). A number of radiation-induced changes are observed. The intensity of the major
peak is significantly diminished due to irradiation, which
can be interpreted as an active decarboxylation of cysteine.
A new peak at ;290.4 eV first appears and then rapidly
disappears again. This energy position is typical of C 1s →
p* (C5O) transitions in dialkylcarbonates (45) or molecular CO2 (46). In our case, it probably corresponds to CO2
in an intermediate stage of desorption. Furthermore, pro-
X-RAY DAMAGE OF AMINO ACIDS: XPS, MS AND NEXAFS
355
FIG. 10. Time evolution of carbon, oxygen and nitrogen K-shell and sulfur L2,3-edge NEXAFS spectra of cysteine, normalized to their edge jumps.
Insets show the first (the spectrum labeled ‘‘pristine’’) and the last spectra of the series as measured without rescaling. *Measured with smaller entrance
and exit slits of the monochromator and higher scanning speed resulting in a total dose reduction by a factor of 10 compared to the spectrum labeled
‘‘pristine’’.
nounced and relatively narrow peaks develop at ;285.8 eV
and ;287.0 eV after 10 min of exposure. This energy range
is typical of 1s → p* transitions of C-C and C-N multiple
bonds (46). It suggests that irradiation is accompanied by
an extensive detachment of hydrogen atoms from the carbon atoms.
The oxygen K-shell edge NEXAFS spectrum of cysteine
is dominated by an intense O 1s → p* (C5O) peak at
;532.4 eV, in agreement with an earlier oxygen K-shell
edge study of glycine chemisorbed on Cu (110) (25). During irradiation, no substantial changes in the shape of the
spectrum occurred, but the signal intensity was reduced by
approximately one order of magnitude after 15–20 min of
exposure (Fig. 10, inset in the second panel from the left).
This means that up to 90% of the total amount of oxygen
in the irradiated sample volume of cysteine was liberated
in the form of gaseous decomposition products (mostly CO2
and H2O).
The pristine nitrogen K-shell edge NEXAFS spectrum
(Fig. 10, second panel from the right) of cysteine shows a
broad 1s → s* contribution around 406 eV, in general
agreement with the respective results for glycine on Cu
(110) (25). However, irradiation with soft X rays results in
dramatic changes in the spectrum. In particular, a series of
new peaks in the range of 399–403 eV arises, most probably attributable to N 1s → p* transitions of N5C and
N[C bonds. For instance, a recent high-resolution nitrogen
K-shell edge NEXAFS study of solid acrylonitrile
(CH25CH-C[N) revealed three N 1s → p* transitions at
about 399, 400 and 402.3 eV (47). A similar set of N 1s
→ p* resonances was observed in a NEXAFS study of
cyano-substituted quinones (48). Therefore, the nitrogen Kshell edge NEXAFS results support formation of C-N mul-
tiple bonds suggested on the basis of the C K-edge NEXAFS data.
In contrast, no significant changes in either the spectral
shape or the signal intensity were detected in the sulfur L2,3edge NEXAFS spectra (Fig. 10, right panel).
DISCUSSION
XPS, mass spectrometry, and NEXAFS give complementary information on the chemical processes occurring
in amino acids under irradiation with soft X rays. The experimental data presented above point to the existence of
several competing routes of radiation-induced decomposition. The following primary processes of decomposition are
suggested:
1. Dehydrogenation due to C-H, N-H, O-H and S-H bond
scission. The most significant experimental manifestations of these processes are the increase in the concentration of H2 in the residual gas and the appearance of
C-C and C-N double and triple bonds. Protonated amino
groups of zwitterionic amino acids are presumably most
sensitive to this process. Dehydrogenation probably does
not stop with the deprotonation of originally protonated
amino groups of zwitterionic amino acids but proceeds
further to form imino (C5NH) and cyano (C[N) derivatives.
2. Dehydration, i.e. a detachment of water molecules due
to C-OH bond scission. The most significant experimental manifestations of this process are the increase in the
concentration of water in the residual gas and a fast
reduction of the relative amount of oxygen in the sample
composition (XPS and NEXAFS). Both hydroxyl groups
at the carbon atoms in the side chains (e.g. in serine)
356
3.
4.
5.
6.
ZUBAVICHUS ET AL.
and in the carboxyl groups are affected by this process,
giving rise to unsaturated derivatives, cyclization and
condensation products such as 2,5-diketopiperazines and
oligopeptides.
Decarboxylation, i.e. a detachment of molecular CO2
due to Calk-Ccarb bond scission. The experimental manifestations of this process are the increase in the concentration of carbon dioxide in the residual gas and the
decrease in the Ccarb/Calk ratio.
Decarbonylation, i.e. a detachment of molecular CO.
The experimental manifestations of this process are the
increase in the concentration of carbon monoxide in the
residual gas and the decrease in the Ccarb fraction, as in
the case of decarboxylation. This process probably becomes important in the late stages of the decomposition
when part of the carboxyl group has been transformed
into keto or carbamido derivatives.
Deamination, i.e. a detachment of molecular ammonia
due to C-N bond scission. This is reflected by the increase in the NH21 concentration in the residual gas and
the reduction of the nitrogen:carbon ratio in the sample
composition.
For cysteine: desulfurization, i.e. a detachment of molecular H2S due to C-S bond scission. This process is
revealed by the detection of H2S1, HS1 and S1 ions in
the residual gas.
The dominating decomposition route depends on the molecular structure of the specific amino acid and probably on
the conditions of the irradiation. Under the conditions used
for XPS data collection (magnesium Ka X-ray source), the
dominating decomposition routes are identified as follows:
deamination for alanine, dehydration (Calk-OH scission) for
serine, deamination/decarboxylation for cysteine, decarboxylation for aspartic acid, and dehydration (Ccarb-OH scission) for asparagine. The overall rates of decomposition are
also different for the five amino acids studied. Since the
same experimental setup was used for irradiation of all five
amino acids, their overall radiation stabilities can be compared. With the current setup, we did not control some important parameters of the samples that could affect the decomposition kinetics, such as the mean particle size of the
amino acid powders and the thickness and homogeneity of
the amino acid layers on indium, but we assume that these
parameters are not crucial and will not distort the results of
a qualitative comparison. Relative changes in the surface
composition for alanine, serine, cysteine, aspartic acid and
asparagine after about 150 min of irradiation with soft X
rays are given in Table 2 along with the respective degassing rates quantified as relative increases in the pressure in
the main vacuum chamber. Therefore, among the five amino acids studied, serine can be considered the most susceptible to the soft X rays, whereas alanine and, even more so,
aspartic acid are more resistant to the radiation-induced decomposition. Cysteine and, in particular, asparagine appeared to be most stable according to our results.
TABLE 2
Changes in the Chemical Composition of Amino
Acids (notations as in Table 1) and Experimental
Chamber Pressure (P) after 150 min of Irradiation
with Respect to the Pristine Amino Acids (Final
Value/Initial Value Ratios)
Ser
Ala
Asp
Cys
Asn
Ccarb
Carbon
Oxygen
Nitrogen
P
0.79
0.73
0.82
0.86
0.97
1.31
1.23
1.19
1.06
1.07
0.66
0.78
0.80
0.83
0.88
1.10
0.87
1.15
1.01
1.04
13.3
13.5
9.4
5.6
2.8
Although we were able to clarify some aspects of the
radiation-induced modifications of amino acids, the basic
events triggering the decomposition remain unclear. In principle, both soft X-ray photons and low-kinetic-energy secondary electrons, which are inevitably produced by inelastic scattering of photoelectrons and Auger electrons in the
sample, can act as initiators of the damage through a number of mechanisms, such as the dissociative electron attachment (DEA) (49) or core hole photoionization followed
by the Auger electron decay (50). The latter process is very
effective for a dissociation since it leads to a double-hole
final state. While the multiple ionization is already sufficient for a bond breakage (Coulomb explosion) (51), the
double hole has the additional effect of a self-localization,
i.e. a localization of two holes in previously bonding molecular orbitals (valence bond depopulation) on the time
scale of fragmentation (52, 53).
It should be mentioned that a pyrolysis of amino acids
at temperatures of 300–5008C is accompanied by processes
analogous to those discussed above (28, 29, 54). Although
we did not observe a significant macroscopic temperature
increase during irradiation of the amino acids, a local heating of surface areas cannot be totally ruled out.
CONCLUSIONS
Summarizing all experimental results, we conclude that
the aliphatic amino acids we studied—alanine, serine, cysteine, aspartic acid, and asparagine—are susceptible to soft
X rays and undergo chemical transformations under prolonged or intense exposure at room temperature. There are
a number of pathways through which the amino acids decompose, such as dehydrogenation (deprotonation), dehydration, decarboxylation, decarbonylation, deamination and
desulfurization. All of the C-H, N-H, O-H and S-H bonds
as well as the Calk-OH, Ccarb-OH, Calk-Ccarb, Calk-N and CalkS bonds in intact amino acid molecules are affected by
radiation. Scission of these bonds is accompanied by a liberation of respective gaseous species, such as H2, H2O, CO2,
CO, NH3 and H2S, as well as by a formation of C-C and
C-N multiple bonds. The driving force of the amino-group
deprotonation observed in N 1s XPS is probably not a
transformation of a zwitterion into a neutral molecule, but
X-RAY DAMAGE OF AMINO ACIDS: XPS, MS AND NEXAFS
rather a formation of low-basicity nitrogen-containing functional substituents, such as imino (C5NH), cyano (C[N),
or amido (CONH2) groups.
The amino acids studied can be arranged in the ascending order of their radiation stability: serine , alanine ,
aspartic acid , cysteine , asparagine. Therefore, since
these amino acids can be formally considered as derivatives
of alanine with respective substituents, destabilizing effects
of functional groups on aliphatic amino acids with respect
to soft X rays decrease as –OH . –COOH ø –NH2 . –
SH . –CONH2.
The processes of radiation damage described here must
to be taken into account in the interpretation of results involving exposure of amino acids or more complex biological polymers to soft X rays, in particular to intense synchrotron beams. These results are also expected to be relevant for studies of other classes of organic molecules containing similar functional substituents.
13.
14.
15.
16.
17.
18.
19.
ACKNOWLEDGMENTS
We are grateful to Dr. M. Zharnikov and Dr. A. Schaporenko (Angewandte Physikalische Chemie, Universität Heidelberg) for technical assistance and valuable discussions. This work was supported by BMBF
(projects No. 05 KS1VHA/3 and 05 KS1WW1/6) and the Fonds der
Chemischen Industrie (MG and EU).
20.
21.
22.
Received: May 27, 2003; accepted: September 15, 2003
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