characterization of nodularin variants innodularia spumigena from the baltic sea using liquid...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2006; 20: 2023–2032

) DOI: 10.1002/rcm.2558
Published online in Wiley InterScience (www.interscience.wiley.com
Characterization of nodularin variants in Nodulariaspumigena from the Baltic Sea using liquid

chromatography/mass spectrometry/mass spectrometry

Hanna Mazur-Marzec1*, Jussi Meriluoto2, Marcin Plinski1 and Janusz Szafranek3

1Department of Marine Biology and Ecology, University of Gdansk, Al. Marszałka Piłsudskiego 46, 81-378 Gdynia, Poland2Department of Biochemistry and Pharmacy, Abo Akademi University, Tykistokatu 6A, 20520 Turku, Finland3Faculty of Chemistry, University of Gdansk, Sobieskiegi 18, 80-952 Gdansk, Poland

Received 13 March 2006; Revised 28 April 2006; Accepted 29 April 2006

*CorrespoBiologyPiłsudskiE-mail: bContract/in PolandContract/BALTDE

Nodularin is a potent hepatotoxic cyclic pentapeptide produced by planktonic cyanobacterium

Nodularia spumigena. Bloom and culture samples of the cyanobacterium collected and isolated from

the Gulf of Gdansk, southern Baltic Sea, were analyzed. Hybrid quadrupole-time-of-flight liquid

chromatography/mass spectrometry/mass spectrometry (TOF-LC/MS/MS) with ionspray (ISP) and

collision-induced dissociation (CID) were used to characterize nodularin and its analogues. The

identification process was based on the comparison of recorded product ion spectra with the

previously reported FAB-MS/CID (high-energy) mass spectra of the corresponding nodularin

variants. Amino acid structures and sequences were derived from the fragmentation pattern of

the [MRH]R ions. Apart from unmodified nodularin with an arginine residue (NOD-R), three

demethylated variants have been found. The sites of demethylation were located on aspartic acid

[Asp1]NOD, the Adda residue [DMAdda3]NOD, and dehydrobutyric acid [dhb5]NOD. In two other

nodularin variants an additional methyl group is located in the Adda [MeAdda]NOD and Glu

[Glu4(OMe)]NOD residues. The linear NOD and the geometrical isomer of NOD-R, reported earlier

inN. spumigena from New Zealand, have also been detected. Two of the total eight nodularin variants

characterized in the present study, [dhb5]NOD and [MeAdda]NOD, have not been described earlier.

Copyright # 2006 John Wiley & Sons, Ltd.

Nodularins (NOD) are cyclic pentapeptide hepatotoxins

produced by brackish water planktonic cyanobacterium

Nodularia spumigena. The cyanobacterium is of public health

significance as it forms blooms in drinking and recreational

waters.1,2 In Australia and in the coastal areas of the Baltic

Sea, nodularin (LD50¼ 50mgkg�1) has been implicated in

poisonings of humans and animals.3–6 In liver cells the toxin

inhibits the activity of protein phosphatase 1 and 2A7 and

acts as a potent tumour promoter and initiator.8 Nodularin

(Fig. 1) was identified by Rinehart et al.9 inN. spumigena from

New Zealand and by Sivonen et al.1 and Sandstrom et al.10 in

N. spumigena from the Baltic Sea. The structure of nodularin

is cyclo[-D-erythro-b-methylAsp(iso-linkage)-L-Arg-Adda-

D-Glu(iso-linkage)-2-(methylamino)-2(Z)-dehydrobutyric

acid], where Adda is the C20b-amino acid, (2S,3S,8S,9S)-3-

amino-9-methoxy-2,6,8,-trimethyl-10-phenyldeca-4(E),6(E)-

dienoicacid.TheAddaaminoacidisuniquetocyanobacterial

hepatotoxins such as nodularins and the structurally and

functionally similar microcystins. The configuration of the

ndence to: H. Mazur-Marzec, Department of Marineand Ecology, University of Gdansk, Al. Marszałkaego 46, 81-378 Gdynia, [email protected] sponsor: State Committee for Scientific Research; contract/grant number: 0366/PO4/2003/25.grant sponsor: UE Project; contract/grant number:R EVK3-CT-2002-80005.

Adda–Glu part of the toxins is essential for their activity.

Studies on the structure-activity relationship showed that

formation of the [6(Z)Adda] stereoisomer, saturation of the

diene in Adda, methylation of glutamic acid or linearization

render the compounds non-toxic or lead to a significant

decrease in toxicity.11–13 On the other hand, the demethyla-

tion of amino acid residues in nodularins and microcystins

exerts little effect on their toxicity.13

In the first structural analyses of nodularins and micro-

cystins fast-atom bombardment (FAB) ionization was used.

Application of this technique combined with accurate mass

measurement and tandemmass spectrometry14,15 resulted in

the identification of over 50 structural variants of micro-

cystins and five variants of nodularins out of over 80

currently known cyanobacterial hepatotoxin analogues. The

introduction of combined liquid chromatography and mass

spectrometry (LC/MS) with electrospray ionization (ESI)

improved the sensitivity and selectivity of quantitative

methods and allowed for the direct identification of

compounds in cell extracts.16 Matrix-assisted laser deso-

rption/ionization time-of-flight mass spectrometry (MALDI-

TOFMS)17and surface-enhanced laser desorption ionization

mass spectrometry (SELDI-TOFMS)18 are the alternative


Figure 1. General structure of nodularins.

2024 H. Mazur-Marzec et al.

ionization techniques that have recently been applied in

structural studies of cyanobacterial toxins. Tandem mass

spectrometry instruments have utilized quadrupole (Q), ion

trap (IT) or time-of-flight (TOF) mass analyzers. Maizels and

Budde19 developed an LC/ESI-TOFMS method for small

cyanobacterial toxic peptides.

In the current study the liquid chromatography ionspray

mass spectrometry (LC/ISP-MS) instrument with a quadru-

pole analyzer associated with time-of-flight (TOF) was

applied to characterize nodularin variants present in bloom

and culture samples ofN. spumigena from the Gulf of Gdansk

(southern Baltic Sea).

Generally, fragmentation of nodularins and microcystins

proceeds according to the mechanism described for peptides

containing an Arg residue.20–22 The presence of a basic Arg

residue in nodularin attracts the proton and determines the

fragmentation pattern. Yuan et al.20 calculated that for the

[MþH]þ ion of microcystin-LR the lowest energy of proto-

nation was obtained when the proton was localized on the

guanidinyl group of the Arg residue. In this case fragmenta-

tion at the bond adjacent to the Arg residue is favoured. In

positive mode, a linear b-ion with a charge on the N-terminus

can be formed. Further cleavage of peptide bonds in NOD

gives a series of sequence ions, which contain four or fewer

amino acid residues and are very useful in determining their

structure and sequence. Loss of the carbonyl group from

b-type ions leads to the formation of a-type ions (immonium

ions).

The application of hybrid Q-TOF LC/MS/MS has enabled

us to deduce the structure of two novel nodularin variants:

[dhb5]NOD and [MeAdda3]NOD. It was also revealed that

apart from [D-Asp1]NOD the N. spumigena from the Baltic

Sea produces the same NOD variants as the strains from

New Zealand: the linear NOD, NOD-R isomer and

[DMAdda3]NOD. [Glu4(OMe)]NOD was also detected.

EXPERIMENTAL

ChemicalsNodularin to be used as an analytical standard was

purchased from Calbiochem (La Jolla, CA, USA). Gradient-


grade acetonitrile and methanol were from Baker (Deventer,

The Netherlands). Trifluoroacetic acid (TFA) of protein-

sequencing grade and acetic acid were obtained from Fluka

(Buchs, Switzerland) and water was purified to 18.2MV cm

(MilliQ water) using an Ultra Pure water system from

Millipore (Milford, USA).

Cyanobacterial material (bloom and culturesamples)N. spumigena strainNSGG-1, originally isolated from theGulf

of Gdansk off Gdynia (5483006500N, 1883405400E) in July 2000,

and maintained in the laboratory of the Department of

Marine Biology and Ecology, University of Gdansk, was

cultured in autoclaved BG-11 (7 psu). The cells, grown at

218C and a light intensity of 30mEm�2 s�1, were harvested in

the early stationary phase of batch culture and filtered onto

47-mm glass-fibre filter discs (Whatman GF/C) using a

Millipore filtration system. In July 2005, field N. spumigena

samples were collected with a 100-mm mesh plankton net

during cruises in the Gulf of Gdansk off the coast of Gdynia

(548580N, 188340E), Sopot (548270N, 188360E) and Gdansk

(548250N, 188380E). Bloom and culture cyanobacterial

material was freeze-dried and stored at �208C prior to toxin

extraction and analyses.

Sample preparationThe procedure used for extraction of nodularins was based

on methods described by Lawton et al.23 and Spoof et al.24

Water extracts of N. spumigena cells harvested in culture or

collected in the Gulf of Gdansk during bloom of the

cyanobacterium (1 g dry weight (d.w.)) were prepared with

15-min bath sonication (Sonorex, Bandelin, Berlin, Germany)

followed by 1-min probe sonication with an HD 2070

Sonopuls ultrasonic disrupter equipped with a MS 72 probe

(Bandelin, Berlin, Germany; 20 kHz, 25% duty cycle). After

centrifugation at 6.140 g for 15min, the supernatant was

applied to a solid-phase Sep-Pak Vac C18 cartridge (500mg;

Waters, Massachusetts, USA) preconditioned with 10mL of

100%methanol andwashedwith 10mL of water. The sample

was adsorbed onto the cartridge at a flow rate of 5mLmin�1.

Then the cartridge was rinsed with 6mL of water and eluted

with 10mL of 100% methanol to give a fraction containing

nodularin and its variants. The solvent was removed by

rotary evaporation at 358C. The residue of the extract was

redissolved in 30% methanol (1mL); then the sample was

vortexed for 3min (REAX top, Heidolph, Germany) and

centrifuged at 6.140 g for 15min in aMiniSpin plus centrifuge

(Eppendorf, Germany). The obtained supernatant was

analyzed by high-performance liquid chromatography

(HPLC) and LC/MS/MS.

Chromatography with photodiode-arraydetectionFirst, bloom and cultureN. spumigena extracts were analyzed

with a HPLC system from Waters (Milford, MA, USA)

equipped with a model 996 photodiode-array detector.

Absorbance at 238 nm and at 200–300 nm was monitored. A

sample injection (10mL) was made using a Waters 917plus

autosampler. Nodularin variants were separated on aWaters


DOI: 10.1002/rcm

Characterization of nodularin variants in Nodularia 2025

Symmetry RP-18 column (5mm; 150mm� 3.9mm i.d.).

Isocratic elution with the mobile phase of acetonitrile/water

(32:68), both containing 0.05% TFA, and at a flow rate of

1mLmin�1, were chosen as optimal for separation. The

mobile phase solvents were degassed by sparging with

helium at 30mLmin�1. The collected data were processed by

Millennium32 Chromatography Manager software.

Mass spectrometryThe bloom and culture N. spumigena samples were analyzed

with a QStar XL hybrid Q-TOF LC/MS/MS instrument

(Applied Biosystems MDS Sciex, Concord, ON, Canada).

Separation of NOD variants was performed on a Waters

Symmetry RP-18 column (5mm; 150mm� 3.9mm i.d.) using

an Agilent 1100 system (Agilent Technologies, Waldbronn,

Germany). Samples of 7mL were injected. Isocratic elution

with acetonitrile/water (32:68), both containing 0.1% acetic

acid, at a flow rate of 0.5mLmin�1, was employed. A mass

range ofm/z 100–1000was coveredwith a scan time of 1 s; the

instrument was operated in positive ion mode. The ionspray

voltage was 5.5 kV with the nebulizer gas nitrogen pressure

and curtain gas nitrogen pressure set at 30 and 20 psi,

respectively (1 psi¼ 6894.76 Pa). Ionspray was operated at

ambient temperature. The structure of nodularin variants

was determined using collision-induced dissociation at

varying collision energies: 50, 64 and 120 eV. Fragmentation

was achieved with nitrogen collision gas at a pressure of

6 psi. The TOF instrument was calibrated for the exact m/z

measurements with a water/acetonitrile (50:50) solution of

renin (Sigma, St. Louis, MO, USA) containing 0.1% acetic

acid. Operational parameters were optimized by direct

infusion of N. spumigena extract dissolved in 30% methanol

containing 0.1% acetic acid. A spectrometer syringe pump

was used at an injection flow rate of 8mLmin�1. The

parameters were held constant during sample analyses,

except for the collision energy (CE), which was set for each

specific run. About 10–30 scans were accumulated and

averaged. The data were recorded and processed with

Analyst QS 111 software.

RESULTS

Most absorption spectra of cyanobacterial peptide hepato-

toxins, such as microcystins and nodularins, give a strong

maximum at 238 nm. This corresponds to the chromophore

of conjugated double bonds in the Adda residue of the toxins.

In the present work, the HPLC/diode-array detection (DAD)

analyses of N. spumigena culture and bloom sample extracts

revealed some compounds with absorption spectra resem-

bling that of nodularin. Studies to characterize the structure

of these unidentified compounds were undertaken. The

identification process was based on the assumption that

the compounds contain the same, known amino acids, which

are included in NOD-R. Sequence and variations in the

individual amino acids’ structure were determined based on

the product ion spectra of [MþH]þ. Both in bloom

and culture N. spumigena cell extracts the fragmentation

patterns of ions at m/z 811, 825, 839 and 843 were analyzed

(Table 1).


Compound I: NOD-R, [MRH]R¼ 825NOD-R (Fig. 1) was detected as the main component among

eight nodularin variants extracted from bloom and culture

N. spumigena samples. Under the experimental conditions

employed in the current study, the molecular ion at m/z 825

([MþH]þ) occurred as a base peak when a CE of 50 eV was

used. The ISP-MS/CID mass spectrum of NOD-R showed

low intensity ions at m/z 807, 797 and 781, generated by the

loss of small molecules of water, carbon monoxide and

ammonia. When a CE of 64 eV was used (Fig. 2), the ions at

m/z 227 and 135 were the most intense. The O-methylphe-

nylacetaldehyde ([PhCH2CH(OMe)]þ) ion at m/z 135 is

formed by the cleavage between C-8 and C-9 in the Adda

residue. Since it is one of the most abundant product ions

detected in MS/MS spectra of all microcystins and

nodularins containing an Adda residue with the methoxyl

group, it is regarded as an important diagnostic ion in

screening for the toxins. The loss of m/z 135 gave a fragment

ion at m/z 691 ([Mþ2H–135]þ). When, additionally, a

cleavage of the C–N bond between Adda and Arg occurred,

a fragment ion ofm/z 163 [C11H15O]þ was formed. This ion is

contained in the important series of fragment ions at m/z 361

([C11H15O-Glu-Mdhb–CO]þ), 389 ([C11H15O-Glu-Mdhb]þ),

466 ([C11H15O-NH2-Arg-MeAspþ2H]þ), 646 ([C11H15O-Glu-

Mdhb-MeAsp-Arg–CO]þ) and 674 ([C11H15O-Glu-Mdhb-

MeAsp-Arg]þ). Further fragmentation of the C2–C3 bond

in the Adda residue gave a low abundance of ions at m/z 568

([C2H4-CO-Glu-Mdhb-MeAsp-ArgþH]þ). In the ESI-MS/

CID spectrum of NOD-R generated at a CE of 64 eV, apart

from the base peak at m/z 227, corresponding to [Glu-

MdhbþH]þ, other b-type ions were detected at m/z 366

([Mdhb-MeAsp-ArgþH–NH3]þ) and 383 ([Mdhb-MeAsp-

ArgþH]þ). These fragment ions, together with the series of

ions containing a [C11H15O]þ unit, constitute a reliable source

of information on the composition and amino acid sequence

in nodularin. The peak atm/z 303 was probably produced by

the [MeAsp-Argþ2HþNH2]þ fragment ion. Two other

product ion peaks of high intensity at m/z 253 and 209 could

represent [CO-Glu-Mdhb–H]þ and [Glu-MdhbþH–H2O]þ,

respectively. At a CE of 120 eV, the number of detected

product ions was dramatically reduced and the m/z 135 ion

became a base peak (Table 1). Since only low m/z value ions

(115, 135, 153, 181, 209, 227, 292) were present, the spectrum

obtained at this CE provided definitely less structural

information.

Compound II: NOD-isomer, [MRH]R¼ 825In the total ion chromatogram (TIC), at least two peaks of the

m/z 825 ion were observed: an intense one at 8.16min

(compound I) and a much smaller one at 6.93min

(compound II) (Fig. 3(A)). Since in both spectra the same

fragmentation patterns were recorded, it might be concluded

that compound II is a NOD-R isomer. As in the ISP-MS/CID

mass spectrum of NOD-R, at a CE of 120 eV, only a few low-

mass ions were recorded.

In the NOD-R spectrum of N. spumigena culture extract

the peak at m/z 825 (at CE of 64 eV) was 1000 times more

abundant than the one in the spectrum of compound II. In

the bloom sample the ratio of the molecular ion peaks in the

spectra of NOD-R and compound II was 60:1.


DOI: 10.1002/rcm

Table 1. Fragment ions observed in ISP-MS/CID mass spectra of nodularin variants (CE values are given as subscripts)

Ion composition NOD-R ILinearNOD III

[D-Asp1]NOD IV

[DMAdda3]NOD V

[dhb5]NOD VI

[MeAdda3]NOD VII

[Glu4(OMe)NOD VIII

[MþH]þ 82550,64 84350 81150,64,120 81150,64,120 81150,64,120 83950,64 83950,64[MþH–H2O]þ 80750,64 — 79364 79350,64 79350,64 — —[MþH–CO]þ 79764 — 78350,64,120 78350,64,120 78350,64,120 81150,64 —[MþH–NH2–CO]þ 78150,64 — 76750 76764 76750 79550,64 79550[MþH–CO–CO2]

þ 75350, 64 — 73950,64,120 73950,64,120 73950,64 76750,64, 76750,64[MþH–NH3]

þ — 82650,64 — — — — —[Mþ2H–135]þ 69150,64 — 67750,64,120 69150,64,120 67750,64 70550 70550,64[M–NH2–135]

þ — 69250,64 — — — —[Mþ2H–135–NH3]

þ 67450,64 — 66050 67450 66050,64,120 68850 68850[MþH–135–NH2–CO]þ 64650,64 — 63250,64,120 64664,120 63264,120 — —[C2H4-CO-Glu-Mdhb-MeAsp-Argþ2HþOH]þ

— 58650,64 — — — — —

[C2H4-CO-Glu-Mdhb-MeAsp-ArgþH]þ

56850,64 — — — — 56850,64 58250

[CO-Glu-Mdhb-MeAsp-ArgþOH]þ

— 55650,64 — — — — —

[Glu-Mdhb-MeAsp-Argþ2HþOH]þ

— 53064 — — — — —

[C11H15O-NH2-Arg-MeAspþ2H]þ

46650,64 — 45250,64 46650,64 46650 — —

[C11H15O-Glu-Mdhb]þ 38950,64 38950,64 38950, 64,120 38950,64,120 37550,64,120 40350,64 40350,64[C11H15O-Glu-Mdhb–CO]þ 36150,64 — — 36150,64 347120 — 37550[C11H15O]þ 16350,64 — 16350, 64,120 16350,64,120 16364,120 17750,64 16364,120[Arg-Adda-GluþH]þ 59964 — 59950,120 58450,64 59950,64 61350,64 —[Mdhb-MeAsp-ArgþH]þ 38350,64 — 36950, 64,120 38350,64,120 36950,64,120 38350,64 —[Mdhb-MeAsp-ArgþH–NH3]

þ36650,64 — 35250, 64,120 36650,64,120 35250,64,120 36650,64 36650

[MeAsp-Argþ2HþOH]þ — 30450,64 — — — — —[MeAsp-Argþ2HþNH2]

þ 30350,64 — 28950 30350, 64,120 30350,64,120 30350 30350[CO-Glu-Mdhb–H]þ 25350,64 25350,64 25350, 64,120 25350, 64,120 239120 25350,64 26750,64[Glu-MdhbþH]þ 22750,64,120 22750,64,120 22750, 64,120 22750, 64,120 21350,64,120 22750,64 24150,64[Glu-MdhbþH–H2O]þ 20950,64,120 — 20950,64,120 20950, 64,120 19550,64 20950,64,120 22350,64,120[ArgþHþNH3]

þ 17450,64 17550,64 17450, 64,120 17450, 64,120 17450,64,120 17450 17450,64[ArgþH]þ 15764 — 15764 — 15764 15750 15764PhCH2CH(OCH3) 13550,64,120 13550,64,120 13550,64 121120 13550,64,120 13550,64,120 13550,64,120


Compound III: linear NOD, [MRH]R¼ 843ISP-MS/CID analyses of bloom and culture N. spumigena

samples revealed the presence of the linear NOD variant

(Fig. 4). In the TIC it occurred at 4.08min. At CE values of 50

and 64 eV, the m/z 843 product ion spectrum gave fragment

ions at m/z 826 ([MþH–NH2]þ), 692 ([MþH–NH2–135]

þ),

586 ([C2H4-CO-Glu-Mdhb-MeAsp-Arg-OHþ2H]þ), 556 [CO-

Glu-Mdhb-MeAsp-Arg-OH]þ) and 530 ([Glu-Mdhb-MeAsp-

Arg-OHþ2H]þ) (Fig. 5). The presence of a base peak at

m/z 826 might indicate that under the conditions used in the

experiment a 17Da fragment, presumably NH3, was lost

from [MþH]þ ion. In the spectrum, high intensity ions at

m/z 175 ([Arg-OHþ2H]þ) and 304 ([MeAsp-Arg-OHþ2H]þ)

were also recorded. As in the NOD-R spectrum the following

fragment ions at m/z 389, 253, 227, 135 were present;

however, their intensities were low. At a CE of 120 eV, only

ions with low m/z were measured (Table 1). Their intensity

increased so that now the ion atm/z 201 ([CO-Arg-OH]þ) was

observed (not seen at 50 and 64 eV) and the ion at m/z 103

became a base peak. In the spectra of cyanobacterial toxins

with themethoxyl group in the Adda residue, the peak atm/z

103 is formed by the loss of methanol from the m/z 135 ion.

The TIC of the Nodularia cell culture extract gave only one

ion at m/z 843, while in the Nodularia bloom sample three


peaks at m/z 843 were present. Two of them have different

fragmentation patterns; however, at lower m/z some frag-

ment ions typical of a nodularin molecule were present

(at m/z 366, 303, 253, 227, 174 and 135).

Compound IV: [D-Asp1]NOD, [MRH]R¼ 811In the TIC of N. spumigena extracts some peaks with m/z 811

occurred, indicating the possible presence of demethylated

NOD variants (Fig. 3(C)). The peak at 7.10min, attributed to

compound IV, was most abundant in the cell culture extract.

The m/z 811 product ion spectrum of compound IV revealed

fragment ions at m/z 389 ([C11H15O-Glu-Mdhb]þ) and 227

([Glu-MdhbþH]þ). This eliminated the possibility of

demethylation on the Mdhb residue. The ions at m/z 135

([Mþ2H–135]þ), 660 ([Mþ2H–135–NH3]þ) and 632 ([Mþ2H–

135–NH3–CO]þ) confirmed the presence of amethoxyl group

on the Adda residue. On the other hand, all the ions that

contain MeAsp in the NOD-R spectrum occurred here at 14

units lower m/z values: 289, 352, 452, 632, 660, 677, 739,

767, 783. These data clearly indicate that Asp not the MeAsp

residue is included in the structure of compound IV. The

presence of [D-Asp1]NOD was revealed both in Nodularia

bloom samples collected in the Gulf of Gdansk and in

cultured cells of N. spumigena strain NSGG-1.


DOI: 10.1002/rcm

Figure 2. ISP-MS/CID mass spectrum of NOD-R acquired at a collision energy of 64 eV.


Compound V: [DMAdda3]NOD, [MRH]R¼ 811In the TIC of theN. spumigena bloom sample the most intense

m/z 811 peak occurred at about 3.94min and was labelled as

compound V (Fig. 3(C)). In the cell culture extracts the

intensity of the peak was lower. The fragment ions atm/z 793

([MþH–H2O]þ), 783 ([MþH–CO]þ), 767 ([MþH–NH2–CO]þ)

and 739 ([MþH–CO–CO2]þ) were 14 mass units less than the

corresponding ions in the NOD-R spectrum, indicating that

one amino acid residue is lacking a CH2 group (Fig. 6(A)).

Fragment ions at m/z 227 ([Glu-MdhbþH]þ), 253 ([CO-Glu-

Mdhb–H]þ), 303 ([MeAsp-Argþ2HþNH2]þ), 366 ([Mdhb-

Figure 3. Selected ion recording chromatograms

(D) m/z 839 (N. spumigena bloom (A, C) and cultu


MeAsp-ArgþH–NH3]þ), 383 ([Mdhb-MeAsp-ArgþH]þ), 389

([C11H15O-Glu-Mdhb]þ), 466 ([C11H15O-NH2-Arg-MeAspþ2H]þ) and 646 ([C11H15O-Glu-Mdhb-MeAsp-Arg–CO]þ)

proved that neither the MeAsp nor the Mdhb residue is

modified. Additionally, fragment ions at m/z 691 ([Mþ2H–

121]þ) and 674 ([Mþ2H–121-NH3]þ) were generated. They

could derive from the cleavage between the C-8 and C-9

bond in the DMAdda residue with a C-9 hydroxyl group

instead of a methoxyl group. A low intensity peak atm/z 121,

which corresponds to [PhCH2CHOH]þ, occurred only at a

CE of 120 eV (Table 1). According to Yuan et al.,25 the m/z 135

of (A) m/z 825, (B) m/z 843, (C) m/z 811, and

re (B, D) extracts).


DOI: 10.1002/rcm

Figure 4. General structure of linear nodularin.


fragment ion is more stable than the m/z 121 ion, due to the

higher electron density around oxygen in the methoxyl

group of Adda rather than around the hydroxyl group in

DMAdda.

A peak at m/z 135.12 was also observed in the spectrum.

However, we do not identify it as the [PhCH2CH(OMe)]þ

ion. In the m/z 811 product ion spectra of other nodularin

variants with an unmodifiedAdda residue an abundant peak

at m/z 135.08 was present. The recorded 0.04Da difference in

the two masses with the same charge is significant and

implies that they derive from two different fragment ions.

We suggest that them/z 135.12 ion derives from the [C10H15]þ

fragment generated by loss of carbon monoxide from the

[C11H15O]þ ion. The ion atm/z 811 was the base peak when a

CE of 50 eV was used. At higher CE values (64 and 120 eV)

the peak at m/z 227 became the dominant one. All the

collected data indicate the structure of compound V to be

[DMAdda3]NOD.

Compound VI: [dhb5]NOD, [MRH]R¼ 811At a CE of 50 eV the protonated molecule of compound VI

(in the TIC at about 7.99min) gave a base peak at m/z 811

(Fig.3(C)).Thisionpeak,togetherwithotherionsatm/z14units

lower value than NOD-R, i.e. 632 ([MþH–135–NH2–CO]þ),

660 ([Mþ2H–135–NH3]þ), 677 ([Mþ2H–135]þ), 739 ([MþH–

CO–CO2]þ), 783 ([MþH–CO]þ) and 793 ([MþH–H2O]þ),

imply a demethylated NOD variant. At CE values of 64 and

120 eV, the m/z 135 ion became a base peak (Fig. 6(B)). The

Figure 5. ISP-MS/CID mass spectrum of linea

64 eV.


presence of product ions at m/z 157 ([ArgþH]þ), 174

([ArgþHþNH3]þ) and 303 ([MeAsp-Argþ2HþNH2]

þ) are

indicative of the unmodified Arg and MeAsp residues.

Fragment ions, which in the NOD-R spectrum contain

the Mdhb residue, occurred here at 14 units lower m/z: 195

([Glu-dhbþH–H2O]þ), 213 ([Glu-dhbþH]þ), 352 ([dhb-

MeAsp-ArgþH–NH3]þ), 369 ([dhb-MeAsp-ArgþH]þ), 375

([C11H15O-Glu-dhb]þ) and 682 ([Arg-Adda-Glu-dhbþH]þ).

The abundant ions at m/z 213, as well as the lack of ions at

m/z 227, confirmed the structure of compound VI as

[dhb5]NOD. This nodularin variant has not been reported

before.

Compound VII: [MeAdda3]NOD,[MRH]R¼ 839The TIC showed the presence of some ion peaks withm/z 839

(Fig. 3(D)). Their product ion spectra were various, but some

ions typical of NOD-R in the [MþH]þ spectrum or 14 units

higher occurred. The most pronounced peak with m/z 839

occurred in the TIC at 5.25min and was labelled as

compound VII. The product ion spectrum of this m/z 839

ion (Fig. 7(A)) showed the presence of ions atm/z 767, 795 and

811, whichmight be generated by the loss of a small molecule

to give [MþH–CO–CO2]þ, [MþH–NH2–CO]þ and [MþH–

CO]þ ions, respectively. The fragmentation of them/z 839 ion

at CE values of 50 and 64 eV gave a base peak at m/z 253. The

intensities of the ions that derived from the m/z 253 ion were

also high. Product ions at m/z 209 ([Glu-MdhbþH–H2O]þ),

227 ([Glu-MdhbþH]þ), 253 ([CO-Glu-Mdhb–H]þ), 366

([Mdhb-MeAsp-Arg-H–NH3]þ), 383 ([Mdhb-MeAsp-

ArgþH]þ) and 568 ([C2H4-CO-Glu-Mdhb-MeAsp-ArgþH]þ)

gave evidence that in compound VII the structures of the four

amino acid residues: Glu, Mdhb, MeAsp and Arg, are not

modified. The ion at m/z 135 [PhCH2CH(OMe)]þ was also

observed in the spectrum. Several fragment ions containing

Adda or [C11H15O]þ were shifted to 14 units higher values

than the corresponding ions in the m/z 825 [MþH]þ product

ion spectrum of NOD-R. They occurred at m/z 177

([C12H17O]þ), 306 ([C12H17O-Glu]þ), 403 ([C12H17O-Glu-

Mdhb]þ) and 710 ([Arg-(AddaþCH2)-Glu-MdhbþH]þ).The

results allow us to conclude that a methyl group has to be

located at some place in the [C11H15O]þ fragment of the Adda

residue. The fragment ion at m/z 568 ([C2H4-CO-Glu-Mdhb-

MeAsp-ArgþH]þ) eliminates the methylation site at C-2

and C-17.

r nodularin variant acquired at a CE of


DOI: 10.1002/rcm

Figure 6. ISP-MS/CID mass spectra of demethylated nodularin variants: (A) [DMAdda3]NOD and (B)

[dhb5]NOD acquired at a CE of 64 eV.


Compound VIII: [Glu4(OMe)]NOD,[MRH]R¼ 839Compound VIII occurred in the TIC at 9.19min (Fig. 3(D))

and gave a fragment ion at m/z 767, which is presumed to

represent [MþH–CO–CO2]þ (Fig. 7(B)). Lack of structural


modification in the Adda residue was proved by the

presence of the m/z 135 ion as well as the m/z 163 and 705

ions corresponding to [C11H15O]þ and [Mþ2H–135]þ. A

fragment ion at 174 ([ArgþHþNH3]þ) confirmed the

presence of an Arg residue. On the other hand, the m/z


DOI: 10.1002/rcm

Figure 7. ISP-MS/CID mass spectra of methylated nodularin variants: (A) [MeAdda]NOD acquired

at a CE of 50 eV and (B) [Glu4(OMe)]NOD acquired at a CE of 64 eV.


values of fragment ions containing Glu were 14 units higher.

Based on this finding, compound VIII was deduced to be a

NOD variant with a methyl derivative of Glu, presumably

glutamic acid methyl ester. The presence of Glu(OMe) was

confirmed in fragment ion peaks at m/z 223 ([Glu(OMe)-

MdhbþH–H2O]þ), 241 ([Glu(OMe)-MdhbþH]þ), 267 ([CO-

Glu(OMe)-Mdhb–H]þ) and 403 ([C11H15O-Glu(OMe)-

Mdhb]þ). In the spectrum of compound VIII, both at 50 and

64 eV, the m/z 135 ion was a base peak. At 120 eV the number

of product ions was reduced to several ions with m/z lower

than 225 and the base peak at m/z 103.

DISCUSSION

So far, eight nodularin variants have been identified

(NOD-R, linear NOD, [D-Asp1]NOD, [DMAdda3]NOD,

[6(Z)Adda3]NOD, [Glu4(OMe)]NOD, [L-Har2]NOD and [L-

Val2]NOD (Har¼homoarginine))9,11,13,14,26–28 and two other


variants have been synthesized ([D-MeAbu5]NOD, [L-

MeAbu5]NOD, (MeAbu¼ 2-(methylamino)butyric acid)).29,30

In nodularin there are three likely demethylation sites. They

can be located at MeAsp, Adda, or Mdhb. In N. spumigena

from Lake Ellesmere in New Zealand the [D-Asp1]NOD

(culture sample) and [DMAdda3]NOD (bloom sample) were

identified.14 The LD50 values in mice (i.p.) for [D-Asp1]

NOD and [DMAdda3]NOD were 75 and 150mgkg�1,

respectively.12,13

The linear nodularin, which is supposed to be a precursor

of the cyclic peptide hepatotoxin, was also found in the

culture of N. spumigena strain L-575 from New Zealand.11,14

Tandem FAB-MS/CID/MS revealed that the amino acid

sequence in the molecule is the same as in the cyclic peptide.

Additionally, the bloom samples of N. spumigena from Lake

Ellesmere contained the nodularin stereoisomer at the C-6

double bond [6(Z)Adda3].13 This nodularin variant was not

toxic to mice at 2mgkg�1. The molecular weights and


DOI: 10.1002/rcm


formulae of the variants were elucidated by FAB-MS

and high-resolution (HR) FAB-MS analyses; presence of

the 2-(methylamino)-2-dehydrobutyric acid (Mdhb), Adda

and 6(Z)Adda was confirmed by 1H NMR spec-

troscopy.9,11,14 The stereochemistry of other amino acid

componentswas determined by chiral GC. Sequence analysis

of nodularins was performed by tandem FAB-MS/CID-MS.

Rinehart et al.13, with reference to a personal communication

from Choi, reported the detection of [Glu4(OMe)]NOD. In

Nodularia PCC7804 (Pasteur Culture Collection) isolated

from a thermal spring in France, [L-Har2]NOD (LD50¼70mg kg�1) was found.26,27 The structure of [L-Har2]NOD

was determined by NMR and HR-FAB-MS. Motuporin ([L-

Val2]NOD) (LD50¼ 1.0mgkg�1), with a hydrophobic valine

amino acid residue instead of a polar arginine, was found in a

marine sponge Theonella swinhoei Gray from Papua New

Guinea.28 Until now, inN. spumigena from the Baltic Sea only

the unmodified nodularin1,10 and the [D-Asp1]NOD var-

iant31 have been identified. These two nodularin forms have

also been found in bluemussels (Mytilus edulis) and fish from

the Gulf of Finland.32 Nodularin has also been detected in the

liver of eiders (Somateria mollissima) feeding on blue

mussels.33 In the latter studies the authors used liquid

chromatography (LC) coupled to a triple-quadrupole mass

spectrometer or an ion trap, both with ESI in positive mode.

In the current study, the recorded product ion spectra of

nodularin variants were compared with the FAB-MS/CID-

MS spectra of the same nodularins isolated and fully

characterized by Rinehart et al.,9 Choi et al.,11 and Namikoshi

et al.14 We found that the fragmentation patterns of the

corresponding variants were similar and, generally, con-

tained the same foremost fragment ions. Yuan et al.20 also

reported that in ISP-MS/CID mass spectra of microcystins-

LR, YR and RR the higher abundance fragment ions were the

same as in the FAB-MS/CID (high-energy) mass spectra of

the toxins.14

The ISP-MS/CID analyses of bloom and culture samples

of the Baltic N. spumigena showed that the variety of NOD

analogues produced by the species was larger than was

previously thought. Methylated and demethylated nodu-

larin variants aswell as linearNODwere present inNodularia

cells in minor amounts when compared to NOD-R. Based on

HPLC with a photodiode detector it has been estimated that

under culture conditions the linear NOD and [D-Asp1]NOD

are the most abundant variants besides NOD-R. In bloom

samples, on average, there was twice as much [DMAdda3]-

NOD as the other two demethylated variants. The higher

contribution of the geometrical isomer of NOD-R in the

bloom sample rather than in cultured N. spumigena seems to

be in agreement with earlier findings on sunlight-induced

isomerization of cyanobacterial cyclic peptide hepatotox-

ins.34 Both in bloom and culture samples [MeAdda]NOD

was the main nodularin variant with the additional methyl

group. [Glu4(OMe)]NOD was the least abundant variant of

all nodularins detected in N. spumigena extracts. Since

methanol was used for the cleanup procedure, it might

not be excluded that the glutamic acid methyl ester of NOD

was an artefact. Generally, the same NOD variants were

produced in culture and in the field, but they were present in

the cells in different proportions. Apparently, environmental


conditions modify the production and/or degradation rates

of different toxin variants.

In some instances, the characterization of amino acid

structure and sequence on the basis of the product ion

spectrum is difficult, and could lead to incorrect conclusions.

This is due to the fact that Glu and MeAsp are isomers and

can give isobaric ions with exactly the samem/z. For instance,

at a CE of 64 eV, the m/z 227 ion can be attributed to both

[Mdhb-MeAspþH]þ and [Glu-MdhbþH]þ, while the frag-

ment ion at m/z 599 could derive either from [Arg-Adda-

GluþH]þ or [MeAsp-Arg-AddaþH]þ. If the structure of Glu

or MeAsp is modified, both the m/z 227 and 599 ions and the

modified ions could be expected in the spectrum.

The fragmentation patterns of the [MþH]þ product ions

obtained at CE values of 50, 64 and 120 eV showed different

stability of the ions. Regardless of the demethylation site all

precursor m/z 811 ions were still observed in the product ion

spectra obtained at a CE of 120 eV (Table 1). Using the same

CE, the m/z 825, 839 and 343 precursor ions fragmented into

smaller ions. It can be concluded that the [MþH]þ ions

generated by the demethylated nodularin variants are more

stable.

In fact, all the modifications in the nodularin structure

have been characterized earlier for microcystins. In this

group of cyanobacterial cyclic peptide hepatotoxins variants

with fewer or more methyl groups are quite common.

According to Diehnelt et al.,35 46 out of 67 microcystins

possess an Asp residue instead of MeAsp. Dehydrobutyrine-

containing microcystins were found in Nostoc spp.36 and

Planktothrix agardhii.37 Glumethyl esters have been identified

using ESI-MS/CID-MS by Namikoshi et al.38 In microcystin-

LR from cyanobacteria ofMicrocystis genera the replacement

of the methoxyl group at position C-9 in the Adda residue by

a hydroxyl group has been reported by Namikoshi et al.39

They also described the structure of microcystins with an

acetoxyl group at C-9 in ADMAdda.40 The list of microcystin

variants published by Sivonen and Jones15 showed that

in cyanobacteria the ADMAdda is more common than

DMAdda. To our knowledge, microcystin or nodularin

variants with a methyl-Adda residue have not been reported

previously.

CONCLUSIONS

The hybrid Q-TOF LC/MS/MS instrument with ionspray

(ISP) and collision-induced dissociation (CID) proved to be a

powerful tool in generating structural information on

nodularin variants. Using positive ionization, sufficient

fragmentation was achieved to assign the structure of eight

NOD variants. The analyses confirmed the presence of

nodularin-R (NOD-R) and the demethylated [Asp1]NOD in

the Baltic Nodularia strain and revealed the presence of three

known variants, [DMAdda3]NOD, the linear NOD and the

geometrical isomer of NOD-R, reported earlier in N. spumigena

from New Zealand.14 In addition, [Glu4(OMe)]NOD was found.

The structures of the two novel variants were deduced to be

[dhb5]NOD and [MeAdda]NOD.

Under the conditions used in the experiments, the m/z 811

[MþH]þ ions of the demethylated nodularin variants were

more stable than the corresponding ions of the other


DOI: 10.1002/rcm


compounds with m/z 825, 839 and 843. The quantitative

contributions of modified nodularin variants were rather

low, compared to NOD-R, and they changed under different

growth conditions. The findings contribute to knowledge of

the production of nodularin variants byN. spumigena, bloom-

forming, filamentous cyanobacterium.

AcknowledgementsThis work was supported by the State Committee for Scien-

tific Research in Poland (project 0366/PO4/2003/25) and UE

project BALTDER EVK3-CT-2002-80005. MS analyses were

done in the Pomeranian Science and Technology Park in

Gdynia. JM thanks the Academy of Finland, RC for Bios-

ciences and Environment (project 207238) for financial

support.

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DOI: 10.1002/rcm

characterization of nodularin variants innodularia spumigena from the baltic sea using liquid...

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