Jørn Smedsgaard

Aligning of single and multiple wavelength chromatographic profiles for chemometric data analysis using correlation optimised warping

The use of chemometric data processing is becoming an important part of modern chromatography. Most chemometric analyses are performed on reduced data sets using areas of selected peaks detected in the chromatograms, which means a loss of data and introduces the problem of extracting peak data from the chromatographic profiles. These disadvantages can be overcome by using the entire chromatographic data matrix in chemometric analyses, but it is necessary to align the chromatograms, as small unavoidable differences in experimental conditions causes minor changes and drift. Previous aligning methods either fail to utilise the entire data matrix or rely on peak detection, thus having the same limitations as the commonly used chemometric procedures. The method presented uses the entire chromatographic data matrices and does not require any preprocessing e.g., peak detection. It relies on piecewise linear correlation optimised warping (COW) using two input parameters which can be estimated from the observed peak width. COW is demonstrated on constructed single trace chromatograms and on single and multiple wavelength chromatograms obtained from HPLC diode detection analyses of fungal extracts1.

Fungal metabolite screening: database of 474 mycotoxins and fungal metabolites for dereplication by standardised liquid chromatography-UV-mass spectrometry methodology

A standardised LC-UV-MS micro-scale method for screening of fungal metabolites and mycotoxins in culture extracts is presented. The paper includes data for detection and dereplication of > 400 fungal metabolites to facilitate detection and identification when standards are not available. The data also shows the types of components that can be analysed by positive electrospray (ESI+) mass spectrometry (MS) along with common fragments and adducts of these, as well as giving suggestions on whether UV or ESI+-MS methods should be used. Examples of dereplication of penitrems and macro-cyclic ichothecenes, and detection of several novel compounds are shown. This was done by UV spectroscopy combined with accurate mass determination of adduct and fragment ions obtained by high-resolution orthogonal time-of-flight MS.

Micro-scale extraction procedure for standardized screening of fungal metabolite production in cultures

A simple and rapid standardized micro-scale extraction procedure has been developed to prepare extracts from fungal cultures for high-performance liquid chromatographic (HPLC) analysis. The method is based on ultrasonic extraction of three 6-mm plugs cut from a culture using 0.5 ml of solvent followed by a simple solvent change, filtration and injection. Approximately 5 min of work is involved in the extraction and work-up process and the extract can prepared for HPLC analysis within 60-70 min. The method has been used for determination of chromatographic metabolite profiles from 395 fungal isolates, including all terverticillate Penicillium species, cultivated on both Czapek Yeast Autolysate agar and Yeast Extract Sucrose agar. The concentration of the extracts proved to be sufficient to determine all secondary metabolites reported to be produced by these species using HPLC with diode array detection. These findings were confirmed by analyses of 132 pure metabolite standards.

Global metabolite analysis of yeast: evaluation of sample preparation methods

Sample preparation is considered one of the limiting steps in microbial metabolome analysis. Eukaryotes and prokaryotes behave very differently during the several steps of classical sample preparation methods for analysis of metabolites. Even within the eukaryote kingdom there is a vast diversity of cell structures that make it imprudent to blindly adopt protocols that were designed for a specific group of microorganisms. We have therefore reviewed and evaluated the whole sample preparation procedures for analysis of yeast metabolites. Our focus has been on the current needs in metabolome analysis, which is the analysis of a large number of metabolites with very diverse chemical and physical properties. This work reports the leakage of intracellular metabolites observed during quenching yeast cells with cold methanol solution, the efficacy of six different methods for the extraction of intracellular metabolites, and the losses noticed during sample concentration by lyophilization and solvent evaporation. A more reliable procedure is suggested for quenching yeast cells with cold methanol solution, followed by extraction of intracellular metabolites by pure methanol. The method can be combined with reduced pressure solvent evaporation and therefore represents an attractive sample preparation procedure for high‐throughput metabolome analysis of yeasts. Copyright

A proposed framework for the description of plant metabolomics experiments and their results

The study of the metabolite complement of biological samples, known as metabolomics, is creating large amounts of data, and support for handling these data sets is required to facilitate meaningful analyses that will answer biological questions. We present a data model for plant metabolomics known as ArMet (architecture for metabolomics). It encompasses the entire experimental time line from experiment definition and description of biological source material, through sample growth and preparation to the results of chemical analysis. Such formal data descriptions, which specify the full experimental context, enable principled comparison of data sets, allow proper interpretation of experimental results, permit the repetition of experiments and provide a basis for the design of systems for data storage and transmission. The current design and example implementations are freely available (http://www.armet.org/). We seek to advance discussion and community adoption of a standard for metabolomics, which would promote principled collection, storage and transmission of experiment data.

Biochemical characterization of ochratoxin A-producing strains of the genus Penicillium.

ABSTRACT In order to explore the biochemical scope of ochratoxin A-producing penicillia, we screened 48 Penicillium verrucosumisolates for the production of secondary metabolites. Fungal metabolites were analyzed by high-pressure liquid or gas chromatography coupled to diode array detection or mass spectrometry. The following metabolites were identified: ochratoxins A and B, citrinin, verrucolones, verrucines, anacines, sclerotigenin, lumpidin, fumiquinazolines, alantrypinones, daldinin D, dipodazine, penigequinolines A and B, 2-pentanone, and 2-methyl-isoborneol. By use of average linking clustering based on binary (nonvolatile) metabolite data, the 48 isolates could be grouped into two large and clearly separated groups and a small outlying group of four non-ochratoxin-producing isolates. The largest group, containing 24 isolates, mainly originating from plant sources, included the type culture of P. verrucosum. These isolates produced ochratoxin A, verrucolones, citrinin, and verrucines and had a characteristic dark brown reverse color on yeast extract-sucrose agar medium. Almost all of a group of 20 isolates mainly originating from cheese and meat products had a pale cream reverse color on yeast extract-sucrose agar medium and produced ochratoxin A, verrucolones, anacines, and sclerotigenin. This group included the former type culture of P. nordicum. We also found that P. verrucosum isolates and threeP. nordicum isolates incorporated phenylalanine into verrucine and lumpidin metabolites, a finding which could explain why those isolates produced relatively lower levels of ochratoxins than did most isolates of P. nordicum.

Using direct electrospray mass spectrometry in taxonomy and secondary metabolite profiling of crude fungal extracts

Important information about sample composition can be optained within a few minutes by injecting a complex mixture like a crude extract of a fungal culture prepared for standard HPLC analysis directly into an electrospray mass spectrometer using a FIA-ESMS type of setup. The limited fragmentation and high sensitivity of ESMS where used in this study to provide ‘mass profiles’ from ethylacetate/methanol/chloroform extracts from cultures of 10 the most common Penicillium species associated with stored cereals. The analytical parameters were optimized to reduce fragmentations and reactions in ESMS hence only the protonated or sodiated ions were observed for most compounds. The analysis demonstrated that ions corresponding to the protonated molecular ions (M + H+) from most of the known secondary metabolites and mycotoxins produced these species by could observed in the ESMS spectra. A number of other distinct species specific ion were observed as well. The 10 different species could be discriminated either by ions corresponding to known or unknown metabolites. By creating a database of mass spectra obtained from analysis of different species using the facility included in standard MS software it was possible to use a simple library search to identify most of the species included in this study on the basis of their mass spectra.

The P450 monooxygenase BcABA1 is essential for abscisic acid biosynthesis in Botrytis cinerea.

ABSTRACT The phytopathogenic ascomycete Botrytis cinerea is known to produce abscisic acid (ABA), which is thought to be involved in host-pathogen interaction. Biochemical analyses had previously shown that, in contrast to higher plants, the fungal ABA biosynthesis probably does not proceed via carotenoids but involves direct cyclization of farnesyl diphosphate and subsequent oxidation steps. We present here evidence that this “direct” pathway is indeed the only one used by an ABA-overproducing strain of B. cinerea. Targeted inactivation of the gene bccpr1 encoding a cytochrome P450 oxidoreductase reduced the ABA production significantly, proving the involvement of P450 monooxygenases in the pathway. Expression analysis of 28 different putative P450 monooxygenase genes revealed two that were induced under ABA biosynthesis conditions. Targeted inactivation showed that one of these, bcaba1, is essential for ABA biosynthesis: ΔBcaba1 mutants contained no residual ABA. Thus, bcaba1 represents the first identified fungal ABA biosynthetic gene.

Identification of an Abscisic Acid Gene Cluster in the Grey Mold Botrytis cinerea

ABSTRACT Like several other phytopathogenic fungi, the ascomycete Botrytis cinerea is known to produce the plant hormone abscisic acid (ABA) in axenic culture. Recently, bcaba1, the first fungal gene involved in ABA biosynthesis, was identified. Neighborhood analysis of bcaba1 revealed three further candidate genes of this pathway: a putative P450 monooxygenase-encoding gene (bcaba2), an open reading frame without significant similarities (bcaba3), and a gene probably coding for a short-chain dehydrogenase/reductase (bcaba4). Targeted inactivation of the genes proved the involvement of BcABA2 and BcABA3 in ABA biosynthesis and suggested a contribution of BcABA4. The close linkage of at least three ABA biosynthetic genes is strong evidence for the presence of an abscisic acid gene cluster in B. cinerea.

Emericella astellata, a new producer of aflatoxin B1, B2 and sterigmatocystin

Aims:  To report on aflatoxin B1 and B2 production from a species of Emericella.