MALDI-TOF mass spectrometry, an efficient technique for in situ detection and characterization of actinomycins

Article abstract of DOI:10.1002/jms.3329

An extensive study of actinomycins was performed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). Actinomycins represent a well-known family of peptidolactone chromopeptides with potent cytostatic and antibiotic properties. Using five well-characterized streptomycete strains, we introduced MALDI-TOF MS as an efficient technique for rapid in situ detection of actinomycins in surface extracts of cells picked from agar plates. By this procedure, actinomycin complexes can be investigated with high sensitivity and accuracy in a minimum of time. These studies were complemented by mass spectrometric investigation of actinomycins obtained from culture filtrate extracts and purified by high-performance liquid chromatography to detect yet unknown actinomycin species. By feeding experiments, C-demethyl-actinomycins from Streptomyces chrysomallus and Streptomyces parvulus as well as hemi-actinomycins from Streptomyces antibioticus lacking one of the two pentapeptide lactone rings were isolated and characterized as novel variants for structure-activity relationship studies. Structural characterization of the investigated actinomycins was performed by post source decay MALDI-TOF MS. The specific features of the fragmentation patterns of the protonated and cationized forms of selected actinomycins were investigated in detail.

Full text of DOI:10.1002/jms.3329

Research article
Received: 30 September 2013
Revised: 10 December 2013
Accepted: 16 December 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3329
MALDI-TOF mass spectrometry, an efficient
technique for in situ detection and
characterization of actinomycins
Joachim Vater,* Ivana Crnovčić, Siamak Semsary and Ullrich Keller
An extensive study of actinomycins was performed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF
MS). Actinomycins represent a well-known family of peptidolactone chromopeptides with potent cytostatic and antibiotic proper-
ties. Using five well-characterized streptomycete strains, we introduced MALDI-TOF MS as an efficient technique for rapid in situ
detection of actinomycins in surface extracts of cells picked from agar plates. By this procedure, actinomycin complexes can be
investigated with high sensitivity and accuracy in a minimum of time. These studies were complemented by mass spectrometric
investigation of actinomycins obtained from culture filtrate extracts and purified by high-performance liquid chromatography to
detect yet unknown actinomycin species. By feeding experiments, C-demethyl-actinomycins from Streptomyces chrysomallus and
Streptomyces parvulus as well as hemi-actinomycins from Streptomyces antibioticus lacking one of the two pentapeptide lactone rings
were isolated and characterized as novel variants for structure–activity relationship studies. Structural characterization of the inves-
tigated actinomycins was performed by post source decay MALDI-TOF MS. The specific features of the fragmentation patterns of the
protonated and cationized forms of selected actinomycins were investigated in detail. Copyright © 2014 John Wiley & Sons, Ltd.
Keywords: actinomycins; MALDI-TOF MS; in situ detection; structural characterization; PSD-MALDI-TOF MS
magnetic resonance methodologies.[2,7,11–19] In earlier work, a wide
spectrum of mass spectrometric techniques has been used for the
Introduction
Actinomycins are a family of closely related chromopeptides with
potent anticancer and antibiotic activities of medical and
biotechnological interest.[1] An overview on naturally occurring
actinomycins was given by Bitzer et al.[2] They are produced by a
variety of streptomycete strains, each synthesizing a specific set
of such compounds. They consist of a 2-amino-4,6-dimethyl-3-ox
o-phenoxazine-1,9-dicarboxylic chromophore to which two
pentapeptide lactone rings are connected each to one of the car-
boxylic groups in positions 1 and 9 of the phenoxazinone core.
Prominent examples are the actinomycins of the C-type,[3–6] G-
type,[7] X-type,[8–10] Y-type[2] and Z-type.[11] The structures of actino-
mycins and related peptidolactone compounds are shown in Fig. 1.
Variations in the actinomycin structure are restricted to amino
acid substitutions in the peptide lactone rings, while the pheno-
xazinone core is essentially unmodified. In some cases, cyclization
is observed between the amino group in position 2 of the chromo-
phore with functional groups of modified threonine residues in the
β-ring, such as chlorine or a hydroxyl group at position 4 of the
threonine component in the β-ring. In biosynthetic studies, we
recently reported mono-demethyl and di-demethyl derivatives of
actinomycin D, which lack one or both methyl groups in 4-position
and 6-position of the phenoxazinone core[12] and which are one of
the first variations directly at the chromophore. Remarkably, actino-
mycins function as intercalators with the DNA double helix. Their
phenoxazinone core fits between two guanine/cytosine base pairs
with the peptidolactone side chains located in the minor grove of
the helix. In this way, these compounds can be used as specific
inhibitors to study the transcription process.
detection and structural characterization of actinomycins using ion-
ization by electron ionization,[13] plasma desorption,[14] fast atom
bombardment[15,16] and more recently by electrospray ionization
(ESI).[7,11,19] In this paper, we present a comprehensive and compar-
ative mass spectrometric study of actinomycins using matrix-assisted
laser desorption/ionization mass spectrometry (MALDI-TOF MS). This
modern method offers the advantage for rapid in situ detection of
actinomycins in surface extracts of streptomycete cells picked from
agar plates. By this procedure, actinomycin complexes can be inves-
tigated with high sensitivity and accuracy in a minimum of time
followed by structural characterization using post source decay
(PSD)-MALDI-TOF MS. Because of their interesting biological proper-
ties and their attractive potential for medical and biotechnological
applications, such research is of high interest to make available novel
actinomycins for structure–activity relationship studies.
Materials and methods
Chemicals
Actinomycin D was purchased from Applichem (Darmstadt, Germany).
The matrix α-cyano-4-hydroxycinnamic acid was from Bruker
Daltonics, Bremen, Germany. Etamycin was isolated from cultures of
*
Correspondence to: Joachim Vater, Institut für Chemie, Technische Universität
Berlin, Müller-Breslau-Straße 10, D 10623 Berlin, Germany. E-mail: Joachim.
Vater@alumni.tu-berlin.de
Several publications have been focused on the structural charac-
terization of actinomycins by mass spectrometric and nuclear
Institut für Chemie, Technische Universität Berlin, Müller-Breslau-Straße 10, D
10623 Berlin, Germany
J. Mass Spectrom. 2014, 49, 210–222
Copyright © 2014 John Wiley & Sons, Ltd.

MALDI-TOF mass spectrometry of actinomycins
Figure 1. Structures of actinomycin, actinomycin derivatives and the heptapeptidelactone antibiotic etamycin. (a) general structure of actinomycin; (b) hemi-
actinomycin D; (c) 4-methyl-3-hydroxybenzoic acid-(4-MHB)-pentapeptide lactone (actinomycin half molecule); (d) etamycin. Xα,β, Yα,β and Zα,β refer to the
different amino acid substitutions between the actinomycins. MeVal: L-N-methylvaline; Sar: L-N-methylglycine; PheSar: phenylsarcosine; β,N-DiMeLeu: L-β,N-
dimethylleucine; D-Hypro: allo-D-hydroxyproline.
Streptomyces griseoviridus according to Schlumbohm and Keller.[20] and
purified by high-performance liquid chromatography (HPLC). All other
chemicals were of the highest purity commercially available.
combination of both. For the preparation of 4-MHB pentapeptide
lactone, S. chrysomallus was grown up to 1 day in liquid CM[21] after
which 4-methyl-3-hydroxybenzoic acid (4-MHB) was added in
4 mM concentration. After shaking at 220rpm and 30 °C for up to
4 days, each flask was extracted with ethyl acetate, and the extracts
were separated by HPLC.
Strains and cultures
Streptomyces chrysomallus ATCC 11523, Streptomyces parvulus
ATCC 12434 and Streptomyces antibioticus ATCC 14888 (IMRU
3720) were from the American Type Culture Collection. Strepto-
myces fradiae DSM 40711 and Streptomyces iakyrus DSM 41873
were from the Deutsche Sammlung von Mikroorganismen und
Zellkulturen (DSMZ, Braunschweig, Germany). All strains were
maintained on complete medium (CM) agar[21] or on GYM Strepto-
myces medium (DSMZ). To produce mycelium actively synthesizing
actinomycins growth of S. chrysomallus and S. parvulus in liquid
culture was either for up to 2 days in liquid CM[21] or for 3 to 4 days
in a glutamate–mineral salts medium.[22] S. antibioticus, S. iakyrus
and S. fradiae were grown exclusively in the glutamate–mineral
salts medium. All cultures were performed with 1% maltose plus
0.1% glucose as carbon source (both sterilized separately) using
500 ml Erlenmeyer flasks containing 200 ml medium. Incubation
was at 28 °C and 220rpm in a New Brunswick G 25 shaker. The
content of actinomycins in liquid cultures was determined by
extraction of culture aliquots with ethyl acetate and evaporation
to dryness and subsequent spectrophotometric determination at
441 nm using authentic actinomycin D as standard. For preparation
of surface extracts, streptomycete cells were picked from agar
plates and extracted with 50 μl of 50% aqueous acetonitrile
containing 0.1% trifluoroacetic acid. Extracted cells were removed
by centrifugation, and the supernatants used for mass spectrometric
investigation of actinomycins.
HPLC separations
Actinomycin mixtures were fractionated using a Waters HPLC sys-
tem or a Pharmacia LKB HPLC system (Pump 2248, WWM 2141).
Separation was performed on a prepacked EnCaPharm 100-RP18
column (Molnar Institute, Berlin, Germany) isocratically with 60%
(by vol.) acetonitrile–water at a flow rate of 1 mlminÀ1. The
detection wavelength was 452 nm.
Mass spectrometric analysis
MALDI-TOF mass spectra were recorded using a Bruker Autoflex
instrument equipped with a 337 nm nitrogen laser for desorption
and ionization. Of surface extracts or HPLC fractions, 2μl samples
were mixed with the same volume of matrix medium (a saturated
solution of α-cyano-4-hydroxycinnamic acid in 50% aqueous
acetonitrile containing 0.1% trifluoroacetic acid), spotted on the
target, air dried and measured. Spectra were recorded by positive
ion detection in reflector mode. The acceleration and reflector volt-
ages were 19 and 20 kV, respectively, in pulsed ion extraction
mode. Most of the matrix ions were suppressed until 350Da. The
structure of actinomycins was investigated by PSD-MALDI-TOF MS.
Preparation of desaminoactinomycin D and test by TLC
Actinomycin D was incubated with 0.5 ml of 10% HCl for 16 h at
40 °C. After incubation, the product was extracted with 1 ml ethyl
acetate, and the extract was evaporated to dryness.[23] The purity
of the desaminoactinomycin D (2-hydroxy-actinomycin D) was tested
by TLC using ethyl acetate : methanol : water : dimethylformamide
(100:5:5:1, by vol.) as the solvent.
Feeding experiments
Feeding experiments with 3-hydroxyanthranilic acid (3-HA) and
4-methyl-3-hydroxyanthranilic acid (4-MHA) were performed as
follows: Mycelium from cultures of a streptomycete actively synthe-
sizing actinomycins was harvested by suction on a Buechner funnel
and after washing with tap water resuspended in 40% of the orig-
inal volume of tap water containing a carbon source (0.5–1% glu-
cose or galactose), 1–2 mM of each L-threonine, L-valine, L-proline,
L-glycine and 100 μM L-methionine. Of this suspension, 5–8 ml por-
tions were placed in 100 ml Erlenmeyer flasks, and 3-HA or 4-MHA
was added at concentrations of 200 μM. The controls contained no
such additive. After shaking at 220 rpm and 30°C for up to 4 h, each
flask was extracted with ethyl acetate, and the extracts were
separated by thin layer chromatography (TLC) or HPLC or by a
Results and discussion
In situ detection of actinomycins in surface extracts of five
streptomycete strains by MALDI-TOF mass spectrometry
The actinomycin complexes produced by five representative
Streptomyces strains, S. chrysomallus, S. parvulus, S. antibioticus, S.
fradiae and S. iakyrus, were investigated by MALDI-TOF MS. For
J. Mass Spectrom. 2014, 49, 210–222
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J. Vater et al.
this purpose, these strains were grown on agar plates using
either the CM-medium or the GYM-medium. The best results
have been obtained with the CM-medium. After growth for
24 h, cells were picked from the agar plates and extracted with
50% acetonitrile/0.1% trifluoroacetic acid. In this way, surface
extracts were prepared, which efficiently can be used for mass
spectrometric investigation of products that are attached to the
outer surface of the cell wall of the tested Streptomyces strains.
For most of these organisms, the actinomycins were the domi-
nating metabolites. All these compounds could be detected in
situ by MALDI-TOF MS with high sensitivity and precision in a
minimum of time, frequently without the need for further
fractionation and purification. The MALDI-TOF mass spectra of
the actinomycins found in surface extracts of the five test strains
are shown in Fig. 2. They exhibit peaks for their protonated forms
as well as for their sodium and potassium adducts, which domi-
nate in the spectra. Monoisotopic mass numbers were obtained.
The resolution of the mass data was in the range of 5000. Devia-
tions from the calculated values were +0.2 Da. The MALDI-TOF
mass spectra of all investigated actinomycins exhibit an unusual
pattern of isotope distribution comprising 5 to 6 peaks, which will
be discussed later in detail. The mass numbers of the detected
[M + H]+ ions and the alkali adducts of actinomycins are listed
in Table 1. They essentially correspond to those that have been
previously reported for the five test strains.[2,7,11–17] Because it is
not possible in our study to discriminate between amino acid
substitutions in the α-ring and β-ring and to distinguish optical
isomers by MALDI-TOF mass spectrometry, we used the knowl-
edge[1–12] on the structure of actinomycins for assignment of
the detected actinomycin product ions.
Detection of C-type and X-type actinomycins
S. chrysomallus forms actinomycins of the C-type comprising C1–C3,
which differ at the D-Val position of their peptide lactone rings. Ac-
tinomycin C1 (IV, D) that yields a [M+ H]+ ion at m/z 1255.3 is the
well-known reference compound actinomycin D containing D-Val
in both rings, while in actinomycins C2 and C3 with [M+ H]+ ions
at m/z 1269.3 and 1283.4, one or both D-valine residues are
replaced by D-allo-Ile, respectively[2,6] (Fig. 2a). S. chrysomallus is also
a potent producer of nonactins, which in this case dominate in the
mass spectrum. Their [M+ H]+ ions were found between m/z 740
and 840 in Fig. 2b.
Figure 2. Rapid in situ detection of actinomycins in surface extracts of cells taken from agar plates of five streptomycete strains by MALDI-TOF MS.
(a and b) Streptomyces chrysomallus, (c) Streptomyces parvulus, (d) Streptomyces antibioticus, (e) Streptomyces fradiae and (f) Streptomyces iakyrus.
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MALDI-TOF mass spectrometry of actinomycins
Table 1. A survey of actinomycins of five representative Streptomyces strains as detected by in situ MALDI-TOF MS
Strain
Actinomycin species
M + [H,Na,K]+ (m/z)
a) In situ detection in surface extracts:
S. chrysomallus
Actinomycin C1 (D, IV)
1255.4/1277.4/1293.4
1241.4/1263.4/1279.4
1269.4/1291.4/1307.4
1283.4/1305.4/1321.4
1255.5/1277.5/1293.6
1241.4/1263.4/1279.5
1269.5/1291.6/1307.5
1271.6/1293.6/1309.6
1255.7/1277.7/1293.7
1269.7/1291.7/1307.6
1271.7/1293.7/1309.6
1301.2/1323.2/1339.2
1286.0/1308.0/1324.1
1284.0/1306.0/1322.1*
1320.0/1342.1/1358.2
1322.3/1344.3/1360.3*
1270.0/1292.0/1306.0
1268.3/1290.3/1308.3*
1304.2/1326.2/1342.2
1306.2/1328.2/1344.2*
1315.3/1337.3/1353.4*
1256.2/1278.3/1294.3
1258.1/1280.1/1296.2*
1274.3/1296.3/1312.3
1276.2/1298.2/1314.3*
1244.3/1266.3/1282.3*
1314.2/1336.3/1352.3*
C-Demethylactinomycin C1
Actinomycin C2
Actinomycin C3
S. parvulus
Actinomycin C1 (D, IV)
C-Demethylactinomycin C1
Actinomycin V
Actinomycin I
S. antibioticus
S. fradiae
Actinomycin C1 (D, IV)
Actinomycin V
Actinomycin I
Actinomycin Z1
Actinomycin Z2
Actinomycin Z3
Actinomycin Z4
Actinomycin Z5
S. iakyrus
Actinomycin G4
Actinomycin G3/G6
b) Detection in culture filtrates from feeding experiments:
S. chrysomallus
4-MHB-peptidolactone C1
616.4/638.4/654.4
630.5/652.5/668.5
4-MHB-peptidolactone C2
S. antibioticus
C-Demethylactinomycin C1
C-di-Demethylactinomycin C1
C-Demethylactinomycin V
C-di-Demethylactinomycin V
C-Demethylactinomycin I
1241.7/1263.7/1279.8
1227.7/ 1249.7/ 1265.7
1255.9/1277.9/1293.9
1241.9/1263.9/1279.9
1257.8/1279.8/1295.8
1243.7/1265.7/1281.8
792.5/814.5/830.6
C-di-Demethylactinomycin I
hemi-Actinomycin C1 (D, IV)
C-Demethyl-hemi-actinomycin C1
C-di-Demethyl-hemi-actinomycin C1
778.5/800.5/816.5
764.5/786.5/802.5
The mass data of the protonated form and the alkali adducts of the detected actinomycins were listed. Unidentified species were indicated by an asterisk.
The main actinomycin in the mass spectrum of the surface
extract of S. parvulus in Fig. 2c is actinomycin D (C1, IV). In addi-
tion, two minor peaks were detected at m/z 1269.5 and 1271.6,
which could be attributed to the [M + H]+ ions of actinomycins
V and I, which hitherto have not been reported in previous char-
acterization of this streptomycete strain. A similar pattern of acti-
nomycins is exhibited by S. antibioticus (Fig. 2d).[2–6] It produces a
mixture of actinomycin D, actinomycins I and V, which belong to
the actinomycin X-complex. They differ from each other in the
proline site of their β-ring. While actinomycin D (IV, C1) is an
iso-actinomycin with two identical pentapeptide lactone rings
each containing proline, the asymmetric actinomycins I and V
(aniso-actinomycins) show a modification at the proline site in
the β-ring. Both are minor products. Actinomycins I and V show
a 4-hydroxyproline or a 4-oxoproline at this position, respectively.
Detection of G-type and Z-type actinomycins
S. fradiae (Fig. 2e) and S. iakyrus (Fig. 2f) produce actinomycins of
the Z-type and G-type, respectively.[11,7] In the surface extract of
S. fradiae, [M + H]+ ions of variants Z2, Z3 and Z4 were detected
at m/z 1284.0, 1320.0 and 1270.0, while for S. iakyrus, [M + H]+ ions
of G-type species were found at m/z 1256.2 and 1274.3, which
could be attributed to G4 and G3/G6. For these two strains, it
was difficult to detect all actinomycin variants so far known by
in situ MALDI-TOF MS and to identify unknown species. Thus,
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J. Vater et al.
Figure 3. Structure analysis of actinomycin D from Streptomyces chrysomallus and hemi-actinomycin D produced by feeding of 4-MHA to Streptomyces
antibioticus by PSD-MALDI-TOF MS. PSD-production spectra of (a) protonated actinomycin D ([M + H]+ m/z 1255.6) and (b) its sodium adduct ([M + Na]+
m/z 1277.5) and (c) protonated hemi-actinomycin D ([M + H]+ m/z 792.6) and (d) its sodium adduct ([M + Na]+ m/z 814.5 ). Peptidolactone rings of acti-
nomycin D: Xα, Xβ = L-N-methylvaline; Yα, Yβ = L-proline; Zα, Zβ = D-valine.
efficient fractionation and further purification of the actinomycin
mixture were needed. Therefore, we separated both the surface ex-
tracts and the ethyl acetate extracts of the culture supernatants of
S. fradiae and S. iakyrus by HPLC (data not shown). By combination
of MALDI-TOF mass spectrometric analysis with fractionation by
HPLC, we succeeded in identifying most of the Z-type and G-type
actinomycins so far described in the literature.[11,7] For example,
in this way also, the [M+ H]+ ions of Z1 and Z3 were detected at
m/z 1301.2 and 1303.1. Z1 and Z3 appeared as minor products in
the ethyl acetate extracts. In addition, some unidentified actinomy-
cins of the G-type and Z-type were found whose structures still
remain to be elucidated. They are marked in Table 1 by an asterisk.
The characterization of these unknown actinomycins needs further
intensive separation and purification.
also detected in traces by MALDI-TOF MS under natural condi-
tions in surface and culture filtrate extracts of unsupplemented
Streptomyces cultures. Using this technique for S. antibioticus,
the mono-C-demethyl and di-C-demethyl derivatives of all its
actinomycin products, actinomycins D, I and V, were found
(Table 1).
Other novel actinomycins were obtained by feeding S.
antibioticus with excess of 4-MHA and 3-HA. The phenoxazinone
moiety of actinomycins is formed in the last step of their biosynthe-
sis by oxidative condensation of two 4-MHA-pentapeptide lactone
precursors. 4-MHA shows a reactive o-aminophenol structure,
which is able to directly condense with 4-MHA-pentapeptide
lactone units resulting in a phenoxazinone moiety with only one
instead of two pentapeptide lactone rings as in actinomycin. In this
way, hemi-actinomycins were created as new compounds, which
were characterized by MALDI-TOF MS (Table 1). External addition
of 3-HA to mycelium of S. antibioticus instead of 4-MHA led to the
formation of C-demethyl-hemi-actinomycins lacking one or both
methyl groups in positions 4 and 6 of the phenoxazinone
core. Such hemi-actinomycins could be detected in minute
quantities by MALDI-TOF MS also under natural conditions
in long-term cultures of S. antibioticus. In this way, novel
actinomycin species were generated that can be used for
structure–activity relationship studies towards medical and
biotechnological applications.
Characterization of novel actinomycins obtained from feed-
ing experiments
Novel actinomycin species such as C-demethylactinomycins and
hemi-actinomycins were obtained by feeding actively growing
mycelia of streptomycete strains with precursors of actinomycin
biosynthesis. S. chrysomallus and S. parvulus were fed with 3-h
ydroxy-anthranilic acid (3-HA), which is incorporated into penta-
peptide lactone precursors in competition with the regular
precursor 4-MHA. In this way, two novel actinomycins were iden-
tified as the mono-C-demethyl and di-C-demethyl derivatives of
actinomycin D lacking either one or both methyl groups in
positions 4 and 6 of the phenoxazinone core. In S. parvulus these
were obtained in a yield of at most 10%. In this way, we gener-
ated biosynthetically actinomycin species with modifications at
the chromophore for the first time.[12] These compounds were
In this paper, we give an overview on the yet identified
species of these new actinomycins. The pathways for the pro-
duction of the C-demethyl derivatives and their biochemical
characterization are described in the study of Crnovčić et al.[12]
For the hemi-actinomycins such data are presented in another
recent paper.[24]
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MALDI-TOF mass spectrometry of actinomycins
Figure 4. Structure analysis of a X-type, Z-type and G-type actinomycin produced by Streptomyces antibioticus, Streptomyces fradiae and Streptomyces
iakyrus, respectively, by PSD-MALDI-TOF MS. PSD-fragment spectra of (a) protonated actinomycin I ([M + H]+ m/z 1272.0), (b) protonated actinomycin Z4
([M+ H]+ m/z 1270.1), (c) the sodium adduct of actinomycin Z4 ([M + Na]+ m/z 1292.2) and (d) protonated actinomycin G4 ([M+ H]+ m/z 1257.8). In all PSD-
MALDI-TOF spectra, the dominant precursor ion is cut off for a better presentation of the fragment peaks. Peptidolactone rings of (a) actinomycin I: Xα, Xβ =
L-N-methylvaline; Yα = L-proline; Yβ = trans-4-hydroxyproline (Hyp); Zα, Zβ = D-valine. (b and c) actinomycin Z4: Xα = L-N-methylvaline; Xβ = L-N-methylalanine;
Yα = cis-5-methylproline; Yβ = cis-5-methyl-4-oxoproline; Zα, Zβ = D-valine. (d) actinomycin G4: Xα = L-N-methylvaline; Xβ = L-N-methylalanine; Yα = trans-3-hy-
droxy-cis-5-methylproline; Yβ = L-proline; Zα, Zβ = D-valine.
Structural characterization of actinomycins by PSD-
MALDI-TOF MS
In the most stable form of the [M+ H]+ ions of actinomycins, the
ionizing proton presumably is mainly located at the amine substit-
uent in position 2 of the phenoxazinone core. To rationalize the
fragmentation pathways, it was assumed that this proton is trans-
ferred to the less basic lactone bond. Roboz et al.[16] proposed an
initial McLafferty rearrangement at the lactone linkage between
the C-terminal amino acid (methylvaline or methylalanine) and
the threonine in position 1. Thomas et al.[18] discussed a charge-site
initiated mechanism for opening of the protonated lactone rings of
actinomycins, which results in open-chain pentapeptides including
a dehydrothreonine residue with the ionizing proton residing at the
C-terminal carboxyl group from which it can readily be shifted to am-
ide linkages in the same pentapeptide chain. It was postulated that
fragmentations start from this arrangement or from related struc-
tures with both rings opened to form linear peptides attached to
the phenoxazinone ring system through dehydrothreonine residues.
Structural characterization of some of the investigated actinomy-
cins and related compounds was performed by fragment analysis
using PSD-MALDI-TOF MS. Examples of the investigated C-type,
G-type, X-type and Z-type actinomycins are presented in Figs. 3
and 4. The obtained product ions are summarized in Tables 2–5.
Each table is divided into two parts: A and B. Part A shows the
product ions of the peptidolactone moieties, while in part B, we
have listed product ions that were formed from losses of amino
acids and peptide constituents of the actinomycin precursor ions,
still containing the phenoxazinone core.
In the PSD-MALDI-TOF product ions spectra of all investigated
actinomycins, the isotope distribution of the product ions was
not well resolved representing average mass data. Generally,
the product ions peaks of the peptide lactone rings are sharp
and symmetric. Here, the experimental m/z data were found
between 0.1 and 0.5 Da higher than the calculated monoisotopic
values (see parts A in Figs. 2–4). In contrast, the product ions
originating from losses of amino acid and peptide residues still
containing the phenoxazinone core are broad. A part of them
shows still some fine structure. Here, the minor first peak visible
for these product ions was found near the calculated average
mass values, but frequently the peaks show only a broad enve-
lope. In this case, their maxima were found approximately 1 Da
higher than the calculated average values because of the high
intensity of the second peak seen in the unusual isotope pattern
of the MALDI-TOF mass spectra of actinomycins discussed later.
Product ion spectra of protonated actinomycin D and hemi-actinomycin D
Figure 3 shows the PSD-MALDI-TOF product ion spectra of the
protonated form of the reference compound actinomycin D
[M + H]+ at m/z 1255.6 (Fig. 3a) and of its sodium adduct
[M + Na]+ at m/z 1277.5 (Fig. 3b). Actinomycin D is an example
of an iso-actinomycin carrying two identical depsipeptide rings
showing only one set of product ions of the peptide lactone
moieties (Table 2A). For the protonated form of actinomycin D,
all dipeptide, tripeptide and tetrapeptide product ions of the
peptidolactone rings were found, among them the Y-ions that
originate from successive decomposition of the linearized
J. Mass Spectrom. 2014, 49, 210–222
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J. Vater et al.
Table 2. Product ions of the [M + H]+ and [M + Na]+ ions of actinomycin D obtained by PSD-MALDI-TOF MS
a) [M + H]+ m/z 1255.6
b) [M + Na]+ m/z 1277.5
m/z
Calc.
Found
Found
Found
A. Product ions of the peptide lactone moieties:
[M + H]+
197.2
169.1
185.1
203.1
268.2
282.2
300.2
381.3
399.3
482.3
[M + H]+
197.3 (l)
[M + H]+
[M + Na]+
VP
197.4 (l)
PMeG
169.2 (m) (1)
185.3 (l)
169.2 (l) (1)
185.2 (l)
MeGMeV
H-MeGMeV-OH
VPMeG
203.4 (m) (2)
268.3 (m)
203.3 (l) (2)
268.3 (l)
PMeGMeV
H-PMeGMeV-OH
VPMeGMeV
H-VPMeGMeV-OH
TVPMeGMeV
282.3 (h) (3)
300.4 (h) (4)
381.4 (l) (5)
399.4 (h) (6)
482.4 (l)
282.3 (l) (3)
300.5 (h) (4)
381.4 (l)
399.7 (l)
482.4 (l)
B. Product ions generated by elimination of parts of the peptide lactone moieties from actinomycin D precursor ions:
Loss of:
V
1157.3
1159.3
1185.4
1143.3
1030.2
1072.3
959.2
1157.6 (l)
1159.7 (l) (13)
1185.6 (l)
1143.7 (l)
—
—
—
P
—
—
MeG
—
—
MeV
—
1164.5 (h) (11)
1051.6 (m) (9)
1093.5 (l) (10)
980.4 (l) (8)
909.6 (l)
—
2 MeV
—
MeVMeG
—
1072.6 (l)
—
MeV + MeVMeG
959.5 (l)
2 MeVMeG
888.2
—
—
MeGP
1088.3
1060.2
975.2
1088.7 (l)
1059.5 (l)
975.5 (l)
—
PV
—
—
PMeGMeV
—
—
H-PMeGMeV-OH
957.2
957.5 (h) (12)
858.6 (h) (11)
775.4 (l) (10)
658.2 (m) (9)
559.3 (l) (8)
460.1 (m) (7)
—
980.4 (l)
879.7 (l)
796.4 (m)
—
H-VPMeGMeV-OH
858.1
858.6 (h) (7)
775.7 (l)
658.0 (l)
559.6 (l) (6)
459.9 (l) (5)
TVPMeGMeV
775.1
H-PMeGMeV-OH from both rings
H-PMeGMeV-OH from one ring and H-VPMeGMeV-OH from the other
H-VPMeGMeV-OH from both rings
658.0
558.9
—
459.8
—
The numbers set in parentheses behind masses of actinomycin D product ions corresponds to the numbers indicating prominent mass peaks in
the PSD-MALDI-TOF fragment spectra of the precursor ions of actinomycin D in Fig. 3a and b. l, m and h mean low, medium and high intensity.
The calculated reference data in part A and part B are monoisotopic and average, respectively.
peptide lactone moieties from their C-terminus. In addition, the
hydrated species of Y2–Y4 (m/z 203.2, 300.3 and 399.4) were visi-
ble, which are the dominant peaks in the lower part of the PSD
spectrum. In the upper part, above m/z 400 product ions were
detected that are generated by elimination of amino acid and
peptide residues from the [M + H]+ ion of actinomycin D still
containing the phenoxazinone core (Table 2B). Complementary
to the product ion pattern of the peptide lactone moieties in part
A in the range above m/z 450, those ions that originate from the
loss of hydrated tripeptide and tetrapeptide portions from one or
both rings dominate (Table 2B).
Figure 3c and d shows the MALDI-TOF product ion spectra of the
protonated form and the sodium adduct of hemi-actinomycin
D found at m/z 792.6 and 814.5, respectively, produced by S.
antibioticus, which is induced by feeding cells in the growth medium
with 4-MHA. Hemi-actinomycins are lacking one of the peptide
lactone rings. A list of product ions of hemi-actinomycin D is shown
in Table 3. Part A shows a complete set of dipeptide, tripeptide and
tetrapeptide product ions of the single peptidolactone ring, which
is identical with that of actinomycin D (Table 2A).
Product ion spectra of protonated actinomycin I, Z4 and G4
Figure 4 exhibits product ion spectra of the [M+ H]+ ion of actino-
mycin I at m/z 1272.0 produced by S. antibioticus as an example of
a X-type actinomycin (Fig. 4a), of the protonated form ([M + H]+at
m/z 1270.1, Fig. 4b) and of the sodium adduct ([M + Na]+ at m/z
1292.2; Fig. 4c) of actinomycin Z4 from S. fradiae as well as of the
[M+ H]+ ion of actinomycin G4 at m/z 1257.8 (Fig. 4d) from S.
iakyrus. All these actinomycins belong to the group of aniso-
actinomycins exhibiting different α-rings and β-rings with specific
amino acid substitutions in each ring (Fig. 1). These variants
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Copyright © 2014 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2014, 49, 210–222

MALDI-TOF mass spectrometry of actinomycins
Table 3. Product ions of the [M + H]+ and [M + Na]+ ions of hemi-actinomycin D obtained by PSD-MALDI-TOF MS
[M + H]+ m/z 792.6
[M + Na]+ m/z 814.5
m/z
Calc.
Found
Found
A. Product ions of the peptide lactone moieties
[M + H]+
[M + H]+
[M + H]+
—
VP
197.2
169.1
185.1
203.1
268.2
282.2
300.2
381.3
399.3
482.3
197.3 (l)
PMeG
169.2 (m) (1)
185.4 (l)
169.2 (l)
185.2 (l)
203.2 (l)
—
MeGMeV
H-MeGMeV-OH
VPMeG
203.3 (m) (2)
268.2 (m) (3)
282.3 (m)
PMeGMeV
H-PMeGMeV-OH
VPMeGMeV
H-VPMeGMeV-OH
TVPMeGMeV
282.2 (l)
300.2 (l)
—
300.2 (h) (4)
381.4 (l) (5)
399.5 (h) (7)
482.3 (l)
—
—
B. Product ions generated by elimination of parts of the peptide lactone moiety from hemi-actinomycin D precursor ions
m/z 792.6
m/z 814.5
m/z 770.5
[M + H]+
Calc.
[M + H]+
Found
[M + Na]+
Found
[M + Na À CO2]+
Loss of:
Found
V
693.3
695.3
721.4
679.3
608.3
590.3
511.2
493.2
596.2
578.2
525.2
507.2
412.1
394.1
311.1
693.5 (l)
695.4 (l)
—
715.6 (m) (8)
—
—
P
—
MeG
—
—
MeV
679.3 (m) (10)
608.3 (l)
590.3 (l)
511.3 (l)
493.2 (h) (8)
596.3 (l)
578.2 (l) (9)
525.3 (l)
507.2 (l)
412.2 (l)
394.2 (h) (6)
311.1 (l)
701.6 (h) (7)
630.4 (h) (5)
612.3 (l)
533.1 (l)
515.2 (l)
—
657.2 (m) (6)
MeGMeV
H-MeGMeV-OH
PMeGMeV
H-PMeGMeV-OH
VP
586.5 (m) (4)
—
489.3 (h) (3)
—
—
H-VP-OH
VPMeG
—
—
—
—
H-VPMeG-OH
VPMeGMeV
H-VPMeGMeV-OH
TVPMeGMeV
530.0 (l)
434.2 (l)
416.1 (l)
334.2 (m) (1)
—
390.4 (h) (2)
—
—
The numbers set in parentheses behind masses of hemi-actinomycin D product ions corresponds to the numbers indicating prominent mass
peaks in the PSD-MALDI-TOF fragment spectra of the precursor ions of hemi-actinomycin D in Fig. 3c and d. l, m and h mean low, medium
and high intensity.
were distinguished by two sets of peptide lactone product ions
according to the composition of both rings (see parts A in Tables 4
and 5). For all tested aniso-actinomycins, almost all peptide lactone
product ions were detected. The general features of the product
ion spectra of these G-type, X-type and Z-type actinomycins were
similar to those of the reference compound actinomycin D. In each
case, the hydrated tripeptide and tetrapeptide production ions
dominate in parts A of Tables 4 and 5, and complementarily, the
product ions originating by the loss of these species from the
[M+ H]+ precursor ions show the highest intensities in parts B.
Generally, the following features have been observed for the frag-
mentation of the cationized actinomycins. In their PSD-MALDI-
TOF product ion spectra, the most abundant ions are those that
are formed by elimination of the C-terminal amino acid residues
MeV and MeA. These product ions usually appear with the highest
intensities followed by those that originate from subsequent elim-
ination of the Sar residues following in the sequence starting from
the C-terminus. Here, it cannot be decided from our measurements,
whether Sar is eliminated in a coordinated manner successively
after the end-standing residue has been split off or as the MeV/
MeA–MeG dipeptides. In contrast, in the mass spectra of the
protonated forms, such peaks were not found or are of minor inten-
sity, like the peak indicating the elimination of one MeV residue.
A schematic illustration of these fragmentation steps is shown
in Fig. 5 for the sodium adducts of the reference compound
actinomycin D and of actinomycin Z4. For actinomycin D as an
iso-form fragments indicating the loss of L-MeV from one and
both rings found at m/z 1164.6 and 1051.6, respectively,
dominate followed by those originating by further elimination
Product ion spectra of cationized actinomycins
The PSD-MALDI-TOF product ions spectra of cationized actinomy-
cins are simpler than those of the protonated forms. Those for the
sodium adducts of actinomycins D and Z4 are shown in Figs 3–5. In
Tables 2, 4 and 5, the obtained product ions of actinomycins D, I
and Z4 are listed and compared with those from their protonated
forms. Similar results were obtained for the potassium adducts.
J. Mass Spectrom. 2014, 49, 210–222
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J. Vater et al.
Table 4. Product ions of the [M + H]+ and [M + Na]+ ions of actinomycin I obtained by PSD-MALDI-TOF MS
a) [M + H]+ m/z 1272.0
b) [M + Na]+ m/z 1294.0
m/z
Calc.
A. Product ions of the peptide lactone moieties: branch 1
Found
Found
[M + H]+
197.2
169.1
185.1
203.1
268.2
282.2
300.2
381.3
399.3
482.3
[M + H]+
197.3 (m)
169.2 (m) (1)
185.4 (h)
203.4 (h) (2)
268.4 (h) (3)
282.4 (h) (4)
300.6 (h) (5)
381.7 (l)
[M + H]+
197.5 (l)
169.4 (1)
185.4 (l)
203.4 (l)
268.6 (l)
282.4 (l)
300.3 (m)
381.7 (l)
399.7 (l)
482.4 (l)
VP
PMeG
MeGMeV
H-MeGMeV-OH
VPMeG
PMeGMeV
H-PMeGMeV-OH
VPMeGMeV
H-VPMeGMeV-OH
TVPMeGMeV
399.5 (l) (7)
482.8 (l)
A. Product ions of the peptide lactone moieties: branch 2
VHyp
HypMeG
MeGMeV
H-MeGMeV-OH
VHypMeG
HypMeGMeV
H-HypMeGMeV-OH
VHypMeGMeV
H-VHypMeGMeV-OH
TVHypMeGMeV
213.2
213.3 (m)
185.4 (h)
185.4 (h)
203.4 (h) (2)
284.4 (m)
298.5 (l)
316.5 (h) (6)
397.6 (l)
213.3 (l)
185.5 (l)
185.5 (l)
203.4 (l)
284.1 (l)
—
185.1
185.1
203.1
284.2
298.2
316.2
397.3
415.3
498.3
316.8 (l)
—
415.7 (m) (8)
498.6 (l)
415.6 (l)
498.8 (l)
B. Product ions generated by elimination of parts of the peptide lactone moieties from actinomycin I precursor ions:
m/z 1272.0
m/z 1294.0
[M + H]+
Calc.
[M + H]+
Found
[M + H]+
Found
—
[M + Na]+
Found
—
Loss of:
V
1173.3
1175.3
1159.3
1201.4
1159.3
1046.2
1088.3
975.2
904.2
1104.3
1086.3
1088.3
991.2
975.2
973.2
957.2
892.1
876.1
874.1
858.1
791.1
775.1
657.8
675.8
558.9
558.9
576.9
459.8
1174.1 (l)
P
Hyp
MeG
MeV
2 MeV
MeVMeG
2 MeV + MeG
2 MeVMeG
PMeG
H-PMeG-OH
HypMeG
PMeGMeV
1175.9 (l)
1160.0 (l)
—
—
—
—
—
—
—
1159.0 (l) (18)
..
—
1181.1 (h)
1067.9 (m)
1109.8 (l)
996.9 (l)
927.5 (l)
—
—
..
..
..
..
—
—
—
—
..
—
—
1089.1 (l)
992.0 (l) (17)
975.2 (l)
973.9 (m) (16)
957.5 (h) (15)
892.9 (l)
876.2 (m)
875.1 (h) (14)
859.1 (h) (13)
791.7 (l)
775.6 (l)
657.7 (m) (12)
675.5 (l)
558.7 (m) (10)
558.7 (m)
576.9 (l) (11)
459.5 (m) (9)
1088.7 (l)
991.2 (l)
—
1109.8 (l)
—
HypMeGMeV
996.8 (l)
994.8 (l)
—
H-PMeGMeV-OH
H-HypMeGMeV-OH
VPMeGMeV
973.4 (l)
—
892.4 (l)
876.0 (l)
874.6 (l)
—
—
VHypMeGMeV
—
H-VPMeGMeV-OH
H-VHypMeGMeV-OH
TVPMeGMeV
—
879.4 (l)
—
791.1 (l)
—
TVHypMeGMeV
796.7 (m)
—
H-PMeGMeV-OH + H-HypMeGMeV-OH
PMeGMeV + H-HypMeGMeV-OH
H-PMeGMeV-OH + H-VHypMeGMeV-OH
H-HypMeGMeV-OH + H-VPMeGMeV-OH
HypMeGMeV + H-VPMeGMeV-OH
H-VHypMeGMeV-OH + H-VPMeGMeV-OH
658.1 (l)
—
—
559.1 (l)
559.1 (l)
577.4 (l)
460.2 (l)
580.6 (l)
—
—
—
The numbers set in parentheses behind masses of actinomycin I product ions corresponds to the numbers indicating prominent mass peaks in
the PSD-MALDI-TOF fragment spectrum of the precursor ions of actinomycin I in Fig. 4a. l, m and h mean low, medium and high intensity.
Hyp, 4-hydroxyproline.
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MALDI-TOF mass spectrometry of actinomycins
Table 5. Product ions of the [M + H]+ and [M + Na]+ ions of actinomycin Z4 from Streptomyces fradiae obtained by PSD-MALDI-TOF MS
a) [M + H]+ m/z 1270.1
b) [M + Na]+ m/z 1292.2
m/z
Calc.
Found
Found
A. Product ions of the peptide lactone moieties: branch 1
[M + H]+
211.2
183.1
185.1
203.1
282.2
296.2
314.2
395.3
413.3
496.3
[M + H]+
211.5 (l)
183.4 (l)
185.4 (l)
203.4 (m) (1)
282.4 (m)
296.7 (m)
314.6 (hh) (3)
395.5 (l)
[M + H]+
—
VMeP
MePMeG
MeGMeV
H-MeGMeV-OH
VMePMeG
MePMeGMeV
H-MePMeGMeV-OH
VMePMeGMeV
H-VPMeGMeV-OH
TVMePMeGMeV
183.3 (l)
185.3 (l)
203.3 (m) (1)
282.2 (l)
296.1 (l)
314.5 (h) (2)
395.2 (l)
413.5 (l)
—
413.7 (m) (4)
496.2 (l)
A. Product ions of the peptide lactone moieties: branch 2
VOMeP
OMePMeG
MeGMeA
H-MeGMeA-OH
VOMePMeG
OMePMeGMeA
H-OMePMeGMeA-OH
VOMePMeGMeA
H-VOMePMeGMeA-OH
TVOMePMeGMeA
225.2
197.1
157.1
175.1
296.2
282.2
300.2
381.3
399.3
482.3
—
—
197.4 (l)
157.7 (l)
—
197.2 (l)
157.7 (l)
175.2 (l)
296.7 (m)
282.4 (m)
300.5 (l) (2)
381.3 (l)
399.6 (l)
482.4 (l)
296.1 (l)
282.2 (l)
300.6 (l)
381.7 (l)
399.5 (l)
—
B. Product ions generated by elimination of parts of the peptide lactone moieties from actinomycin Z4 precursor ions
m/z 1270.1
m/z 1292.2
[M + H]+
Calc.
[M + H]+
Found
[M + H]+
Found
—
[M + Na]+
Found
—
Loss of:
V
MeP
OMeP
MeG
MeV
MeV + MeG
MeA
MeA + MeG
MeV + MeA
MeV + MeA + MeG
MeV + MeA + 2MeG
MePMeG
H-MePMeG-OH
OmePMeG
MePMeGMeV
OmePMeGMeA
1171.3
1159.3
1145.3
1199.4
1157.3
1086.3
1185.4
1114.4
1072.3
1001.3
930.3
1088.3
1070.3
1074.3
975.2
989.3
957.2
1171.2 (l)
1160.1 (l) (12)
1145.2 (l)
—
—
—
—
—
—
—
—
—
1179.2 (h) (10)
1108.9 (l)
1207.1(h) (11)
1136.2 (m) (9)
1094.4 (h) (8)
1022.8 (m) (7)
952.6 (m) (6)
1110.9 (l)
—
—
—
1185.9 (l)
—
—
—
—
—
—
—
930.1 (m)
1088.7 (l)
1070.4 (l)
—
—
—
—
—
—
975.9 (l)
989.9 (l)
957.2 (h)
971.1 (h) (11)
—
—
—
—
—
H-MePMeGMeV-OH
H-OMePMeGMeA-OH
VMePMeGMeV
—
979.2 (l)
—
971.3
876.1
890.2
858.1
872.1
775.1
—
876.2 (l)
898.2 (m)
—
VOMePMeGMeA
—
—
857.8 (l)
872.6 (m) (5)
—
H-VMePMeGMeV-OH
H-VOMePMeGMeA-OH
TVMePMeGMeV
858.2 (h) (9)
872.2 (h) (10)
—
—
894.2 (l)
—
TVOMePMeGMeA
789.2
658.1
676.1
559.0
559.0
577.0
459.9
789.6 (l)
658.7 (m) (8)
676.3 (l)
559.7 (h) (6)
559.7 (h)
577.7 (l) (7)
460.3 (h) (5)
—
—
H-MePMeGMeV-OH + H-OMePMeGMeA-OH
MePMeGMeV + H-OMePMeGMeA-OH
H-MePMeGMeV-OH + H-VOMePMeGMeA-OH
H-OMePMeGMeA-OH + H-VMePMeGMeV-OH
OMePMeGMeA + H-VMePMeGMeV-OH
H-VOMePMeGMeA-OH + H-VMePMeGMeV-OH
658.3 (l)
—
—
697.6 (l)
—
559.1 (l) (4)
559.1 (l)
—
—
—
460.3 (l) (3)
—
The numbers set in parentheses behind the masses of actinomycin Z4 product ions corresponds to the numbers indicating prominent mass peaks
in the PSD-MALDI-TOF fragment spectra of the precursor ions of actinomycin Z4 in Fig. 4b and c. l, m and h mean low, medium and high
intensity.
MeP, cis-5-methylproline; OMeP, cis-5-methyl-4-oxoproline.
J. Mass Spectrom. 2014, 49, 210–222
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J. Vater et al.
activated ions and is the basis of a method for identification of
the C-terminal amino acid.[26]
MALDI-TOF mass spectra exhibit an unusual isotope pattern of the
actinomycin mass peaks.
The MALDI-TOF mass spectra of all investigated actinomycins ex-
hibit an unusual pattern of their isotope distribution (Fig. 6a and
b). Normally, the first peak indicating a molecule containing only
12C-atoms shows the highest intensity followed by peaks of
species including one or two 13C-atoms of decreasing intensities.
For example, nonactins produced by S. chrysomallus (Fig. 3b) and
peptidolactone compounds, such as the 4-MHB-pentapepti
dolactone derivative of actinomycin[28] (Fig. 6e) as well as the
heptapeptide lactone etamycin[29] (Fig. 6f), are distinguished by
such a normal pattern. In contrast for the MALDI-TOF mass spectra
of actinomycins, peak ensembles have been obtained, which
exhibit five to six peaks, the second of which always shows the
highest intensity. This is shown in Fig. 6a and b for the MALDI-
TOF mass spectra of actinomycin D and the corresponding hemi-
actinomycin in a surface extract of S. antibioticus. These effects are
specific for MALDI-TOF MS. Using ESI-MS, normal isotope patterns
were observed for actinomycins (Fig. 6c and d). The unusual patterns
observed in MALDI-TOF mass spectra are specifically related to the
phenoxazinone core of actinomycins. Peptidolactone compounds,
like the 4-MHB-peptidolactone and etamycin, which contain a
benzoic or a 3-hydroxypicolinic core, do not show such an effect. In
a similar way, in the PSD-MALDI-TOF product ion spectra of actino-
mycins, only those ions still containing the phenoxazinone chromo-
phore revealed such an anomalous pattern, while for the peptide
product ions of the peptidolactone rings, only three isotope peaks
were observed. Resuming these results, we conclude that the
described anomalous behavior occurs as a result of the use of
MALDI-TOF MS and depends specifically on the presence of the
phenoxazinone core of actinomycins.
The reason for this anomaly may be the overlap of the mass
spectra of two actinomycin species, which differ in molecular
mass by 1 Da. For example, such a situation could occur when
the amino group in position 2 of the phenoxazinone core is
converted into a hydroxyl group, because it is known that such
a replacement can be achieved by mild acidic hydrolysis.[23]
Therefore, we tested whether actinomycins would be modified
in this manner during sample preparation for MALDI-TOF MS,
because the matrix α-cyano-4-hydroxy-cinnamic acid used in
our study is a carboxylic acid and the matrix medium contained
0.1% trifluoroacetic acid.
In this case, we expected a normal isotope distribution in the
MALDI-TOF mass spectrum of pure 2-hydroxy-actinomycin D
(desaminoactinomycin D). To prove this assumption, we
prepared this species as the reference compound[23] and tested
by TLC, if this compound was formed in our samples for MALDI-
TOF MS. Using ethyl acetate : methanol : water : dimethyl
formamide (100 : 5 : 5 : 1, by vol.) as the solvent, the 2-hydroxy
derivative showed a quite different Rf-value of 0.09 as the orig-
inal actinomycin D (Rf = 0.61), but a spot at this position was not
found in our samples for mass spectrometry. Obviously, the
putative conversion of 2-amino-actinomycin D to 2-hydroxy-
actinomycin D did not occur in our sample preparation. In addi-
tion, the MALDI-TOF mass spectrum of 2-hydroxy-actinomycin
D showed a similar anomalous isotope pattern of the mass
peaks as the mass spectra of the other actinomycin compounds.
As the consequence, other reasons must be responsible for the
Figure 5. Fragmentation scheme of the sodium adducts of (a) actinomy-
cin D ([M + Na]+ m/z 1277.5) and (b) actinomycin Z4 ([M + Na]+ m/z
1292.2). The dominant product ions in the PSD-MALDI-TOF mass spectra
can be attributed to the loss of the MeV and MeA residues involved in
peptide lactone formation.
of the L-Sar-residues to form the medium intensity peaks at
m/z 1093.5 and 980.4 (Fig. 3b). Similar features were observed
also for the hemi-actinomycins. For example, the dominating
peaks in the product spectrum of the sodium adduct of
hemi-actinomycin D [M + Na]+ at m/z 814.5 in Fig. 3d are
those that originate from the loss of MeV at m/z 701.6 and
of MeV/MeG at m/z 630.4. A specific feature of the sodium
adduct of hemi-actinomycin D is the decarboxylation of the
free carboxyl group at the phenoxazinone core leading to the
loss of CO2. Interestingly, the decarboxylated species showed a
successive elimination of MeV, MeG, P and V. The corresponding
product ions appeared in high intensity (Fig. 3d and Table 3).
In the case of actinomycin Z4 as an example for an aniso-form
with different α-rings and β-rings, the product ions pattern is
more complex. Here, the product ions resulting from the loss of
L-MeV from the α-ring (at m/z 1179.2) and L-MeA from the β-ring
(at m/z 1207.1) as well as that obtained by the simultaneous
expulsion of both L-MeV and L-MeA from either ring m/z 1094.4
represent the prominent peaks in the PSD spectrum of the
sodium adduct of actinomycin Z4. Thereafter, the L-Sar residues
were successively eliminated from both rings. Single elimination
of the other amino acid residues of the peptidolactone rings
either was missing or occurs only in minor extent. Generally, also
for the alkali adducts, many of the product ions that had been
observed for the protonated forms were found at minor intensi-
ties. In particular, the peptide product ions obtained for the
peptidolactone rings showed much lower intensities or even
were completely missing.
Our results are consistent with previous mass spectrometric
studies on the degradation of cationized peptides.[25–27]
Although the degradation process is complex, frequently the
dominating pathway is the elimination of the C-terminal amino
acid. The alkali cations bind primarily to the C-terminus promot-
ing the loss of the C-terminal residue. This process involves
migration of an oxygen atom inducing the loss of the terminal
amino acid by expulsion of CO and an imine. This mechanism is
observed in general for both metastably and collisionally
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J. Mass Spectrom. 2014, 49, 210–222

MALDI-TOF mass spectrometry of actinomycins
Figure 6. Isotope patterns of the mass peaks of MALDI-TOF mass spectra for actinomycin D (a); hemi-actinomycin D (b); 4-methyl-3-hydroxybenzoic
acid-pentapeptide lactone (e) and etamycin (f); and the ESI-MS mass spectra for actinomycin D (c) and hemi-actinomycin D (d).
Figure 7. Mesomeric forms of the semiquinone radical of the phenoxazinone core.
observed anomalous behavior. Because the phenoxazinone
core of actinomycins shows a quinoid structure, we infer the
following mechanism to explain this anomaly. During the mass
spectrometric measurements by laser excitation of actinomy-
cins, phenoxazinone radicals are induced. Their anionic form
(Fig. 7) is able to accept a proton leading to an increase of its
molecular mass by 1 Da. Consequently, the anomaly in the
MALDI-TOF mass spectra would originate from a superposition
of the mass spectra of the non-modified actinomycin molecule
and its radical anion.
the detection and structural characterization of actinomycins in situ.
By feeding experiments with the precursors 3-HA and 4-MHA, C-de
methyl derivatives and hemi-actinomycins were obtained as novel
actinomycin variants. Fragmentation of the protonated and
cationized forms of selected actinomycins was performed by
PSD-MALDI-TOF MS. The specific features of the fragmentation
patterns of these species were investigated in detail. The most
intense peaks in the PSD spectra of the protonated forms were
attributed to the hydrated tripeptide and tetrapeptide ions of the
pentapeptide lactone moieties and those that originate from the
loss of these portions from the [M+ H] + ions of actinomycins. In
contrast, in the PSD spectra of the cationized actinomycins, those
product ions that arose by elimination of the two C-terminal amino
acid residues of the linearized peptide lactone side chains domi-
nate. MALDI-TOF mass spectra of all investigated actinomycins
exhibit an unusual pattern of isotope distribution comprising five
Conclusions
MALDI-TOF MS of surface extracts of five well-characterized Strepto-
myces strains was demonstrated as a rapid, efficient technique for
J. Mass Spectrom. 2014, 49, 210–222
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J. Vater et al.
[13] A. Mauger, E. Katz. Two distinct site-dependent biosynthetic path-
ways for the incorporation of sarcosine into actinomycins. Arch.
Biochem. Biophys. 1976, 176, 181.
[14] A. Mauger, O. Stuart, J. Ferretti, J. Silverton. Conformation and dimer-
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to six mass peaks, which can be interpreted by the formation of
phenoxazinone anion radicals in the course of laser excitation of
the actinomycin samples. Our results represent an extensive study
of actinomycins by MALDI-TOF MS, and we consider that they are a
valuable contribution for the understanding of actinomycins on the
molecular level.
Acknowledgements
[16] J. Roboz, E. Nieves, J. F. Holland, M. McCamish, C. Smith. Collisional
activation decomposition of actinomycins using tandem mass
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We thank Professor D. Naumann and Dr. P. Lasch from the Robert
Koch-Institut Berlin for making available for us the Bruker
Autoflex instrument to perform the MALDI-TOF measurements.
We thank Professor R. Süssmuth from the Technische Universität
Berlin for making available for us the Exactive ESI-Orbitrap-MS to
perform the ESI-MS experiments.
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J. Mass Spectrom. 2014, 49, 210–222