Impurity determination for hepcidin by liquid chromatography-high resolution and ion mobility mass spectrometry for the value assignment of candidate primary calibrators.

In metrology institutes, the state-of-the-art for purity analysis of peptides/proteins mainly addresses short and unfolded peptides. Important developments are anticipated for the characterization of nonlinear peptides or proteins. Hepcidin 1-25 is an interesting model system because this small protein contains four disulfide bridges with a particular connectivity that is difficult to reproduce and could induce a bias in quantification. Hepcidin 1-25 is involved in iron-related disorders and anemia, in an inflammatory context, and its clinical relevance in neurodegenerative disorders is under investigation. It is also an emerging biomarker. Recent inter-laboratory studies showed a need for standardization of hepcidin assay and the need to produce certified reference materials. This paper discusses two hepcidin standards from different synthesis pathways that have been characterized by high-resolution mass spectrometry and ion mobility mass spectrometry.

The peptaibiotics database--a comprehensive online resource.

In this work, we present the 'Peptaibiotics Database' (PDB), a comprehensive online resource, which intends to cover all Aib-containing non-ribosomal fungal peptides currently described in scientific literature. This database shall extend and update the recently published 'Comprehensive Peptaibiotics Database' and currently consists of 1,297 peptaibiotic sequences. In a literature survey, a total of 235 peptaibiotic sequences published between January 2013 and June 2014 have been compiled, and added to the list of 1,062 peptides in the recently published 'Comprehensive Peptaibiotics Database'. The presented database is intended as a public resource freely accessible to the scientific community at peptaibiotics-database.boku.ac.at. The search options of the previously published repository and the presentation of sequence motif searches have been extended significantly. All of the available search options can be combined to create complex database queries. As a public repository, the presented database enables the easy upload of new peptaibiotic sequences or the correction of existing informations. In addition, an administrative interface for maintenance of the content of the database has been implemented, and the design of the database can be easily extended to store additional information to accommodate future needs of the 'peptaibiomics community'.

The comprehensive peptaibiotics database.

Peptaibiotics are nonribosomally biosynthesized peptides, which - according to definition - contain the marker amino acid α-aminoisobutyric acid (Aib) and possess antibiotic properties. Being known since 1958, a constantly increasing number of peptaibiotics have been described and investigated with a particular emphasis on hypocrealean fungi. Starting from the existing online 'Peptaibol Database', first published in 1997, an exhaustive literature survey of all known peptaibiotics was carried out and resulted in a list of 1043 peptaibiotics. The gathered information was compiled and used to create the new 'The Comprehensive Peptaibiotics Database', which is presented here. The database was devised as a software tool based on Microsoft (MS) Access. It is freely available from the internet at http://peptaibiotics-database.boku.ac.at and can easily be installed and operated on any computer offering a Windows XP/7 environment. It provides useful information on characteristic properties of the peptaibiotics included such as peptide category, group name of the microheterogeneous mixture to which the peptide belongs, amino acid sequence, sequence length, producing fungus, peptide subfamily, molecular formula, and monoisotopic mass. All these characteristics can be used and combined for automated search within the database, which makes The Comprehensive Peptaibiotics Database a versatile tool for the retrieval of valuable information about peptaibiotics. Sequence data have been considered as to December 14, 2012.

Heterochromatin influences the secondary metabolite profile in the plant pathogen Fusarium graminearum.

Chromatin modifications and heterochromatic marks have been shown to be involved in the regulation of secondary metabolism gene clusters in the fungal model system Aspergillus nidulans. We examine here the role of HEP1, the heterochromatin protein homolog of Fusarium graminearum, for the production of secondary metabolites. Deletion of Hep1 in a PH-1 background strongly influences expression of genes required for the production of aurofusarin and the main tricothecene metabolite DON. In the Hep1 deletion strains AUR genes are highly up-regulated and aurofusarin production is greatly enhanced suggesting a repressive role for heterochromatin on gene expression of this cluster. Unexpectedly, gene expression and metabolites are lower for the trichothecene cluster suggesting a positive function of Hep1 for DON biosynthesis. However, analysis of histone modifications in chromatin of AUR and DON gene promoters reveals that in both gene clusters the H3K9me3 heterochromatic mark is strongly reduced in the Hep1 deletion strain. This, and the finding that a DON-cluster flanking gene is up-regulated, suggests that the DON biosynthetic cluster is repressed by HEP1 directly and indirectly. Results from this study point to a conserved mode of secondary metabolite (SM) biosynthesis regulation in fungi by chromatin modifications and the formation of facultative heterochromatin.

Marker peptide selection for the determination of hazelnut by LC-MS/MS and occurrence in other nuts.

The aim of this work was identifying and selecting hazelnut marker peptides and subsequently developing a complementary method of common immunoassay for the detection of hazelnut. For this purpose, at first, an in silico digestion of three major hazelnut allergens (Cor a 8, Cor a 9 and Cor a 11) was performed to get information about expected peptides. After extraction and trypsin digestion of hazelnut proteins, the samples were measured with tandem mass spectrometry (MS/MS) by direct infusion, which led to identification of 14 peptides. Eight of them with the highest MS signal were synthesized and used as standards for developing a liquid chromatography (LC)-MS/MS method in selected reaction monitoring (SRM) mode. Since almost all food allergens derived from nuts belong to the seed storage protein family and have homologue structure, a Basic Local Alignment Search Tool (BLAST) search was performed to identify the hazelnut specificity of the developed method. According to BLAST, only one peptide occurs in three other nuts, and the remaining seven selected peptides are hazelnut specific. Additionally to hazelnut, the eight other listed nuts in Directive 2003/89/EC as allergen were extracted, digested and measured with the developed method to prove the BLAST results. The analytical data confirmed that six peptides are hazelnut specific, on the contrary to anti-hazelnut antibodies, which showed cross-reactivities to all other nut extracts. Comparing these results, it could be shown that with this LC-MS/MS method in SRM mode, the specific detection of hazelnut is possible.

Selection of possible marker peptides for the detection of major ruminant milk proteins in food by liquid chromatography-tandem mass spectrometry.

The aim of this work was the determination of peptides, which can function as markers for identification of milk allergens in food samples. Emphasis was placed on two casein proteins (α- and ß-casein) and two whey proteins (α-lactalbumin and ß-lactoglobulin). In silico tryptic digestion provided preliminary information about the expected peptides. After tryptic digestion of four milk allergens, the analytical data obtained by combination of reversed-phase high performance liquid chromatography and quadrupole tandem mass spectrometry (LC-MS/MS) led to the identification of 26 peptides. Seven of these peptides were synthesized and used for calibration of the LC-MS/MS system. Species specificity of the selected peptides was sought by BLAST search. Among the selected peptides, only LIVTQTMK from ß-lactoglobulin (m/z 467.6, charge 2+) was found to be cow milk specific and could function as a marker. Two other peptides, FFVAPFPEVFGK from α-casein (m/z 693.3, charge 2+) and GPFPIIV from ß-casein (m/z 742.5, charge 1+), occur in water buffalo milk too. The other four peptides appear in the milk of other species also and can be used as markers for ruminant species milk. Using these seven peptides, a multianalyte MS-based method was developed. For the establishment of the method, it was applied at first to different dairy samples, and then to chocolate and blank samples, and the peptides could be determined down to 1 ng/mL in food samples. At the end, spiked samples were measured, where the target peptides could be detected with a high recovery (over 50%).

Identification and profiling of volatile metabolites of the biocontrol fungus Trichoderma atroviride by HS-SPME-GC-MS.

In the present study we describe a method, which is based on solid phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) and which can be used for the profiling of microbial volatile organic compounds (MVOCs) in the headspace (HS) of cultures of filamentous fungi. The method comprises the following successive steps: 1. growth of the fungus on a solid culture medium directly in headspace vials, 2. measurement of volatiles by HS-SPME-GC-MS, 3. deconvolution of mass spectra, 4. identification of volatiles by comparison of measured, deconvoluted mass spectra and linear temperature programmed retention indices (LTPRI) on two stationary GC phases with database entries and LTPRI published in the literature, and 5. profiling of the identified MVOCs. The developed method was successfully applied to cultures of the biocontrol fungus Trichoderma atroviride. An in-house library consisting of mass spectra and LTPRI values of fungal VOCs was established and used to study the profiles of MVOCs of this fungus. In total, 25 different MVOCs were identified by applying strict criteria (spectral match factor at least 90% and a maximum relative deviation of LTPRI of +/-2% from literature values). The MVOCs were assigned to the compound classes of alcohols, ketones, alkanes, furanes, pyrones (mainly the bioactive 6-pentyl-alpha-pyrone), mono- and sesquiterpenes, 13 of which have never been reported to be produced by Trichoderma spp. before. Eleven of these volatiles have been additionally confirmed using authentic standards. Finally, time course experiments and cultivation of T. atroviride in the presence of the mycotoxin fusaric acid demonstrated the potential of the method to study the dynamics of MVOC profiles as well as the effect of different environmental/biological conditions on the expression of MVOCs of filamentous fungi.

Characterisation of the peptaibiome of the biocontrol fungus Trichoderma atroviride by liquid chromatography/tandem mass spectrometry.

The present study describes the liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based screening and characterisation of linear antibiotic alpha-aminoisobutyric acid (Aib)-containing non-ribosomal peptides (NRP) in culture samples of the filamentous fungus Trichoderma atroviride ATCC 74058. Fungal culture filtrates were enriched by solid-phase extraction (SPE) and separated by reversed-phase high-performance liquid chromatography (HPLC), prior to mass spectrometric (MS) and tandem mass spectrometric (MS/MS) analysis on a triple quadrupole-linear ion trap tandem mass spectrometer. A workflow consisting of two alternative screening strategies was applied to search for NRP. Various MS full scan and MS/MS measurement modes led to the identification of 16 trichorzianines and diagnostic in-source fragment ions of another four trichorzianines. Furthermore, we detected 15 novel Aib-containing peptides with putative molecular weights ranging from 951.7 to 1043.7 g/mol (monoisotopic masses), composed of up to 9 amino acids. While the amino acid sequences of the novel peptaibiotics showed typical microheterogeneity and consisted of the amino acids Leu/Ile, Aib, Ser, Val/Iva, Gly, Ac-Aib, Tyr and Phe, the mass increments at the C-termini of the peptides were not assignable to any residues described in the literature. The amino acid sequences were confirmed and structure proposals made for both molecule termini by high-resolution MS and MS/MS analysis. We propose the group name 'trichoatrokontins' for the newly identified peptaibiotics. As no other peptaibiotics were found in the culture samples, the peptaibiome of the investigated strain of T. atroviride consists of at least 20 trichorzianines and 15 trichoatrokontins.

Profiling of trichorzianines in culture samples of Trichoderma atroviride by liquid chromatography/tandem mass spectrometry.

Peptaibols are bioactive linear peptides of 5-20 amino acid residues and contain specific non-proteinogenic amino acids such as alpha-aminoisobutyric acid (Aib). They are antibiotic secondary metabolites of moulds belonging predominantly to the genus Trichoderma, some species of which are successfully used as biocontrol organisms to fight against plant diseases. In the present study we developed a profiling method for the relative quantification of 16 trichorzianine peptaibols in culture samples of T. atroviride and the comparison of their expression patterns by liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS). The method is based on selected reaction monitoring (SRM) in a triple-quadrupole tandem mass spectrometer using three SRM transitions per compound. The trichorzianines were enriched by solid-phase extraction (SPE) on C(18) cartridges. SPE recoveries were evaluated for diluted trichorzianine standard solutions and ranged from 72-97%. Suppression of the ionisation of the peptaibols in the ESI source ranged from 67-128% for most of the trichorzianines in culture filtrates of two different strains of T. atroviride and in spiked culture medium. In the case of trichorzianines TA Vb, TA VIa and TA VIb the presence of matrix components in the fungal culture samples caused a reduction of the SRM signal, with intensities between 34% and 56% relative to pure standard solutions. Finally, the profiling method was successfully applied to culture samples of T. atroviride P1 wild-type and two deletion mutants showing different trichorzianine expression patterns characteristic for the investigated fungal strains. This is the first LC-SRM profiling method for peptaibols for the investigation of peptaibol expression patterns in fungal culture samples.

Signaling via the Trichoderma atroviride mitogen-activated protein kinase Tmk 1 differentially affects mycoparasitism and plant protection.

Trichoderma atroviride is a mycoparasite of a number of plant pathogenic fungi thereby employing morphological changes and secretion of cell wall degrading enzymes and antibiotics. The function of the tmk 1 gene encoding a mitogen-activated protein kinase (MAPK) during fungal growth, mycoparasitic interaction, and biocontrol was examined in T. atroviride. Deltatmk 1 mutants exhibited altered radial growth and conidiation, and displayed de-regulated infection structure formation in the absence of a host-derived signal. In confrontation assays, tmk 1 deletion caused reduced mycoparasitic activity although attachment to Rhizoctonia solani and Botrytis cinerea hyphae was comparable to the parental strain. Under chitinase-inducing conditions, nag 1 and ech 42 transcript levels and extracellular chitinase activities were elevated in a Deltatmk 1 mutant, whereas upon direct confrontation with R. solani or B. cinerea a host-specific regulation of ech 42 transcription was found and nag 1 gene transcription was no more inducible over an elevated basal level. Deltatmk 1 mutants exhibited higher antifungal activity caused by low molecular weight substances, which was reflected by an over-production of 6-pentyl-alpha-pyrone and peptaibol antibiotics. In biocontrol assays, a Deltatmk 1 mutant displayed a higher ability to protect bean plants against R. solani.