Norbert Stoppacher

Required metrological tools, such as higher order reference measurements procedures, pure substance and matrix certified reference materials, are established for small well defined molecules. Difficulties still remain in the provision of SI traceable standards in the area of larger biomolecules such as peptides/proteins. The provision of Primary Calibration Reference Services has been identified as a core technical competency for National Measurement Institutes (NMIs). A concept has been elaborated by the Focus Group I on peptide/protein purity for the strategic planning of ongoing Key Comparisons of the Protein Analysis Working Group (PAWG) within the Comité Consultatif pour la Quantité de Matière (CCQM). The assignment of the mass fraction content of high purity peptide/protein materials will be the subject of the CCQM-K115/P55.2 comparison series to directly support NMI services and certified reference materials currently being provided by NMIs.

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 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.

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'.

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 a...

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.

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.

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.

It is common practice to quantify the mass concentration of a peptide solution through quantitative determination of selected chemically stable amino acids produced following complete hydrolysis of the parent peptide. This is because there is generally an insufficient quantity of material available to allow for the obvious alternative of a direct purity analysis characterization of the parent peptide, and the subsequent constitution of a calibration solution. However, selected accurately characterized pure peptide reference materials are required to establish reference points for the dissemination of metrologically traceable measurements and to develop reference measurement systems for laboratory medicine. In principle, purity assignment of a peptide can be performed by using the so-called mass balance approach, by employing a range of analytical techniques to obtain an estimate of the mass fraction content of all impurities present in the intact peptide, and by utilizing the difference from the theoretical limit value to assign the mass fraction content of the main peptide. Liquid chromatography-high-resolution tandem mass spectrometry (LC-hrMS/MS) is a key technique for the detection, identification, and determination of structurally related impurities present in a peptide material, and experiments characterizing the model peptide hormone angiotensin I (ANG I) are described in the present work. Degradation products that were generated from ANG I after storage at elevated temperatures were screened. The formation of peptide fragments such as ANG II or ANG III was determined by comparison of measured mass values with calculated mass values. The use of a data-dependent acquisition technique enabled the detection and structural characterization of ANG II and other peptide fragments as major impurities in the same LC-hrMS/MS analysis run. Subsequent quantification using external calibration allowed the mass fraction of the major impurities in a candidate reference material to be estimated as 10.4 mg/g. Failure to correct for these impurities would lead to a 1% error in the determination of the concentration of the peptide in solution by amino acid analysis techniques.