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Peptaibols of Trichoderma atroviride: screening, identification, and structure elucidation by liquid chromatography-tandem mass spectrometry

R. Schuhmacher*,1, N. Stoppacher1, and S. Zeilinger2 1Center for Analytical Chemistry, Department for Agrobiotechnology (IFA-Tulln), University of Natural

Resources and Applied Life Sciences Vienna, Konrad Lorenz Strasse 20, A-3430 Tulln, Austria 2Institute of Chemical Engineering, Research Area of Gene Technology and Applied Biochemistry,

Working Group Molecular Biochemistry of Fungi, Vienna University of Technology, Getreidemarkt 9/1665A, A-1060 Vienna, Austria

Trichoderma spp. are filamentous fungi which frequently colonise soil and plant roots. As an alternative to chemical pesticides, some Trichoderma species such as T. harzianum, T. atroviride and T. virens are being used as biocontrol agents against plant pathogenic fungi. Biocontrol strains of Trichoderma show both, induction of resistance in plants and direct mycoparasitism of plant pathogenic fungi. For both of these processes, the production and secretion of antifungal secondary metabolites such as peptaibols plays a key role. Peptaibols show antimicrobial activity and constitute a large group of more than 300 known different linear peptides with a length of up to 20 amino acid residues, carrying non-proteinogenic amino acids such as α-aminoisobutyric acid (Aib) as well as a modified N-terminus (e.g. acetylation) and a C-terminal amino alcohol (e.g. leucinol). Due to their bioactive properties, peptaibols are interesting target compounds for the development of biocontrol agents as well as for the study of the interactions between Trichoderma, pathogenic fungi and plants.

In this contribution we describe the development and application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) based methods for the analysis of this specific group of peptides. Different analytical approaches were used for screening, identification and elucidation of amino acid sequences of peptaibols. The paper presents the rationales of these analytical approaches and the results which we obtained for culture samples of T. atroviride.

Keywords Trichoderma; peptaibols; liquid chromatography (LC); tandem mass spectrometry (MS/MS)

1. Introduction

1.1 Biological background of the study

Biological control agents to fight plant diseases become more and more widespread. Several mycoparasitic Trichoderma species are used in biocontrol against plant pathogenic fungi such as Botrytis cinerea and Fusarium spp. [e.g. 1, 2]. The mechanisms involved in biological plant pathogen control by Trichoderma are complex and include competition for nutrients and root exudates, inactivation of pathogen´s enzymes, direct attack and lysis of the plant pathogen (mycoparasitism), induction of systemic resistance in plants [3, 4] and the production of antibiotic secondary metabolites. Mycoparasitic interaction of Trichoderma with the pathogen starts with sensing of the pathogenic fungus. This usually involves the binding of pathogen-derived elicitors, e.g. cell wall degradation products, proteins or toxins to receptor proteins of Trichoderma (membrane- or cytosolic proteins) and is followed by signal transduction and response pathways (e.g. secretion of lytic enzymes and antifungal secondary metabolites). An important type of receptor proteins are G protein coupled receptors (GPCRs). GPCRs are transmembrane proteins, to which heterotrimeric G-proteins (each consisting of α-, β- and γ- * Corresponding author: e-mail: [email protected]

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subunits) are attached inside the cell. Either the Gα or the Gßγ subunits transmit the signal via stimulation of adenylyl cyclase or the mitogen-activated protein (MAP) kinase cascade. Fungal Gα subunits are highly conserved and can be divided into three subgroups. Several mutants of T. atroviride P1 bearing deletions in genes encoding signal transduction pathway components have been constructed with the aim to study the relevance of the respective genes for mycoparasitism and biocontrol of this fungus. Deletion mutants missing G protein-α-subunit (∆tga1, ∆tga3) - or MAP kinase (∆tmk1) -encoding genes revealed that both, heterotrimeric G proteins and a MAP kinase of T. atroviride are involved in biocontrol-relevant processes. We showed that there are substantial differences between the T. atroviride wild-type strain and the deletion mutants with respect to vegetative growth, conidiation, chitinase formation and growth inhibition of host fungi [5-7]. As antifungal secondary metabolites are known to be important for successful biocontrol [e.g. 29], we were interested to investigate and compare the production of peptaibols between the T. atroviride wild-type strain and the ∆tga1, ∆tga3 and ∆tmk1 mutants.

1.2 Peptaibols, antibiotic secondary metabolites of Trichoderma

Peptaibols are non-ribosomal peptides, synthesised by non-ribosomal peptide synthetases [31] and are composed of 5 to 20 amino acid residues. They contain characteristic non-proteinogenic amino acids such as α-aminoisobutyric acid (Aib) or isovaline (Iva). Their N-terminal amino acids are acylated (usually acetylated) and the C-Termini are reduced to alcohols, e.g. phenylalaninol (Pheol) or trytophanol (Trpol) [8]. Peptaibols often occur as micro-heterogeneous mixtures of up to 20 individual substances with very similar chemical structures. The type of peptaibol families produced by Trichoderma obviously is species-specific. The different families of peptaibols reported to be produced by T. harzianum for example, have been summarized by Szekeres et al. [8]: harzianins, trichokinidins, trichorozins, trichorzianines and trichorzins. T. atroviride, which was investigated in the present study, has been reported to produce atroviridins A-C and neoatroviridins A-D [9]. More than 300 peptaibols are listed in a database [10] and recently many more of these compounds have been discovered [e.g. 30]. The biological activity of peptaibols is closely linked to their three dimensional α-helical structures and their potential to form pores in bilayer lipid membranes. Peptaibols display antibacterial, antiviral and antifungal activities [8] and were shown to inhibit the growth of fungal pathogens such as B. cinerea and A. niger [11, 8]. Additionally, peptaibols may also elicit plant resistance to pathogens by inducing metabolic activities like ethylene emission and biosynthesis of volatile compounds [12].

TrpolGluGlnIleAibLeuProAibLeuSerAibAibAibGlnAibAibAlaAlaAcAib

19181716151413121110987654321

TrpolGluGlnIleAibLeuProAibLeuSerAibAibAibGlnAibAibAlaAlaAcAib

19181716151413121110987654321

O

CH CH2

NH2 O NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

N NH

NH

NH

NH N

HNH

O O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

CH2CH2

CH2OH

CH2

OHCH2CH

CH2

CH2CH 2

CH2OHN

H

OAcNH

CHCH3CH2

O OHNH2O

Fig. 1 Sequence and structure of trichorzianine TB IIa, representing a typical peptaibol containing α-amino-isobutyric acid, acetylated N-terminus and reduced C-terminus. Ac: acetyl; Aib: α-amino-isobutyric acid; Trpol: tryptophanol

1.3 Analytical methods for the detection of peptaibols in fungal culture samples

Sample preparation: The analysis of peptaibols in fungal cultures usually includes extraction of the analytes from culture medium or mycelium, purification from matrix components, analyte enrichment, and eventually chromatographic separation and detection. For the extraction from fungal mycelium or culture media different organic solvents, e.g. dichloromethane [13]; methanol [14] or a mixture of methanol and chloroform [15] were used. Further purification was achieved by liquid chromatography

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(LC) using different polar and non-polar stationary phases such as Sephadex LH 20, silica gel [15], RP-C18 or RP-C8 cartridges [16-18]. Chromatographic separation and detection of peptaibols: As thin layer chromatography (TLC) is fast and cheap without the need for sophisticated instrumentation but lacks of sensitivity and selectivity, this technique was used mainly for monitoring peptaibol formation during fermentation of Trichoderma cultures [19]. Moreover, gas chromatography (GC) with flame ionization detection (FID) or MS has also been applied to analyse peptaibols. However, since the peptaibols are non-volatile amphiphilic peptides, the use of gas chromatography is not straight forward but requires hydrolytic cleavage of the peptide bonds and subsequent derivatisation of the amino acids to obtain volatile derivatives. GC methods can be regarded as valuable tools to obtain complementary information on peptaibol structures. They have been used successfully to elucidate the chirality of the amino acid residues and amino alcohols in the peptaibol chain [e.g. 18] as well as to determine the relative composition of individual amino acid residues [e.g. 20]. Most frequently, chromatographic end separation of peptaibols is carried out by reversed phase high performance liquid chromatography (RP-HPLC) in combination with ultraviolet (UV) - or MS-detection. In consideration of the high similarity of individual members of a given mixture of peptaibols, MS can be regarded as the most powerful technique for the selective and sensitive detection and structural characterisation of these secondary metabolites [13, 16, 19, 21-23]. Online coupling of LC with MS for the analysis of peptaibols usually includes electrospray ionisation and subsequent MS and MS/MS detection. Information typically derived from MS full scan spectra are for example the molecular mass of the intact peptaibol (derived from [M+H]+, [M-H]-, molecular ions in combination with typical adduct ions (e.g. [M+Na]+, [M+NH4]+, [M+K]+ and multiple charged ions) and the charge states of the ion species formed in the ion source. In MS/MS experiments the intact analytes can be separated by RP-HPLC prior to electrospray ionisation (ESI) - online MS collision induced fragmentation, which leads to the cleavage of the specific peptaibol marker Aib from the peptide chain. The presence of Aib can be monitored by fragments showing a specific mass difference of 85 amu in the MS/MS spectrum. Moreover, the amino acid sequence can be obtained from peptide fragment ladders. The more or less random cleavage of peptide bonds by collision induced dissociation (CID) leads to a mixture of fragments which belong to two different classes of ions, a) fragments containing the N-terminus plus one (b1-ion) or more amino acid residues (bn-ions) and b) fragments carrying the C-terminus (termed yn-ions). The mass differences between ions bn and bn+1 or yn and yn+1 directly correspond to the mass increment of one single amino acid [32]. Valine and isovaline or leucine and isoleucine can generally not be distinguished by MS since they have the same molecular masses. Comprehensive structure elucidation of the components of micro-heterogeneous peptaibol mixtures usually is achieved by a combination of different chromatographic separation techniques, MS and other spectroscopic techniques such as nuclear magnetic resonance (NMR) [14, 24].

2. Material and methods

2.1 Chemicals

Acetonitrile and methanol (both LC gradient grade) were purchased from J.T. Baker (Deventer, The Netherlands) and ammonium acetate (MS grade) was obtained from Sigma-Aldrich (Vienna, Austria). Water was purified successively by reverse osmosis and a Milli-Q plus system from Millipore (Molsheim, France). Trichotoxin standards were kindly provided by Prof. Brückner (University of Giessen, Germany). Trichorzianine standard mixtures consisting of the groups TA and TB were used for structure confirmation and were kindly provided by Prof. Rebuffat (National Museum of Natural History, Paris, France). For each group of peptaibols, 1 mg of solid material was dissolved in 1 ml of methanol and stored as stock solution at 4°C. Working standards were prepared from these stock solutions by dilution with methanol and HPLC mobile phase.

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2.2 HPLC

Chromatographic separation of the peptaibols was carried out at 25°C on a BDS Hypersil C18 column, 150 x 2.1 mm, 5 µm particle size (Thermo Electron Corp., Waltham, MA, USA), using an HPLC series 1100 (Agilent Technologies, Waldbronn, Germany). Injection volume: 5 µl, flow rate: 0.3 or 0.6 ml/min. Different linear elution gradients were used for the separation of the peptaibols. HPLC eluent systems consisted either of water + acetonitrile (9+1, v+v) containing 5 mM ammonium acetate; water + acetonitrile (5+95, v+v) containing 5 mM ammonium acetate or water + acetonitrile (9+1, v+v) containing 0.1 % formic acid; 0.1 % formic acid in acetonitrile. A sixport switching valve (VICI Valco Instruments, Houston, TX, USA) was used to transfer the column effluent either to the mass spectrometer or to the waste.

2.3 Mass spectrometry

There are numerous types of tandem mass spectrometers which can be employed for the analysis of peptaibols. The instrument used in this study is a tandem mass spectrometer equipped with an electrospray ion source and a so called Q Trap triple quadrupole mass analyser, which consists of three quadrupole devices connected in series (Fig. 2). The first quadrupole (Q1) can be operated as mass filter, alternatively scanning over a definded m/z range or specifically filtering selected ions having certain m/z ratios. The second quadrupole (Q2) is filled with an inert gas (e.g. N2) and may serve as a collision cell for the fragmentation of ions passing Q1. The third quadrupole can be either used as a quadrupole mass filter (Q3), similar to Q1, or as linear ion trap (LIT). In the latter mode, ions passing Q1 and Q2 are collected and stored (trapped) in the LIT before being successively scanned to the detector. This so called Q Trap instrument allows for numerous unique and highly sensitive operation modes for the detection and structure characterisation of linear peptides such as the peptaibols. As the detailed description of the operation methods of this MS instrument goes beyond the scope of this article the interested reader is referred to recent literature [25-26].

Mass filter Q3 / LIT

Collision cellN2 gasMass filter

Q1ESIcapillary

DetectorMass filter

Q3 / LIT

Collision cellN2 gasMass filter

Q1ESIcapillary

Detector

filtering of selected ions

fragmentationscanningPrecursor ion scan

trapping, scanning

fragmentationfiltering of selected ions

Enhanced production scan (EPI)

ion transferion transferscanningQ1 full scan

Q3 / LITCollision cellQ1Scan mode

filtering of selected ions

fragmentationscanningPrecursor ion scan

trapping, scanning

fragmentationfiltering of selected ions

Enhanced production scan (EPI)

ion transferion transferscanningQ1 full scan

Q3 / LITCollision cellQ1Scan mode

The Q Trap 4000 LC-MS/MS system (Applied Biosystems, Foster City, CA, USA) was equipped with a TurboV ESI source and was operated in the positive ionisation mode. For the results illustrated below the following ion source parameters were used: curtain gas: N2@69 kPa; ion spray voltage +4000 V; source temperature: 450°C or 515°C; sheath gas: N2@345 kPa; drying gas: N2@345 kPa; declustering potential (DP): 50V. For Q1-MS full scan measurements, spectra were recorded between m/z 200 and 2000; scan time 3s. EPI spectra were recorded with a collision energy (CE) 50 V; Q1 low resolution; linear ion trap (LIT) settings: scan rate 4000 amu/s; filltime 50 ms; Q3 entry barrier 8 V. Precursor ion scan settings were as follows: Q1 precursor m/z 355.4; Q3 scan range m/z 945 – 1000; scan time 1 s; Q1, Q3 unit resolution; CE 68V.

2.4 Sample preparation

Culture filtrates were either diluted 1+1 with HPLC mobile phase and directly analysed by LC-MS(/MS) or concentrated using C18 solid phase extraction (SPE) columns. SPE was carried out according to [16]

Fig. 2 Ion path of the Q Trap instrument. In Q1 full scan mode, Q1 is scanning over a defined m/z range, all ions are guided through Q2 and Q3 to the detector. In an EPI scan, only selected target ions may pass Q1, which then will be fragmented in Q2. Subsequently, all fragment ions are trapped in Q3 (operated as LIT) before being scanned to the detector. In a precursor ion scan, Q1 is scanned over a defined m/z range (covering the masses of potential precursor ions), in Q2 precursors are fragmented and only selected fragments may pass Q3.

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after slight modifications. In brief, Bakerbond C18 cartridges (500mg, 3 ml) were preconditioned subsequently with 10 ml of methanol and 10 ml of water + methanol (2+1, v+v). Then, 15 ml of the culture filtrate (diluted with methanol 2+1, v+v) were applied to the SPE columns, the column was washed with 10 ml of water and 10 ml of 33% of aqueous methanol before eluting the peptaibols with 4 ml of methanol. The eluate was dried under a gentle stream of nitrogen and reconstituted in 0.5 ml of HPLC mobile phase.

2.5 Trichoderma strains and culture conditions

T. atroviride wild-type strain P1 (formerly classified as T. harzianum ATCC 74058) and the mutants ∆tga1, ∆tga3 and ∆tmk1 were cultivated in three replicates in sterilised synthetic medium (containing KH2PO4, KNO3, CaCl2, MgSO4, MnSO4, CuSO4 and FeSO4 and glucose) as described in [5]. The stationary cultures were incubated at 28 °C for 10 days and the mycelia separated from the fermentation broth by filtration. For prevention of microbial growth one spatula tip of sodium azide was added to each of the culture filtrates. The filtrates and dried mycelia were stored at 4°C until analysis.

3. Results and dicussion

3.1 Screening for peptaibols

In a first step we investigated the LC-MS(/MS) properties of the trichotoxins which served as model peptaibols in this study. The Q1 full scan mass spectrum of trichotoxin A40/III (Fig. 3) illustrates some general properties of peptaibols with respect to their behaviour in the electrospray ion source. According to their chain length of 18 amino acids, the doubly protonated molecular ions [M+2H]2+ dominate over the [M+H]+ species. Additionally, singly and doubly charged sodium adducts of the peptaibol were formed in the ESI source. It has been described in the literature that the peptide bond between the peptaibol specific Aib and proline (Pro) generally tends to be labile under the conditions occurring in an ESI source (so called in-source fragmentation) [22]. As trichotoxin A40/III contains one Aib-Pro motif, the preferential cleavage of the Aib-Pro bond in the ion source of the MS instrument resulted in two prominent MS/MS fragments with m/z 627.8 and m/z 1078.9. The signal at m/z 1078.9 corresponds to the b12-ion of the b-ion series, an acylium ion consisting of 12 amino acids including the acetylated N-terminus. The ion with m/z 627.8, termed y6, consists of the remaining 6 amino acid residues including the C-terminus. Both fragments can be used as diagnostic species for further MS/MS experiments to elucidate the amino acid sequence of the compound.

Ac-Aib-Gly-Aib-Leu-Aib-Gln-Aib-Aib-Ala-Ala-Aib-Aib-Pro-Leu-Aib-Iva-Glu-Valolb12 (m/z 1078.9) y6 (m/z 627.8)

Ac-Aib-Gly-Aib-Leu-Aib-Gln-Aib-Aib-Ala-Ala-Aib-Aib-Pro-Leu-Aib-Iva-Glu-Valolb12 (m/z 1078.9) y6 (m/z 627.8)

853.5627.8

864.4

1705.5

1727.5

600 800 1000 1200 1400 1600 1800m/z (amu)

1078.9y6[M+2H]2+

[M+Na+H]2+

b12

[M+H]+

[M+Na]+

1.0e62.0e63.0e6

4.0e6

5.0e6

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sity (

cps)

0.0

853.5627.8

864.4

1705.5

1727.5

600 800 1000 1200 1400 1600 1800m/z (amu)

600 800 1000 1200 1400 1600 1800m/z (amu)

1078.9y6[M+2H]2+

[M+Na+H]2+

b12

[M+H]+

[M+Na]+

1.0e62.0e63.0e6

4.0e6

5.0e6

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sity (

cps)

0.0

1.0e62.0e63.0e6

4.0e6

5.0e6

Inten

sity (

cps)

0.0

Fig. 3 Q1 full scan MS spectrum and amino acid sequence of trichotoxin A40/III. The trichotoxins were used as model peptaibols to study their ionisation and fragmentation properties in ESI-MS and ESI-MS/MS. Since the T. atroviride wild-type strain P1 formerly was classified as T. harzianum ATCC 74058, we also included peptaibols, which have been described to be produced by T. harzianum (see 1.2).

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According to the results obtained for the trichotoxins, target screening was carried out by programming the MS instrument to search for chromatographic peaks corresponding to the masses (i.e. m/z signals) of known peptaibols and their doubly protonated molecular ions as well as their corresponding y- and b- fragments resulting from the cleavage of the Aib-Pro bond(s). These measurements were carried out in the full scan MS and product ion (EPI) modes (see 2.3 and Fig. 2). Fig. 4 shows a typical total ion current (TIC) chromatogram obtained for a culture sample of the signal transduction mutant ∆tmk1 (missing the MAP kinase -encoding gene tmk1). The TIC represents the sum of ions obtained from three consecutive MS/MS experiments (each of which taking 0.4-0.8 seconds): fragmentation of ion species corresponding to [M+H]+ (m/z 1911), [M+2H]2+ (m/z 956) and y7 fragment (m/z 803) of trichorzianine TB VIb. The prominent peak in the chromatogram indicated that at least one of the precursor ions was present in the ∆tmk1 sample.

3.2 Elucidation of amino acid sequences and screening for common substructures

The screening of culture filtrates of the T. atroviride P1 parental strain and the mutants for the occurrence of peptaibols known to be produced by T. atroviride and T. harzianum resulted in TIC chromatograms, for which the underlying MS and MS/MS spectra were further evaluated. In agreement with other studies on peptaibols [e.g. 19], we also observed ions of the b-series and, to a much lesser extent, of the y-series after MS/MS determination of the T. atroviride culture samples. A typical result is shown in Fig. 5. The mass differences between the product ions correlate to distinct amino acids and identify the investigated molecules as peptides. Furthermore, the mass increment of 85 amu, which is characteristic for Aib, was frequently observed in the EPI spectra, thus confirming the compounds to be peptaibols. The combination of the EPI spectra of the precursors [M+H]+, [M+2H]2+ and the respective yn fragment that was already formed in the ion source by cleavage of the Aib-Pro bond, enabled us to assign most of the amino acids of the detected peptaibols. A comparison of the found sequences with the entries in the database [10] and the literature indicated that each of the T. atroviride strains produced peptaibols of the trichorzianine subgroups TA and TB [27, 28] (see Tab. 1), although these peptaibols have only been assigned to T. harzianum so far. This finding was also confirmed by the LC-MS/MS analysis of standard mixtures of trichorzianines TA and TB. Additionally, we applied the precursor ion scan mode (Fig. 2) to detect peptaibols which yield fragments of the same m/z after CID. EPI scan experiments showed that all trichorzianines formed fragments of m/z 355, corresponding to the N-terminal partial sequence Ac-Aib-Ala-Ala-Aib (Tab. 1). Therefore, we screened the T. atroviride culture samples for all molecules (precursors) which formed ions of m/z 355.4 in the collision cell of Q2. Fig.6 illustrates a typical result of the precursor ion scan mode. In addition to the trichorzianines already found by EPI scans, we detected other peptaibols in the culture samples which we have not entirely characterised yet. Moreover, the MS/MS measurements revealed that each of the mutants ∆tga1, ∆tga3 and ∆tmk1 produced significantly elevated concentrations of peptaibols compared to the parental strain.

Fig. 4 TIC of a culture filtrate sample of mutant ∆tmk1. The peak with the retention time of 12.98 min represents the sum of ions generated in three consecutive EPI experiments: fragmentations of m/z 1911.3, 956.2 and 803.0 corresponding to [M+H]+, [M+2H]2+ the y7 fragment of trichorzianine TB VIb

Time (min)8 10 12 14 16 18

Inten

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cps)

0.0

2.0e8

4.0e8

6.0e8

8.0e8

1.0e9 12.98

Time (min)8 10 12 14 16 18

Time (min)8 10 12 14 16 18

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4.0e8

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4.0e8

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8.0e8

1.0e9 12.98

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Ac-Aib-Ala-Ala-Aib-Aib-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Val-Aib-Ile-Gln-Glu-Pheolb12 (m/z 1108.8) y7 (m/z 803.0)

Ac-Aib-Ala-Ala-Aib-Aib-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Val-Aib-Ile-Gln-Glu-Pheolb12 (m/z 1108.8) y7 (m/z 803.0)

a)

m/z (amu)800 1000 1200 1400 1600 1800

Inten

sity (

cps)

1911.3823.6

910.6 1760.4738.6 1023.21.0e6

2.0e6

3.0e6

0.0

1108.8[M+H]+

Aib Ser Leu PheolAib

+EPI (1911.3) b9b8 b10 b11 b12 b18

m/z (amu)800 1000 1200 1400 1600 1800

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sity (

cps)

1911.3823.6

910.6 1760.4738.6 1023.21.0e6

2.0e6

3.0e6

0.01.0e6

2.0e6

3.0e6

0.0

1108.8[M+H]+

Aib Ser Leu PheolAibAib Ser Leu PheolAib

+EPI (1911.3) b9b8 b10 b11 b12 b18

b) +EPI (956.2)

200 400 600 800 1000m/z (amu)

803.6440.51108.8653.6 738.7355.3 823.6270.3199.2

910.6567.7 1023.81.0e72.0e73.0e74.0e7

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0.0128.1

b1

Ala AibAibAla GlnAib Aib AibAib LeuSerb12b9b8b7b6b5b4b3b2 b11b10+EPI (956.2)

200 400 600 800 1000m/z (amu)

200 400 600 800 1000m/z (amu)

803.6440.51108.8653.6 738.7355.3 823.6270.3199.2

910.6567.7 1023.81.0e72.0e73.0e74.0e7

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0.01.0e72.0e73.0e74.0e7

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0.0128.1

b1

Ala AibAibAla GlnAib Aib AibAib LeuSerAla AibAibAla GlnAib Aib AibAib LeuSerb12b9b8b7b6b5b4b3b2 b11b10 b12b9b8b7b6b5b4b3b2 b11b10

c) +EPI (803.0)

200 400 600 800m/z (amu)

282.3

197.2 395.4652.5523.5

803.6GlnIleAib PheolGlu

4.0e6

8.0e6

1.2e7

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y7/b2 y7/b3 y7/b4 y7/b5 y7/b6 y7p

Pro + Val

+EPI (803.0)

200 400 600 800m/z (amu)

200 400 600 800m/z (amu)

282.3

197.2 395.4652.5523.5

803.6GlnIleAib PheolGlu

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8.0e6

1.2e7

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y7/b2 y7/b3 y7/b4 y7/b5 y7/b6 y7p

Pro + Val

Fig. 5 MS/MS spectra (EPI mode) obtained for the chromatographic peak at 12.98 min of Fig. 4. The selected precursor ions correspond to the predicted m/z values of a) [M+H]+, b) [M+2H]2+ and the y7 ion of trichorzianine TB VIb. The bn ion fragments dominated the EPI spectra for each of the precursor ions.

a) b)

2 4 6 8Time (min)

0.04.0e58.0e51.2e61.6e62.0e6

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34

5,67

13

11

9,108

12

1615

14

2 4 6 8Time (min)

0.04.0e58.0e51.2e61.6e62.0e6

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cps)

2 4 6 8Time (min)

2 4 6 8Time (min)

0.04.0e58.0e51.2e61.6e62.0e6

Inten

sity (

cps)

0.04.0e58.0e51.2e61.6e62.0e6

Inten

sity (

cps)

12

34

5,67

13

11

9,108

12

1615

14

950 960 970 980 990 1000m/z (amu)

975.4

986.4983.9

1.0e4

2.0e4

3.0e4

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sity (

cps)

0.0

[M+2H]2+

[M+Na+H]2+

[M+NH4+H]2+

950 960 970 980 990 1000m/z (amu)

975.4

986.4983.9

1.0e4

2.0e4

3.0e4

Inten

sity (

cps)

0.0

1.0e4

2.0e4

3.0e4

Inten

sity (

cps)

0.0

[M+2H]2+

[M+Na+H]2+

[M+NH4+H]2+

Fig. 6 a) TIC chromatogram after precursor ion scan of trichorzianines sharing the common substructure with m/z 355: a) TIC of an SPE-enriched culture filtrate of mutant ∆tmk1. 1: trichorzianine TB IIa, 2: TB IIIc, 3: TB IVb, 4: TB Vb, 5: TB VIa, 6: TB VIb, 7: TA IIa, 8: TB VII, 9: TA IIIb, 10: TA IIIc, 11: TA IIIa, 12: TA IVb, 13: TA Vb, 14: TA VIb, 15: TA VIa, 16: TA VII. b) mass spectrum of the peak at 6.0 min (9, 10) showing the precursor ion species corresponding to the isobaric peptaibols TA IIIb & TA IIIc.

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Table 1 Amino acid sequences of trichorzianines as published by El. Hajji et al. and S. Rebuffat et al. [27-28]. Note the high similarity of amino acid sequences. They only differ in the amino acids illustrated in bold. The groups of TA and TB differ in one single residue at position 18: Members of TA contain a Gln, whereas all members of TB carry a Glu. Moreover, all trichorzianines share common structure elements such as amino acids 1-4 leading to the same MS/MS fragment of m/z 355. Aib: α-aminoisobutyric acid, Iva: isovaline, Trpol: tryptophanol, Pheol: phenylalaninol, MW: molecular weight.

Trichor-zianine

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

MW (g/mol)

TA IIa AcAib-Ala-Ala-Aib-Aib-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Leu-Aib-Ile-Gln-Gln-Trpol 1962.1 TA IIIa AcAib-Ala-Ala-Aib-Iva-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Leu-Aib-Ile-Gln-Gln-Trpol 1976.2 TA IIIb AcAib-Ala-Ala-Aib-Aib-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Val-Aib-Leu-Gln-Gln-Trpol 1948.1 TA IIIc AcAib-Ala-Ala-Aib-Aib-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Val-Aib-Ile-Gln-Gln-Trpol 1948.1 TA IVb AcAib-Ala-Ala-Aib-Iva-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Val-Aib-Ile-Gln-Gln-Trpol 1962.1 TA Vb AcAib-Ala-Ala-Aib-Aib-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Leu-Aib-Ile-Gln-Gln-Pheol 1923.1 TA VIa AcAib-Ala-Ala-Aib-Iva-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Leu-Aib-Ile-Gln-Gln-Pheol 1937.1 TA VIb AcAib-Ala-Ala-Aib-Aib-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Val-Aib-Ile-Gln-Gln-Pheol 1909.1 TA VII AcAib-Ala-Ala-Aib-Iva-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Val-Aib-Ile-Gln-Gln-Pheol 1923.1 TB IIa AcAib-Ala-Ala-Aib-Aib-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Leu-Aib-Ile-Gln-Glu-Trpol 1963.1 TB IIIc AcAib-Ala-Ala-Aib-Aib-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Val-Aib-Ile-Gln-Glu-Trpol 1949.1 TB IVb AcAib-Ala-Ala-Aib-Iva-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Val-Aib-Ile-Gln-Glu-Trpol 1963.1 TB Vb AcAib-Ala-Ala-Aib-Aib-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Leu-Aib-Ile-Gln-Glu-Pheol 1924.1 TB VIa AcAib-Ala-Ala-Aib-Iva-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Leu-Aib-Ile-Gln-Glu-Pheol 1938.1 TB VIb AcAib-Ala-Ala-Aib-Aib-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Val-Aib-Ile-Gln-Glu-Pheol 1910.1 TB VII AcAib-Ala-Ala-Aib-Iva-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Val-Aib-Ile-Gln-Glu-Pheol 1924.1

4. Conclusion

We could demonstrate by HPLC-MS/MS analysis that cultures of T. atroviride produce complex mixtures of peptaibols, sixteen members of which belonged to the trichorzianine group. It was possible to separate most of the peptaibols by HPLC and to characterise them using MS and MS/MS techniques. We found that each of the tested T. atroviride strains (wild-type P1 as well as the mutants ∆tga1, ∆tga3 and ∆tmk1) produced all 16 trichorzianines. The findings were confirmed by comparison with trichorzianine standard mixtures. Moreover, the results of this study indicate that each of the mutants produces higher levels of trichorzianines compared to the wild-type strain P1. Finally, new peptaibols were found in the T. atroviride cultures. In the near future, we intend to develop a semiquantitative peptaibol-profiling method for a detailed comparison of the peptaibol expression levels of different T. atroviride strains and mutants. We also plan to investigate the amino acid sequences of the newly found peptaibols in more detail.

Acknowledgements The authors thank Prof. Hans Brückner and Prof. Sylvie Rebuffat for providing the peptaibol standards. Moreover, the financial support by the Government of Lower Austria and the Austrian Science Fund (FWF project P18109-B12) is gratefully acknowledged.

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