Probe Compound for Detecting and Isolating Enzymes and Means and Methods Using the Same - DocsLib

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(11) EP 2 230 312 A1

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 22.09.2010 Bulletin 2010/38 C12Q 1/00 (2006.01) G01N 33/542 (2006.01)

(21) Application number: 09003977.7

(22) Date of filing: 19.03.2009

(84) Designated Contracting States: • Waliczek, Agnes AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 38100 Braunschweig (DE) HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL • Ferrer, Manuel PT RO SE SI SK TR 28007 Madrid (ES) Designated Extension States: • Beloqui, Ana AL BA RS 28049 Madrid (ES) • Guazzaroni, Maria E. (71) Applicants: 28049 Madrid (ES) • Helmholtz-Zentrum für Infektionsforschung • Vieites, Jose M. GmbH 28049 Madrid (ES) 38124 Braunschweig (DE) • Pazos, Florencio • Institute of Catalysis (CSIC) 28049 Madrid (ES) 28049 Madrid (ES) • Lopez de Lacey, Antonio 28049 Madrid (ES) (72) Inventors: • Fernandez, Victor M. • Golyshin, Peter N. 28049 Madrid (ES) 38302 Wolfenbüttel (DE) • Golyshina, Olga V. (74) Representative: Forstmeyer, Dietmar et al 38302 Wolfenbüttel (DE) Boeters & Lieck • Timmis, Kenneth N. Oberanger 32 38300 Wolfenbüttel (DE) D-80331 München (DE) • Chernikova, Tatyana 38102 Braunschweig (DE)

(54) Probe compound for detecting and isolating enzymes and means and methods using the same

(57) The present invention relates to a probe com- tion which leads to reproducible enzymatic reactions with pound that can comprise any substrate or metabolite of the substrates. These specific enzymatic reactions trig- an enzymatic reaction in addition to an indicator compo- ger the indicator (e.g. a fluorescence signal) and bind the nent, such as, for example, a fluorescence dye, or the enzymes to the respective cognate substrates. Moreo- like. Moreover, the present invention relates to means ver, the invention relates to means for isolating enzymes for detecting enzymes in form of an array, which com- in form of nanoparticles coated with the probe compound prises any number of probe compounds of the invention of the invention. The immobilisation of the cognate sub- which each comprise a different metabolite of intercon- strates or metabolites on the surface of nanoparticles by nected metabolites representing the central pathways in means of the probe compounds allows capturing and all forms of life. Moreover, the present invention relates isolating the respective enzyme, e.g. for subsequent se- to a method for detecting enzymes involving the appli- quencing. cation of cell extracts or the like to the array of the inven- EP 2 230 312 A1

Printed by Jouve, 75001 PARIS (FR) EP 2 230 312 A1

Description

Background of the Invention

5 [0001] The present invention relates to a probe compound for detecting and isolating enzymes, to a method for producing the probe compound, to means for detecting enzymes and means for isolating enzymes, to a method for producing the means for detecting and isolating enzymes, and to a method for detecting and isolating enzymes using the same. [0002] In more detail, the present invention relates to a probe compound that can comprise any substrate or metabolite 10 of an enzymatic reaction in addition to an indicator component, such as, for example, a fluorescence dye, or the like. Moreover, the present invention relates to means for detecting enzymes in form of an array. The array according to the present invention comprises any number of probe compounds of the invention which each comprise a different metabolite of interconnected metabolites representing the central pathways in all forms of life. Moreover, the present invention relates to a method for detecting enzymes involving the application of cell extracts or the like to the array of the invention 15 which leads to reproducible enzymatic reactions with the substrates. These specific enzymatic reactions trigger the indicator (e.g. a fluorescence signal) and bind the enzymes to the respective cognate substrates. Moreover, the invention relates to means for isolating enzymes in form of nanoparticles coated with the probe compound of the invention. The immobilisation of the cognate substrates or metabolites on the surface of nanoparticles by means of the probe compounds allows capturing and isolating the respective enzyme, e.g. for subsequent sequencing. In short, the probe compound 20 of the invention provides two new aspects: first, that only an active protein (enzyme) triggers the indicator signal, and second, that the active protein subsequently binds to the probe compound. [0003] In recent years, the new discipline of "functional genomics" has greatly accelerated the research on the genomic basis of life processes in health and disease, and has significantly improved our understanding of such processes, their regulation and underlying mechanisms. However, until relatively recently, functional genomics has been sequence- 25 centric, that is, functional assignments and metabolic network reconstructions have mostly depended on the genome sequence of the organism in question, combined with bioinformatic analyses based on homologies to known gene/ protein-relationships. In addition to the fact that a significant fraction of genes in databases available today has a questionable annotation, many are not annotated at all, which adds further uncertainties to analyses and predictions based on them. With the recent advent of "metabolomics", functional insights into the metabolic state of a cell became 30 possible independently of sequence information. However, problems of metabolite identification and quantification still exist, and the link with the cognate metabolic pathways is still heavily dependent upon sequence-based metabolic reconstructions. Furthermore, it is currently very difficult to derive global metabolic overviews of non-sequenced organisms or communities of organisms existing in an individual habitat or biotop (biocoenosis). [0004] Therefore, there exists an urgent need to solve the twin problems of the identification of metabolites and the 35 enzymes involved in their transformation and, simultaneously, to begin the critical process of rigorous annotation of yet unannotated and incorrectly annotated open reading frames (genes), and thereby improving the utility of the exponentially growing body of genome sequence information. Thus, an activity-based, annotation-independent procedure for the global assessment of cellular responses is urgently needed. [0005] "Microarrays" or "biochips" have proven to be an important and indispensable tool for the fast gain and process- 40 ing of information required in the field. Here, the term ’array’ refers to a collection of a large number of different test compounds preferably arranged in a planar plane, e.g. by attachment on a flat surface, such as a glass slide surface, or by occupying special compartments or wells provided on a plate, such as a micro-titre plate. The test compounds, which are also referred to as probes, probe compounds or probe molecules, are usually bound or immobilised on the flat surface or to the walls of a compartment, respectively. The use of arrays allows for the rapid, simultaneous testing 45 of all probe molecules with respect to their interaction with an analyte or a mixture of analytes in a sample. The analytes of the sample are often referred to as target molecules. The advantage of a planar array over a test (assay) having immobilised probe molecules on mobile elements, such as, for example, beads, is that in an array the chemical structure and/or the identity of the immobilised probe molecules is precisely defined by their location in the array surface. A specific local test signal, which is produced, for example, by an interaction between the probe molecule and the analyte molecule, 50 can accordingly be immediately assigned to a type of molecule or to a probe molecule. As evidence of an interaction between a probe molecule and an analyte molecule, it is also possible to use the enzymatic conversion of the probe by the biomolecule, with the result that a local test signal can also disappear and accordingly serves as direct evidence. Particularly in miniaturised form, arrays having biological probe molecules are also known as "biochips". [0006] Usually, the surface of the microarray having the bound probe molecules is brought into contact, over its entire 55 area, with the solution of the analyte molecules from a sample. Then, the solution is usually removed after a predetermined incubation time. Alternatively, appropriate amounts of the sample solution are filled into the respective compartments (wells) of the array. When the specific and selective interaction between the probe molecule and an analyte molecule is complete, a signal is generated at the location of the probe molecule. That signal can either be produced directly, for

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example by binding of a fluoresence-labelled biomolecule, or can be generated in further treatments with detection reagents, for example in the form of an optical or radioactive signal. Many different technical details relating to procedure and detection are well known and completely described in the art. There are numerous array protocols and processes which are adapted for automatic handling by corresponding (robotic) apparatuses, thus allowing for high reliability and 5 reproducibility of information gain and processing. [0007] Examples of known arrays in the prior art are nucleic acid arrays of DNA fragments, cDNAs, RNAs, PCR products, plasmids, bacteriophages and synthetic PNA oligomers, which are selected by means of hybridisation, with formation of a double-strand molecule, to give complementary nucleic acid analytes. In addition, protein arrays of anti- bodies, proteins expressed in cells or phage fusion proteins (phage display) play an important part. Furthermore, com- 10 pound arrays of synthetic peptides, analogues thereof, such as peptoids, oligocarbamates or generally organic chemical compounds, are known, which are selected, for example, by means of binding to affinitive protein analytes or other analytes by means of enzymatic reaction. Moreover, arrays of chimaeras and conjugates of the said probe molecules have been described. [0008] DNA microarray technology has a vast potential for improving the understanding of microbial systems. Micro- 15 array-based genomic technology is a powerful tool for viewing the expression of thousands of genes simultaneously in a single experiment. While this technology was initially designed for transcriptional profiling of a single species, its applications have been dramatically extended to environmental applications in recent years. The use of microarrays to profile metagenomic libraries may also offer an effective approach for characterizing many clones rapidly. As an example, a fosmid library was obtained and further arrayed on a glass slide. This format is referred to as a metagenome microarray 20 (MGA). In the MGA format, the ’probe’ and ’target’ concept is a reversal of those of general cDNA and oligonucleotide microarrays: targets (fosmid clones) are spotted on a slide and a specific gene probe is labelled and used for hybridization. This format of microarray may offer an effective metagenome-screening approach for identifying clones from metagenome libraries rapidly without the need of laborious procedures for screening various target genes. [0009] However, one of the greatest challenges in using microarrays for analyzing environmental samples is the low 25 detection sensitivity of microarray-based hybridization in combination with the low biomass often present in samples from environmental settings. Microarrays for expression profiling can be divided into two broad categories, microarrays based on the deposition of preassembled DNA probes (cDNA microarrays) and those based on in situ synthesis of oligonucleotide probes (e.g. Affymetrix arrays, oligonucleotide microarrays). Applications employing DNA microarrays include, for example, the characterization of microbial communities from environmental samples such as soil and water. 30 Various types of DNA microarrays have been applied to study the microbial diversity of various environments. Those include, for example, oligonucleotides, cDNA (PCR amplified DNA fragments), and whole genome DNA. [0010] One of the major problems associated with nucleic acid-based micro-arrays is derived from the short half-life of mRNA, and that mRNA in bacteria and archaea usually comprise only a small fraction of total RNA. Moreover, the study of the gene expression from an environmental sample using DNA microarrays is a challenging task. First, the 35 sensitivity may often be a part of the problem in PCR-based cDNA microarrays, since only genes from populations contributing to more than 5% of the community DNA can be detected. Second, samples often contain a variety of environmental contaminants that affects the quality of RNA and DNA hybridization and makes it difficult to extract undegraded mRNA. The specificity of the extraction method plays a central role and should vary depending on the site of sampling, as there must be sufficient discrimination between probes. However, there is a promising perspective for 40 microarrays in determining the relative abundance of a microorganism bearing a specific functional gene in a complex environment. [0011] However, specificity is a key issue, since one needs to distinguish the differences in hybridization signals due to population abundance from those due to sequence divergence. Furthermore, annotation and the comprehensive functional characterization of proteins or RNA molecules remain difficult, error-prone processes, but systems microbi- 45 ology relies heavily on a thorough understanding of the functions of gene products. [0012] At the moment, after DNA micro-arrays, the peptide arrays are the most popular. In this kind of arrays, peptides with different chemical composition are synthesised and immobilized on glass slides. The peptides may also contain a marker, such as a fluorescence dye marker (e.g. a fluorescent cyanine dye known under the name ’Cy3’), but here the detection method is only based on the lowered fluorescence obtained with a protein bound to the molecule. There is no 50 enzymatic reaction necessary for the signal, so un-specific bindings may occur and trigger a signal, which may lead to incorrect assignments. Further, there is no possibility to reconstruct metabolic networks. [0013] Another array alternative is to bind proteins to a slide. Such system is usually not based on the detection of a fluorescence signal, but rather on the utilization of surface Plasmon resonance. This system has been exploited for the analysis of molecular interactions, i.e. protein-protein or molecule-protein interactions. 55 [0014] In view of the problems encountered in the prior art, the present invention is therefore based on the object of providing a novel probe compound, which allows for the testing of a reactive interaction of an enzyme with a small molecule or enzymatic substrate. The probe compound should allow for the easy linkage of all small molecules or substrates necessary for the life functions of an organism or communities living in a habitat (biocoenosis). Thus, a

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plurality of probe compounds should allow for the construction of a ’reactome array’ or microarray which allows for the testing of all life supporting enzymatic reactions of an organism or community simultaneosly. Particularly, the probe compound should provide a highly sensitive, accurate, reproducible, and robust high-throughput tool for a genome-wide analysis of the metabolic status of an organism or community. In this context, the term "genome-wide analysis" means 5 an analysis that is independent of genome sequence. Moreover, the probe compound should also allow for use in the isolation of an enzyme so that said enzyme may be further analysed or identified in a subsequent step. Moreover, the probe compound should also allow for the identification of small molecules, substrates and/or metabolites which are metabolised by an organism or community, thus allowing the identification of biologic pathways or the direct comparison of the reactomes of different organisms, which might be applied in the search for new targets for drug-screening. 10 Summary of the Invention

[0015] The object of the invention is solved by a probe compound comprising a transition metal complex and a reactive component comprising a test component and an indicator component, wherein the test component and the indicator 15 component are linked to form the reactive component, and wherein the reactive component is linked to the transition metal complex. The probe compound of the invention provides a means for testing of a reactive interaction of an enzyme with a small molecule or enzymatic substrate. The probe compound may be readily used in combination with all small molecules or substrates necessary for the life functions of an organism or communities living in a habitat (biocoenosis). Further, the probe compound provides a highly sensitive, accurate, reproducible, and robust high-throughput tool for a 20 genome-wide analysis of the metabolic status of an organism or community. It allows the fast and reliable detection of a substrate specific enzyme interaction. Moreover, the probe compound may be readily used to detect the involvement of one enzymatic substrate in different metabolic pathways. Moreover, the probe compound provides a means to im- mobilise a substrate-specific enzyme, which can be advantageously used to isolate this enzyme from a sample. [0016] A preferred embodiment of the probe compound of the invention can be illustrated by the following general 25 formula (1):

30

35 wherein AC represents an optional anchoring component, MC represents the transition metal complex, TC represents the test component, IC represents the indicator component, and LAC-MC,LMC-TC,LTC-IC and LMC-IC each independently represents an optional linker moiety between the respective components indicated by the subscripts. [0017] In more detail, the present invention relates to a probe compound that can comprise any substrate or metabolite 40 of an enzymatic reaction in addition to an indicator component, such as, for example, a fluorescence dye, or the like. In short, the probe compound of the invention provides two new aspects: first, that only an active protein (enzyme) triggers the indicator signal, and second, that the active protein subsequently binds to the probe compound. [0018] The object of the invention is also solved by a method for preparing the probe compound of the invention. The inventive method provides a versatile method for preparing all embodiments of the probe compound. Moreover, the 45 method of the invention can be used for the identical and reproducible production of probe compounds comprising different enzymatic substrates, which allows for the ready use in automatic processes, such as parallel synthesis or the like. [0019] The object is also solved by an array which comprises a plurality of different probe compounds of the invention. The array (which is sometimes referred to as "reactome array" in the following) can be used for the simultaneous detection 50 of all reactive interactions between the probe compounds and analyte molecules (enzymes) from a sample. The array also provides a fast and reliable way to detect all metabolic pathways active in an organism or community, and may be used advantageously for an activity-based, annotation-independent procedure for the global assessment of cellular responses. The array can include a number of interconnected metabolites representing central pathways in all forms of life. The application of cell extracts to the array leads to reproducible enzymatic reactions with substrates that trigger 55 the indicator signal and bind enzymes to cognate substrates. [0020] The invention also provides a method for producing an array according to the invention, which allows for a versatile, fast and reproducible production of arrays according to the invention. [0021] Moreover, the object is also solved by an isolation means comprising a probe compound according to the

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invention and a nanoparticle, preferably a magnetic nanoparticle. The isolation means according to the invention allows for the substrate specific interaction and binding of an enzyme which can then be isolated by means, such as, for example, filtration, gravitation force (centrifugation), an external magnetic force, or the like. The invention also provides a method for producing an isolation means according to the invention. The immobilisation of the cognate substrates or 5 metabolites on the surface of nanoparticles by means of the probe compounds allows capturing and isolating the respective enzyme, e.g. for subsequent sequencing. [0022] Moreover, the object is solved by a method for detecting enzymes using the probe compound according to the invention, or the array according to the invention, as well as by a method for isolating enzymes, using the isolation means according to the invention. 10 [0023] The particular subject-matter of the invention and its preferred embodiments will be described in more detail in the following description as well as in the examples and figures attached thereto.

Brief Description of the Figures

15 [0024]

Figure 1 is a schematic representation of the reactome strategy, comprising three major parts:

steps 1 and 2: extensive data and synthetic mining effort to produce a library of metabolites that can be included 20 in the probe compound of the invention and arrayed in glass slides in a spatial addressable manner; steps 3 and 4: detection and analysis of chemical reactions through application of cell lysates of microbial cultures, or libraries of metagenomic clones of microbial communities; step 5: detection of proteins acting against particular metabolites immobilized in a gold nano-particle followed by protein sequencing.

25 The reactome reaction principle is shown in the central part of the figure (step 3). The general strategy showing the successive synthetic steps is displayed in Figure 2(B).

Figure 2: Part A shows a schematic representation of an example of the probe compound according to the invention,

wherein the transition metal complex is represented by the cobalt (II) complex of Nα,Nα-bis-(carboxymethyl)-L-lysine 30 (ANTA-Co(II)); the anchoring component is represented by a Poly(A) chain linked via a phosphoric acid amide bond to the transition metal complex; the test component (SM) is represented by N,N-dimethylamine which is linked via aminobutyric acid as a linker moiety to a Cy3 fluorescence dye molecule representing the indicator component (Cy3), wherein the so-formed reactive component is linked via two histidine residues (His) to the central cobalt atom of the transition metal complex by two individual coordination bonds (illustrated by dashed double lines); wherein 35 one of the histidines is linked to the to test component and the other is linked to the indicator component, respectively. The dashed lines between test compound and histidine indicate two different attachments of histidine to the same substrate, which allow for its detection in two different metabolic pathways; Part B is a schematic representation of the synthetic strategy for the production of the probe compound comprising Cy3-labelled metabolites: the production of a Cy3-metabolite-probe compound having a poly(A) tail (bottom left), 40 and the linkage of a Cy3-metabolite-probe compound to gold nanoparticles (bottom right).

Figure 3 shows Dose-response curves determined with pure E. coli β-Gal and Cy3-linked X-Gal. (A) Substrate dose response with fixed amount of β-Gal (5 ng/ml); (B) β-Gal dose response with fixed amount of Cy3-modified X-Gal (2.52 pmol/ml). For each experiment, normalized intensity values and fit curves were scaled relative to the 45 maximum asymptotic values of the fit.

Figure 4 shows the Receiver Operating Characteristic (ROC) curve of the array. The ROC shows the capacity of the array to discriminate compounds potentially metabolised by P. putida from those which are not metabolised. The "true positive rate" (TRP) is plotted on the Y-axis against the "false positive rate" (FRP) on the X-axis. The 50 diagonal line represents the discriminative power of a random method.

Figure 5 shows an overall comparison of metabolites transformed by lysates of the three communities KOL, VUL and L’A. (A) Pairwise comparisons of the compounds metabolized by lysates of the KOL, VUL and L’A metagenome libraries. (B) Overall comparison of compounds metabolized by the three libraries. (C) Pairwise comparisons of the 55 compounds metabolized by lysates of the individual metagenome libraries and that of P. putida.

Figure 6 shows dose-response curves determined with purified P. putida KT2440 proteins (A) and metagenomic proteins (B). Left and right figures represent protein and molecule dose responses. Results shown are the average

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of three independent assays, and were corrected for background signal. Results are not fitted to any model. The spotting process was carried out using a MicroGrid II micro-arrayer (Biorobotics) by spotting 0.25 nL droplets of SMs-Cy3 solutions (spot size 400 mm diameter with concentrations ranging from 0 to 0.25 pmol/ml) and further arrayed with 60 ml of a solution of pure enzyme (from 16 to 90 ng/ml in PBS buffer, depending on the enzyme used) 5 (left column) or by spotting 0.25 nL droplets of SMs-Cy3 solution (spot size 400 mm diameter with concentration of 0.4 pmol/ml) and further arrayed with 60 ml of solution of pure enzyme at different concentrations (from 0 to 6000 mg/ml in PBS buffer). Signals were analyzed and quantified using GenePix pro 4.1 software (Axon). As shown, Cy3 fluorescence emission increased with increasing the amount of both protein and substrate, whereas inactive proteins did not (see below). (C) FTIR spectrum of L’A62 hydrogenase. Inset shows the H2-uptake activity using methyl 10 viologen as acceptor.

Detailed Description of the Invention

[0025] The present invention provides a probe compound comprising a transition metal complex and a reactive com- 15 ponent comprising a test component and an indicator component, wherein the test component and the indicator component are linked to form the reactive component, and wherein the reactive component is linked to the transition metal complex. The probe compound of the invention thus comprises the following components: a transition metal complex and a reactive component. The reactive component in turn comprises the following components: a test component and an indicator component, which are linked to form the reactive component. Further, the reactive component is linked to 20 the transition metal complex. [0026] A preferred embodiment of the probe compound of the invention can be illustrated by the following general formula (1):

25

30

wherein AC represents an optional anchoring component, MC represents the transition metal complex, TC represents the test component, IC represents the indicator component, and LAC-MC,LMC-TC,LTC-IC and LMC-IC each independently 35 represents an optional linker moiety between the respective components indicated by the subscripts. [0027] Herein, the term "linked" means that two components are connected with each other by means of at least one chemical bond, preferably by one, two, or three chemical bonds, and most preferred by exactly one chemical bond. A chemical bond indicates any chemical bond known in the art, such as, for example, an ionic bond, a covalent bond, including a single bond, a double bond, a triple bond, or another suitable multiple bond, and a hydrogen bond, and the 40 like. Preferably, the "link" or chemical bond between two components is a direct bond between the two components; but it may also comprise a suitable linker atom or molecule, if necessary. In some cases, one or both of the components to be linked will have to be activated in order to allow for the formation of a chemical bond or link. The procedures and means to be applied for such activation and formation of chemical bonds are standard knowledge of the field, and any skilled person will immediately know how to proceed. 45 [0028] Since the individual components are required to be linked (or chemically bonded) to each other as specified, the term "component" is used to indicate the respective parts of the probe compound which are characterised by their respective function as determined in the following. However, for reasons of consistency, the term "component" will also be used to refer to the respective molecules before they are reacted to form the respective part (component) of the probe compound, or after they are released from the probe compound, respectively. A person skilled in the art will readily 50 understand the exact nature of the respective molecules, as well as their contribution to the probe compound. [0029] The term "linked" indicates the presence of at least one chemical bond between the corresponding components. A chemical bond can be a hydrogen bond, an ionic bond or a covalent bond, including a bond between a transition metal atom and its surrounding ligand atoms in a coordination compound (also referred to as "coordination bond" in the following). Preferably, the chemical bond(s) between the components of the probe molecule are covalent bonds, wherein 55 the bond(s) between the reactive component and the transition metal complex preferably is a coordination bond. The probe compound of the invention is required to have bonds between the transition metal complex and the reactive component, as well as between the test component and the indicator component forming the reactive component. However, the respective components may also be linked by additional bonds, as long as such bonds do not hinder the

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function of the probe compound. For example, the reactive component may be linked to the transition metal complex by at least one coordination bond formed between the test component moiety of the reactive component and the central metal atom of the transition metal complex and/or by at least one coordination bond formed between the indicator component moiety of the reactive component and the central metal atom of the transition metal complex. In a preferred 5 embodiment, the reactive component is linked to the transition metal complex by exactly one coordination bond formed between the test component moiety of the reactive component and the central metal atom of the transition metal complex and by exactly one coordination bond formed between the indicator component moiety of the reactive component and the central metal atom of the transition metal complex. The respective coordination bonds can be direct bonds between the metal atom and a suitable atom of the test or indicator components, or a bond formed via a suitable linker moiety, 10 which is linked to the test and/or indicator component. A preferred linker moiety is a histidine residue. By forming links via a linker moiety, it is possible to obtain a reproducible binding property for all test and indicator components. [0030] The term "test component" is preferably used to indicate a so-called "small organic molecule" that can interact with an enzyme. Preferably, the test component is a known substrate of at least one enzyme. Moreover, the test component can also be a pseudo-substrate or inhibitor of a known enzyme. However, the test component may also be 15 a molecule suspected to interact with the active site of an enzyme. The test component may also be a small organic molecules for the search for pharmaceutical active ingredients. According to one embodiment, the "test component" does not comprise a polymeric compound based on nucleic acids, such as DNA, cDNA, RNA, or the like, or a polymeric compound based on amino acids, such as peptides, proteins, or the like. According to another embodiment, a test compound may comprise at least one nucleic acids and/or amino acids, if necessary. Preferably, the test compound 20 comprises one or two nucleic acids, or one or two amino acids. Moreover, the test component can be preferably functionalised with a moiety suitable for binding to the transition metal complex. Such functionalisation is especially advantageous for test components comprising substrates, which do not readily form coordination bonds. In a preferred embodiment, the test component is functionalised with a histidine molecule (sometimes referred to as a "His-tag" in the following). The amino acid histidine was found to be especially versatile, because it may be linked to a test component 25 or indicator component by either its amine function or its carboxylic acid function, while the imidazole ring provides for a good coordination bond to the transition metal atom. A skilled person will know how to link a histidine residue to the desired site of a test or indicator component, whether a special activation or linker moiety will be required, as well as the starting materials and conditions necessary therefor. A His-tag has the additional advantage to ensure an identical binding property of all possible substrates to the transition metal complex. Moreover, it was found that a link including 30 a His-tag may be advantageously broken upon enzymatic reaction of a test component comprising a His-tag. Moreover, it was found that by selecting the site of histidine binding to the test component, it is possible to prepare probe compounds which allow for the identification of different metabolic pathways and the enzymes involved therein. [0031] The term "indicator component" is used to indicate a molecule which can generate a signal, thus indicating the location of the probe molecule on an array. That signal can either be produced directly, for example by adsorbance or 35 fluorescence, or can be generated in further treatments with detection reagents, for example in the form of an optical or radioactive signal. Preferably, the indicator component comprises a dye, further preferred a fluorescence dye. The term "fluorescence dye" indicates a molecule showing fluorescence upon irradiation with a suitable light source. Pref- erably, an indicator component comprises a fluorescent azo compound or a cyanine compound, or the like. Especially preferred is a cyanine compound, which is known in the art under the name of "Cy3". If necessary, the moiety having 40 the fluorescence property is further modified, e.g. by addition of a suitable linker moiety, in order to allow the binding to the test component, and/or to the transition metal complex. For example, a Cy3 dye available as its Cy3-NHS-ester may be reacted with both histidine and a 4-amino-3-butyric acid linker to allow for linking with both the transition metal complex and the test component, respectively. An additional linker moiety has the advantage to ensure an identical and reproducible binding to the indicator component and/or the transition metal complex, which allows for the ready use in parallel 45 synthesis or the like. [0032] The test component is linked to the indicator component to form the reactive component. Preferably, the test component is linked to the indicator component by at least one covalent chemical bond, further preferred by one, two, or three covalent bonds, and still further preferred by exactly one covalent bond. A preferred example of such a covalent bond is a carbon-oxygen single bond, which can be formed between the test component and the indicator component 50 by various chemical reactions, such as, for example, a condensation reaction, an addition reaction, an oxidation reaction, and the like. A preferred example is a condensation reaction between a carboxylic acid and an alcohol, or between two alcohols, or an addition reaction wherein the oxygen atom of an alcohol function is added to an aliphatic or aromatic carbon atom, or the like. It should be noted that such bond forming reaction is not restricted to the examples given above, and that there is no prejudice regarding which individual function should be present in the respective molecules to 55 become the respective components, etc. A person skilled in the art will immediately know which combinations of functional groups will be required to form a corresponding chemical bond between the individual molecules to become the respective components of the probe compound, and also which starting materials and conditions etc. are required to form the desired chemical bond between the components. Another preferred covalent bond is a carbon-nitrogen single bond.

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The link or bond between the test component and the indicator component may also be formed between the test component and a linker moiety previously attached to the indicator component, or vice versa. In a preferred embodiment, the indicator component comprises an amino butyric acid linker moiety, and a bond or link to the test component is formed using the carboxylic acid function of this linker moiety. 5 [0033] The reactive component comprising the test component and the indicator component, which are linked to each other, optionally by a linker moiety or molecule, is in turn linked to the transition metal complex. Preferably, the reactive component is linked to the transition metal complex by at least one coordination bond, further preferred by one, two, three or four coordination bonds, and still further preferred by exactly two coordination bonds. That means that at least one atom of the reactive component is a direct ligand atom of the transition metal atom of the transition metal complex, 10 thus being part of the immediate coordination sphere of the central transition metal atom of the transition metal complex. The term "at least one atom of the reactive component" also includes an atom of a linker moiety, which can optionally be attached to either the test component or the indicator component. For example, in a preferred embodiment, a histidine molecule is attached as a linker moiety (His-tag) to the reactive component. In this case, the "at least one atom of the reactive component" can also indicate an atom of the His-tag, preferably a nitrogen atom of the imidazole ring system. 15 The at least one atom of the reactive component being a ligand atom can be one atom of the test component or one atom of the indicator component. In the case of more than one coordination bonds between the reactive component and the transition metal complex, one, two, three or more ligand atom(s) can be an atom of the test component, and/or one, two, three or more ligand atom(s) can be an atom of the indicator component. Preferably, the reactive component is linked to the transition metal complex by two coordination bonds, wherein one ligand atom is an atom of the test 20 component and the other ligand atom is an atom of the indicator component. In a preferred embodiment, the reactive component comprises a His-tag linked to the test component and a His-tag linked to the indicator component. In this case, the reactive component is linked to the transition metal complex by two coordination bonds, wherein each bond includes one atom of one of the respective His-tags. [0034] A preferred coordination bond between the reactive component and the transition metal complex is a M-O-R- 25 bond, wherein M indicates the transition metal atom and O-R indicates an oxygen atom or function of a molecule R constituting the reactive component. Preferred examples of suitable oxygen functions are an alcoholate function (R-O-), a carboxylate function (RCOO-), a peroxide function (R-O-O-), or the like. Another preferred coordination bond between the reactive component and the transition metal complex is a M-N-R-bond, wherein M indicates the transition metal atom and N-R indicates a nitrogen atom or function of a molecule R constituting the reactive component. An example 30 for such nitrogen function is an aliphaptic amine function, including a primary, secundary and tertiary amine function. The nitrogen atom can also be part of an aromatic or (partially) saturated ring system, such as, for example, a pyrrol, imidazole, diazole or triazole ring, or the like. A specially preferred coordination bond is a coordination bond comprising a nitrogen atom of an imidazole ring, which may be part of a histidine moiety or His-tag. Another preferred coordination bond is a M-S-R or a M-C-R single bond, wherein M indicates the transition metal atom and S and C, respectively, 35 indicate a sulphur or carbon function of a molecule R constituting the reactive component. It should be noted that the coordination bond between reactive component and transition metal complex is not restricted to the examples given above, and that there is no prejudice regarding which individual function should be present in the respective molecules to form the desired coordination bond, etc. A person skilled in the art will immediately know which functional groups will be required to form a corresponding coordination bond, and also which starting materials and conditions etc. are required. 40 [0035] The transition metal complex preferably comprises at least one transition metal atom and at least one ligand molecule, wherein at least one coordination bond is formed between the ligand molecule and the transition metal atom. The at least one ligand molecule preferably comprises one or more atoms or funtions which can form a coordination bond with a transition metal atom. Preferred examples for atoms or function are an oxygen atom, a nitrogen atom, a phosphorous atom, a sulphur atom, or the like. These atom or functions all can form a coordination bond with a transition 45 metal atom, and are the same as exemplified above. Preferably, a ligand molecule comprises more than one atom or function that can form a coordination bond with a transition metal atom, further preferred two to eight of such atoms or functions, still further preferred three to five of such atoms or functions, and most preferred four or five of such atoms of functions. In a preferred embodiment of the present invention, the transition metal complex comprises exactly one transition metal atom and exactly one ligand molecule comprising more than one atom or function that can form a 50 coordination bond with a transition metal atom. Herein, the term "transition metal atom" is used to indicate the central transition metal atom of the transition metal complex, which is linked to both the ligand molecule(s) and the reactive component by coordination bonds as defined above. Examples of suitable transition metal atoms are Ti, V, Mn, Fe, Co, Ni, Cu, Zn, Mo, W, Pt, Au, or the like. Preferred examples are Co, Ni, and Cu. The transition metal atom means any transition metal atom irrespective of its oxidation state. The term transition metal ion is also used for a transition metal 55 atom that carries a net charge, which is sometimes referred to as a "transition metal ion" in the art, wherein a formal charge or oxidation state is assigned to the transition metal atom or ion. Preferred oxidation states of transition metal atoms of the present invention are from 0 to +4, preferably +2 to +3, and especially preferred +2. Preferred examples of transition metal atoms are Co(II), Ni(II), and Cu(II). Accordingly, the coordinating atoms or functions of the ligand

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molecule may be assigned with a formal charge, wherein preferred charges range from 0 to -2, wherein a charge of 0 or -1 is especially preferred. As used herein, the term "ligand molecule" refers to a molecule that comprises at least one atom or function which forms a direct coordination bond to the central atom of a transition metal complex, preferably one, two, three, four, five, six, seven, or eight such atoms or functions. It should be noted that the coordination bond 5 between ligand molecule and transition metal atom is not restricted to the examples given above, and that there is no prejudice regarding which individual function should be present in the ligand molecule to form the desired coordination bond, etc. A person skilled in the art will immediately know which functional groups will be required to form a corresponding coordination bond, and also which starting materials and conditions etc. are required. [0036] The term "probe compound" indicates a compound or molecule that can interact with an enzyme (or analyte 10 molecule) in a reactive manner, as outlined in the following. The term "interaction with an enzyme in a reactive manner" indicates an interaction wherein the reactive component is not only bound to the enzyme, but also transformed in a reaction catalysed by the enzyme (metabolised). The reaction catalysed by the enzyme can be any reaction catalysed by an enzyme, such as, for example, an oxidation or reduction reaction, an addition reaction, a hydrolytic bond cleaving reaction, an elimination reaction, an isomerisation reaction, or a condensation reaction. 15 [0037] Preferably, the reaction catalysed by the enzyme is a hydrolytic cleaving reaction or an elimination reaction, wherein the enzyme cleaves the link between the test component, or its reaction product, respectively, and the indicator component and/or the transition metal complex. [0038] Preferably, the interaction with the enzyme results in a cleavage of the link between the test component and the transition metal complex by the enzyme, whereupon a reaction product comprising the metabolised test component 20 and the indicator component together. Further, the interaction with the enzyme may also result in a cleavage of both the link between the test component and the transition metal complex and the link between the test component and the indicator component by the enzyme, whereupon more than one reaction products comprising the metabolised test component and/or the indicator component in separate molecules, may be released from the probe compound. For example, in a preferred embodiment wherein the test component is linked to the transition metal complex by a histidine 25 moiety (His-tag), the link between the histidine is cleaved by the enzymatic reaction. As a result, the reaction product comprising both the test component and the indicator component remains linked to the transition metal complex, and thus to the remainder of the probe compound. The test component or the reaction product thereof may remain bound to the active site of the enzyme, thus immobilising the enzyme to the probe compound, or its reaction product, respectively. Moreover, the signal characteristic of the indicator component, which is triggered by the enzymatic reaction, also remains 30 at the probe compound. [0039] Alternatively, the interaction with the enzyme results in a cleavage of the link between the indicator component and the test component by the enzyme, resulting in a reaction product comprising the indicator component alone, or a reaction product comprising the metabolised test component and the indicator component together, or more than one reaction products comprising the metabolised test component and/or the indicator component in separate molecules. 35 Here, the term "reaction product comprising the indicator component (or test component)" indicates a compound or molecule based on the indicator component or test component, respectively, which is metabolised and/or released by the corresponding reaction catalysed by the enzyme. [0040] Alternatively, the interaction with the enzyme results in a cleavage of the both the link between the test component and the transition metal complex and the link between the test component and the indicator component by the 40 enzyme, whereupon a reaction product comprising the metabolised test component, or more than one reaction products comprising the metabolised test component and/or the indicator component in separate molecules, may be released from the probe compound. For example, in a preferred embodiment wherein the test component is linked to the transition metal complex by a histidine moiety (His-tag), the link between the histidine is cleaved by the enzymatic reaction, and the link to the indicator component is cleaved as well. 45 [0041] Preferably, the reaction product comprising the indicator component is not released from the probe compound, i.e. the reaction product comprising the indicator component remains part of the probe compound, or its reaction product, respectively. For example, in a preferred embodiment wherein the indicator component is linked to the transition metal complex by a histidine moiety (His-tag), the histidine remains linked to both the transition metal complex and the indicator component upon enzymatic cleavage of the link between the test component and the indicator component. As a result, 50 the reaction product comprising the indicator component remains linked to the transition metal complex, and thus to the remainder of the probe compound. Thereby, the signal characteristic of the indicator component, which is triggered by the enzymatic reaction, also remains at the probe compound. [0042] Preferably, the reaction product comprising the test component is not released from the probe compound. For example, in a preferred embodiment wherein the test component is linked to the transition metal complex by a histidine 55 moiety (His-tag), only the link between the histidine and the test component is cleaved by the enzyme. As a result, the reaction product comprising both the test component and the indicator component remains linked to the transition metal complex, and thus to the remainder of the probe compound. Thereby, the test component or the reaction product thereof may remain bound to the active site of the enzyme, thus immobilising the enzyme to the probe compound, or its reaction

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product, respectively. Moreover, the signal characteristic of the indicator component, which is triggered by the enzymatic reaction, also remains at the probe compound. [0043] It was found that the probe compound of the present invention allows for the detection of enzyme concentrations which are as low as 1.5 ng/ml protein or 2.5 pmol/ml substrate, respectively. 5 [0044] It was found that the probe compound of the invention advantageously allows for the testing of a reactive interaction of an enzyme with a small molecule or enzymatic substrate. The presence of the central transition metal complex further allows to provide a probe compound wherein all small molecules or substrates necessary for the life functions of an organism or communities living in a habitat can be readily included. The probe compound of the invention was shown to be highly versatile for the identification of the reactive interaction with any small molecule or substrate 10 tested so far. The key characteristic of the probe compound is that a productive reaction with a cognate enzyme releases the indicator component, producing a detectable signal, e.g. a fluorescent signal, and simultaneously results in the capture of the reacting enzyme through coordination with the transition metal complex. Non-productive interactions of proteins with the probe compound, such as, for example, binding without chemical reaction, do not lead to the release of the indicator component and the associated production of a detectable signal. An example of this reactional behaviour 15 is shown in Figure 1. The probe compound provides a highly sensitive, accurate, reproducible, and robust high-throughput tool for a genome-wide analysis of the metabolic status of an organism or community. Advantageously, the complete reactome (i.e. the complement of metabolic reactions of an organism) can be provided without prior knowledge of its sequence in as little time as 30 minutes or less. [0045] Preferably, the transition metal complex comprises a cobalt or copper atom, and most preferred a cobalt atom. 20 It was found that a cobalt or copper complex shows an advantageous binding property to all reactive components of interest, thus allowing for a most versatile use of the probe component of the invention. Especially preferred is a cobalt complex wherein the central cobalt atom is assigned a formal oxidation state of +2. [0046] Preferably, the transition metal complex comprises a multidentate ligand molecule, i.e. a ligand molecule comprising two or more ligand atoms bound to the central transition metal atom. Further preferred, the transition metal 25 complex comprises a multidentate ligand molecule comprising two, three, four, five, or six ligand atoms bound to the central transition metal atom. Especially preferred, the transition metal complex comprises a multidentate ligand molecule whose coordinating ligand atoms do not occupy all possible coordination positions of the central transition metal atom. For example, in the case of a central cobalt atom (Co2+), which usually exhibits coordination numbers of five or six, the multidentate ligand may occupy five, four or three of the potential coordination (binding) positions. Thus, a preferred 30 ligand molecule for a central cobalt atom should provide three, four, or five coordinating ligand atoms, especially preferred four coordinating ligand atoms. A preferred example for a ligand molecule is a molecule comprising at least one coordinating nitrogen atom, such as, for example, a nitrogen atom of an aliphatic amine function, and at least one coordinating oxygen atom, such as, for example, an oxygen of a carboxilic acid function. A preferred example for a ligand molecule comprises nitrotriacetic acid. 35 [0047] For example, in the case of a central cobalt atom, a ligand molecule based on nitrotriacetic acid will occupy four of the five or six potential coordination or binding positions, thus leaving one or two free binding positions for binding of the reactive component. However, a person skilled in the art will readily know which other ligand molecules exhibit similar properties and thus can also be used in the present invention. [0048] Preferably, the transition metal complex comprises an anchoring component. The term "anchoring component" 40 indicates a molecule or function that can be used to attach the probe compound to a solid surface, or to another component or molecule, or the like. Thus, the transition metal complex, and thereby the whole probe compound, can be attached to any suitable solid surface, or component or molecule, known in the art. Suitable solid surfaces may be porous surfaces, such as, for example, paper or cellulose substrates or the like, or non-porous surfaces, such as, for example, glass surfaces, or surfaces of polymeric materials, such as a surface of polycarbonate, or the like. For example, the anchoring 45 component can be used to attach the probe compound to the surface of a glass slide in order to form an array thereon. Alternatively, the anchoring component can be used to attach the reactive compound to another component, such as, for example, a nanoparticle. The anchoring component may be any molecule or function known in the art which has the desired function to allow for the attachment to a solid surface, another component or molecule, or the like. A person skilled in the art will readily know which molecule or function should be used for a desired attachment. Preferably, the 50 anchoring component comprises a polymeric compound, further preferred a polymeric compound of a biomolecule. A specially preferred anchoring component comprises a poly A chain, i.e. a polymer of adenosin. Any poly A chain known in the art may be used. Preferably, the poly A chain has a molecular weight of from 10 kDa to 150 kDa, further preferred of from 50 kDa to 120 kDa, and most preferred of about 100 kDa. A poly A chain is known in the art for binding to glass surfaces, activated silica, or the like. A poly A chain can be easily attached to a glass surface or the like, which might 55 be optionally activated, and polymerised thereon using UV radiation according to standard protocols. [0049] In another preferred embodiment of the anchoring component comprises at least one thiol function, which allows for the attachment to metal clusters, such as, for example, gold nanoparticles. However, another function which is known to bind to a desired metal cluster or nanoparticle can also be used. A compound which has a carboxylate group

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or a phosphate group in one moiety and a thiol group in the other moiety is preferably used. A preferred example for an anchoring component comprising thiol functions is an α-dihydrolipoic acid residue (or a 6,8-dithioctic acid residue, TA). Depending on the nature of the surface of the metal cluster or nanoparticle to be used, i.e. either depending on the nature of the metal surface itself, or depending on the nature of a first activation or coordination layer, a skilled person 5 will know which anchoring compound should to use to form an anchoring link in the sense of the invention. [0050] Optionally, the anchoring component is linked to the transition metal complex by a suitable spacer component or moiety, such as, for example, an aliphatic hydrocarbon chain having at least one suitable functional group(s) for linking, such as, for example, a pentyl moiety having an amino group, or the like. [0051] The anchoring component is directly linked to the transition metal complex. Preferably, the anchoring component 10 is linked to the transition metal complex by a covalent bond, which is formed between an atom of the anchoring component and the ligand molecule of the transition metal complex. The anchoring component can also be linked to the transition metal complex by a coordination bond, which is formed between an atom of the anchoring component and the central transition metal atom of the transition metal complex. [0052] In a preferred embodiment of the probe compound of the present invention, the transition metal complex 15 comprises a nitrotriacetic acid Co(II) complex. It was found that the presence of this transition metal complex allows for the most versatile adaptation of the probe compound for the identification of the reactive interaction with any small molecule or substrate tested so far. A preferred transition metal complex comprising an anchoring component is provided

by using the compound Nα,Nα-bis-(carboxymethyl)-L-lysine, or (S)-N-(5-amino-1-carboxypentyl)-imino-diacetic acid, respectively, as a ligand molecule for forming a cobalt(II) complex. In the so-formed cobalt complex, the aminopentyl 20 residue, which is attached to one of the acetic acid groups of the ligand molecule, constitutes an anchoring component or a spacer moiety for attaching another anchoring component. The amine function of this anchoring component itself can be readily used to attach the transition metal complex, and thus the whole probe compound, to another component or molecule, such as, for example, a nanoparticle. Moreover, the amine function of this anchoring component can also be used to further attach a polymeric chain, such as, for example, a poly A chain, which can be used to attach the 25 transition metal complex, and thus the whole probe compound, to a solid surface, such as, for example, a glass slide, in order to advantageously manufacture an array comprising corresponding probe compounds. [0053] Thus, the term "probe compound" indicates a compound or molecule that can be immobilised on a supporting base by the anchoring component. [0054] Preferably, the indicator component comprises a fluorescence dye, further preferred a fluorescent azo com- 30 pound or a cyanine compound, examples of which are known in the art under the names of "Cy3" or "Cy5" or the like. By using this well-established dyes, it is possible to use the corresponding hardware available commercially, such as, for example, wave-length optimised reader apparatuses, corresponding software solutions, and the like. However, a person skilled in the art will understand that the present invention can easily be adopted to other indicator components and/or detection systems, if required. A person skilled in the art will also know how to carry out such adaptation. 35 [0055] Preferably, the test component comprises a known substrate, a metabolite, a pseudo-substrate, or an inhibitor of an enzyme. Further preferred, the test component comprises a molecule identified as a substrate of at least one metabolic reaction in the Kyoto Encyclopedia of Genes and Genomes (KEGG Database), the University of Minnesota Biocatalysis and Biodegration Database (UM-BBD), PubMed, or the like. Further preferred, the test component comprises a compound selected from the group listed in Table 1, 2 and 3, respectively. 40 [0056] By using such test components, it is possible to prepare probe compounds according to the invention which collectively form most of the central metabolic networks of cellular systems. However, the test component may also comprise additional metabolites characteristic of microbial metabolic activities not yet assigned or included in these databases, as well as [0057] In a specially preferred embodiment of the probe compound of the present invention, the transition metal 45 complex is represented by the Nα,Nα-bis-(carboxymethyl)-L-lysine cobalt(II) complex, which may further comprise an anchoring component comprising a poly A chain or a 6,8-dithioctic acid (TA) linked to the free amine function of the ligand molecule. In this embodiment, an indicator component comprises a Cy3 fluorescent dye, which comprises a histidine moiety (His-tag) for linking to the transition metal complex and a aminobutyric acid moiety as an optional linker moiety for linking to the test component. The test component may be any enzymatic substrate or other suitable small 50 organic molecule, for example, one of the test components or substrates listed in Table 1. The test component is linked to the indicator component via the optional aminobutyric acid linker moiety and further comprises a histidine moiety (His- tag) for linking to the transition metal complex. By choosing the appropriate position for introducing the histidine moiety, the involvement of one substrate in different metabolic pathways can be detected by the probe compound. The so- formed reactive component is linked to the transition metal complex by two coordination bonds, one of which comprises 55 the His-tag linked to the test component, while the other comprises the His-tag of the indicator component. It was surprisingly found that, if the reactive component is linked to the transition metal complex, the characteristic fluorescence activity of the indicator component is no longer observed. In other words, the probe compound remains silent with respect to the characteristic fluorescence signal. This would result in a dark spot in an assay or array position. When the probe

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compound is brought into contact with an enzyme having a function specific to the test component or substrate comprised in the probe compound, the test component or substrate is metabolised. It was surprisingly found that this enzymatic reaction has two effects. First, the characteristic fluorescence signal of the indicator component is observed again, and second, the enzyme remains bound to the transition metal compound. Thus, the probe compound allows for the unam- 5 biguous detection of the specific enzymatic reaction by flurescence detection. Moreover, the probe compound allows for an immobilisation of the substrate-specific enzyme, which can be advantageously used to isolate this enzyme from a sample. [0058] The probe compound according to the above-discussed preferred embodiment of the invention can be illustrated by the following general formula (2): 10

15

20 wherein AC represents an optional anchoring component, preferably poly A or TA, MC represents the Nα,Nα-bis-(carboxymethyl)-L-lysine cobalt(II) complex, TC represents the test component, IC represents the fluorescence dye Cy3, His-tag represents a histidine moiety as described above, and LAC-MC and LTC-IC each represent optional linker moieties between the respective components indicated by the subscripts. [0059] Although the exact mechanisms involved in these reactions are not completely understood yet, it is believed 25 that the linking of the reactive component to the transition metal complex influences the indicator component in such a manner that its electronic structure necessary for its characteristic fluourescence signal is disturbed. The reaction of the enzyme with the test component or substrate is believed to alter or break down the reactive component, which influences the binding properties of the reactive component or its respective metabolised products, respectively, to the transition metal complex. Thereby, the adverse effect on the indicator component is no longer present so that the indicator com- 30 ponent can again exhibit its characteristic fluorescence signal. Moreover, this enzymatic alteration or break down of the reactive component may also create new binding sites at the reactive component and/or the transition metal complex, which result in the binding of the enzyme. [0060] The present invention also provides a method for preparing the probe compound. The method for preparing the probe compound of the invention comprises the following steps: 35 a) Preparing a transition metal complex by reacting a suitable salt of a transition metal with a desired ligand molecule, optionally comprising an anchoring component. Herein, the appropriate conditions of solvent, pH, temperature and the like can be chosen according to known procedures. If necessary, the thus-obtained transition metal complex can be further purified by standard procedures, such as, for example, filtration, re-crystallisation, or the like. In a 40 preferred embodiment, Nα,Nα-bis-(carboxymethyl)-L-lysine is reacted with cobalt(II) chloride to form the corresponding complex.

b) If an anchoring component is desired, it may also be incorporated at this stage. For example, in a preferred

embodiment, the above cobalt(II) complex of the Nα,Nα-bis-(carboxymethyl)-L-lysine ligand may be reacted with a 45 suitably activated poly A chain or with 6,8-dithioctic acid, or the like.

c) In a separate reaction, a substrate molecule to be incorporated in the test component is coupled to an indicator component, optionally including a linker moiety. If necessary, either one or both of the test component and indicator component is previously transferred into an activated form suitable for coupling, according to standard techniques. 50 Herein, the appropriate conditions of solvent, pH, temperature and the like can be chosen according to known procedures. For example, a Cy3 dye available as its Cy3-NHS-ester may be reacted with a 4-amino-3-butyric acid linker to allow for linking with the test component. If necessary, the thus-obtained reactive component can be further purified by standard procedures, such as, for example, filtration, re-crystallisation, or the like.

55 d) Optionally, the test component and/or the indicator component is functionalised with a moiety suitable for binding to the transition metal complex, either before or after formation of the reactive component. Such functionalisation is especially advantageous for indicator components or test components, which do not readily form coordination bonds. In a preferred embodiment, the test component and/or the indicator component is functionalised with a

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histidine molecule (sometimes referred to as a "His-tag" in the following). A His-tag has the additional advantage to ensure an identical binding property of all possible test components (substrates) and/or indicator components (dyes) to the transition metal complex.

5 e) In a subsequent step, the so-prepared reactive component comprising the test component and the indicator component is linked to the transition metal complex to form the probe compound according to the present invention. Herein, the appropriate conditions of solvent, pH, temperature and the like can be chosen according to known procedures. If necessary, the thus-obtained probe compound can be further purified by standard procedures, such as, for example, filtration, re-crystallisation, or the like. 10 [0061] The method comprises the steps a), c) and e), and the optional steps b) and/or d), if desired. The optional steps b) and d) can be carried out at any stage of the method. For example, step d) may be carried out before step c), or subsequent to step b), and step b) may be carried out before step a), subsequent to step a), or subsequent to step e), respectively. In a preferred embodiment, the individual steps are carried out in the order of steps a), b), d), c, and 15 e). Alternatively, the steps may be carried out in the order of steps b), a), c), d), and e), or in the order of steps a), d), c), e), and b). A person skilled in the art will know which order and conditions are most suitable for the respective task. [0062] The present invention also provides an array for detecting enzymes comprising a plurality of different probe compounds according to the invention. The array comprises a plurality of different probe compounds according to the invention (sometimes referred to as library of probe compounds in the following), i.e. probe compounds according to 20 the invention which differ at least with respect to the test component comprised therein. Preferably, the probe compounds only differ with respect to the test component comprised therein. A plurality of different probe compounds can be prepared using automatic procedures, for example, parallel synthesis protocols and apparatuses, or the like. The term "comprising a plurality of different probe compounds" means at least two different probe compounds, and may comprise up to several thousands different probe compounds. For example, apparatuses and protocols, which are known and commercially 25 available at the moment, allow the standard production of arrays based on glass slides wherein from 5000 to 15000 different probe compounds may be deposited on a single glass slide by micro-spotting techniques, or the like. In a preferred embodiment, about 2500 different probe compounds are included in the array (cf. example). Alternatively, the array may be constructed using a plate providing separate compartments or wells, such as, for example, a micro-titre plate, which is commercially available with, e.g., 384 wells, or different numbers of wells. A complete array may comprise 30 one or more slides or plates depending on the actual number of probe compounds included in the array, as well as on the density available on the respective medium. Preferably, an array comprises more than one copy of a library of probe compounds on one or more supports. For example, a library or sub-library of probe compounds may be provided in duplicate or triplicate on one support or slide. [0063] In one embodiment of the array, the plurality of probe compounds is attached to a planar surface of a suitable 35 support or carrier. A support may be any support used in the art, preferred examples of which are glass surfaces, preferably of glass slides, surfaces of polymeric materials, such as polyacetate surfaces, or the like, or surfaces of cellulose or paper materials, or the like. In order to be attached to a solid surface, such as, for example, the glass surface of a glass slide, the probe compounds are provided with a suitable anchoring component. A preferred anchoring component known from the art is a poly A chain or tail, which can be readily attached onto a glass surface or the like following 40 standard protocols using irradiation by UV light. Other suitable solid surfaces and corresponding anchoring components as well as protocols for their use are known in the art, and a person skilled in the art will be able to select an appropriate combination. For example, when using polymeric supports, it is advantageous for the bonds between the probe molecules and the polymeric support to be chemical bonds, especially covalent chemical bonds, which allow a long-lasting, stable bond between the probe molecules and the polymeric support. 45 [0064] In the case of using a well plate, the probe compounds do not necessarily require an anchoring component because they can be held in the respective wells without attachment to the wall. Preferably, probe compounds are attached to the walls of a well of a well plate. Therefor, all methods known in the art may be used. [0065] The array of the invention (which is sometimes referred to as "reactome array") can be used for the simultaneous detection of reactive interactions between the probe compounds and all analyte molecules (enzymes) from a sample. 50 In particular, the array provides a solution to the problem of the identification of metabolites and the enzymes involved in their transformation. The array provides a fast and reliable way to detect all metabolic pathways active in an organism or community, and may be used advantageously for an activity-based, annotation-independent procedure for the global assessment of cellular responses. [0066] The invention also provides a method for producing an array according to the invention. An array may be 55 produced using well plates, such as commercially micro-titre plates, wherein each probe compound of the plurality of different probe compounds according to the invention is filled into an individual well, together with appropriate amounts of solvent, cofactors, cations, supplements, or the like, which are known or predicted to be required for the expected enzyme reaction. The filling of the wells may be carried out automatically, e.g. by a suitable robotic apparatus, or the

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like. Alternatively, the array is preferably produced using different probe compounds, which all comprise the same anchoring component. The different probe compounds having an anchoring component are then arranged onto an appropriate support surface, e.g. the surface of a glass slide, and subsequently bound thereto with the anchoring component. The arranging and binding of the different probe components may be carried out automatically, e.g. using 5 a suitable robotic apparatus, or the like, and following established procedures and protocols. In a preferred embodiment, different probe compounds comprising a poly A chain as an anchoring component are spotted onto a glass slide by using a robotic apparatus, and subsequent fixation is achieved by cross-linking the poly A tails according to an established protocol using UV radiation. However, several other methods for arranging and fixing the different probe molecules onto a suitable support surface are known in the art. Using the method of the invention allows for a versatile, fast and 10 reproducible production of arrays according to the invention. [0067] Moreover, the invention also provides an isolation means comprising a nanoparticle and a probe compound according to the invention. A probe compound is preferably provided with a suitable anchoring component and attached to a nanoparticle. The term "nanoparticle" means a particle having a maximum dimension of less than 500 nm, preferably of less than 300 nm, and most preferred of about 100 nm. A minimum dimension is about 10 nm, preferably about 50 15 nm. A maximum or minimum dimension of a nanoparticle refers to the diameter in the case of a spherical nanoparticle. Examples for suitable nanoparticles are known in the art, as well as methods for producing the same, or anchoring components for linking the probe compound to the same. The probe compound may be linked to any nanoparticle known in the art, preferably a magnetic nanoparticle. Preferably, a nanoparticle comprises one or more metallic elements, including the transition metal elements. Preferred examples of nanoparticles comprise metallic elements, either pure 20 metallic elements, such as, for example gold nanoparticles, or alloys comprising different metallic elements, or oxides of metallic elements, such as, for example, iron oxides, or the like. Preferred oxidic nanoparticles may comprise silicon, e.g. in form of silicon oxide. Moreover, preferred nanoparticles may also have a layered structure, e.g. an oxidic core coated by a metallic layer, such as a gold-coated silicon oxide nanoparticle, or a metallic core coated by an oxidic layer, such as a cobalt core coated with an iron oxide layer. For example, the synthesis and application of suitable magnetic 25 gold nanoparticles is described by Abad et al. in J. Am. Chem. Soc. 127, 5689 (2005). Preferably, a nanoparticle has a magnetic property. A preferred anchoring component for linking a probe molecule to a magnetic gold nanoparticle is 6,8-dithioctic acid or α-dihydrolipoic acid, respectively, which advantageously may be linked to a transition metal complex comprising the cobalt(II) complex of Nα,Nα-bis-(carboxymethyl)-L-lysine (ANTA-Co(II)) by forming an amide bond between the carboxylic acid function of the 6,8-dithioctic acid and the amine function of the ligand molecule. The isolation 30 means according to the invention allows for the substrate specific interaction and binding of an enzyme which can then be isolated by means of filtration, gravitation force (centrifugation), an external magnetic force in case of a magnetic nanoparticle, or the like. It was found that, owing to the substrate specific binding of an enzyme by the probe compound, the isolation means of the invention allows for the directed isolation of an enzyme because of its substrate specificity. [0068] The invention also provides a method for producing a isolation means according to the invention. In this method, 35 a probe compound comprising a suitable anchoring component is prepared according to the method of producing a probe compound according to the invention, and subsequently attached to a suitable nanoparticle prepared according to known methods. Preferably, the probe compound is attached to a magnetic nanoparticle. The method according to the invention provides an easy access to an isolation means having specific binding sites for an enzyme. [0069] Moreover, the invention also provides a method for detecting enzymes in a substrate specific manner, using 40 the probe compound according to the invention or the array according to the invention. An array comprising a plurality of different probe compounds is prepared according to the method of the invention. The array can preferably comprise more than one copy of the respective library of probe compounds, either by providing the library on one support or slide in duplicate or triplicate or in more copies, or by providing more than one support or slide comprising identical sets or libraries of probe compounds. The array is then brought in contact with a sample comprising the analyte molecules, i.e. 45 the enzymes whose substrate specific activity is to be tested. This step is also referred to as incubation with a sample. A sample in the context of the method includes any kind of solution of analyte molecules (enzymes) that can enter into an enzymatic reaction with the probe compounds on the array. These include especially biological samples obtained from the lysis of cells of bacteria, archeae, or higher organisms, but also from biological fluids such as blood, serum, secretions, lymph, dialysate, liquor, sap, body fluid from insects, worms, maggots, etc. Also included is extraction from 50 natural sources such as biopsies, animal and plant organs or parts, cell, insect, worm, bacteria, microbe and parasitic matter as well as supernatants of cell cultures and of bacterial, microbial or parasitic cultures. A sample may also be a chemical-synthetic sample containing, for example, synthetic proteins, or the like. After incubation is complete, the sample is removed from the array. In order to obtain a complete removal of the sample, the array may be washed one or more times. Then, the array is analysed for the presence of the signal characteristic for the indicator component. 55 Preferably, a fluorescence signal is read out and processed by a suitable apparatus, which is available in the art. All protocols and procedures known in the art may be used, including automatic processing by robotic apparatuses and the like. In a preferred embodiment, using an array comprising probe compounds comprising a Cy3 fluorescence dye, the fluorescence signal is preferably measured using a laser scanning system.

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[0070] The method of the invention using the array of the invention allows to assess the complete reactome of an organism or community in one step. That is, all substrate specific enzymatic reactions which are performed within the metabolic pathways necessary for the individual life form(s) is readily accessible. This advantageously allows for the accurate reconstruction of metabolic pathways. 5 [0071] Moreover, the invention also provides a method for isolating enzymes using the isolation means according to the invention. According to the method, the nanoparticles are used to isolate enzymes which are bound to the isolation means by the probe molecules after the substrate specific enzymatic reaction. The reaction can be carried out under the same conditions as described above for the array of the invention, including all processes and protocols that are known in the art. Preferably, the isolation means is brought into contact with a sample comprising analyte molecules 10 (enzymes) that can enter into an enzymatic reaction with the probe compounds on the isolation means. The isolation means carrying the enzymes are then isolated by magnetic means or by filtration. The isolated enzymes can then be analysed using standard techniques, such as, for example, sequence analysis, mass spectrometry, or the like. Identi- fication of enzymes whose function is verified by the specific reaction with the probe compound of the invention allows for the detection of metabolic pathways as well as for the correct annotation of unsequenced genes and/or unknown 15 proteins or enzymes. The method can be used to complete the metabolic pathway map of all life forms, as well as specialised parts thereof. The method can also be used to reconstruct the global metabolism of complete communities living in distinct natural (microbial) communities. [0072] Moreover, the method of the invention can be advantageously used in the search for new drugs, in particular in screening for potential new targets: here the guiding principle is selective toxicity, so the search is for molecular targets 20 in the target organism that do not exist in a human. The method of the invention is also useful for the identification of drugs that are specific for certain pathogens, so that treatment of an infected patient would not result in the elimination of a major part of the body flora, as is currently the case with common broad spectrum antibiotics, and all the negative consequences of this that ensue. Using the array to obtain reactome profiles of the representatives of the major phyla of life allows their comparison and identification of individual reactions/enzymes that are specific for each branch or 25 clusters of branches. Some of these phylum-specific functions will be known already, but others may be new: such metabolic reactions/enzymes may then serve as newly-discovered targets for drug screening. For example, the comparison of the human reactome with the composite reactome of bacteria and archaea may identify new targets for broad spectrum antibiotics, or the comparison of the reactomes of the various bacterial phyla may reveal potential targets specific for individual phyla, such as those to which, e.g., Neissseria, Pseudomonas, Mycobacterium, Vibrio, Staph, 30 Strep, Pneumo, coliforms belong, providing a route to more specific, narrow spectrum antibiotics. Moreover, the comparison of reactomes of photosynthetic organisms may identify new targets for herbicides specific for, e.g., moss, or algae, or monocots, or dicots etc. that would allow the development of new generation anti-moss treatments that would not affect other plants, anti-algal treatments not active against other plants, etc., etc. Moreover, the comparison of the reactomes of insect vectors of disease (mosquito, black fly, etc) with those of other insects, humans etc. could identify 35 vector-specific targets. [0073] In other words, the present invention provides a versatile tool for obtaining information on the reactomes of all major branches of the tree of life which can be advantageously used in future drug development. Through the selective inhibition of pathogens, there become available a lot of applications involving the selective inhibition of specific microbial populations, either because they are known to be problematic, or simply to test experimentally their contribution to a 40 particular process (e.g. the role of a particular GI tract organism in a particular GI physiological role, like folate production, the role of a particular rhizosphere organism in protection from fungal infections, etc.). Thus the array provides the means of identifying phylum-specific metabolic targets for inhibitors that, in turn, can be used to inhibit specifically those phyla in microbial communities to assess their role in a particular process. [0074] The use of the reactome array of the invention for identifying new targets can be applied from human medicine 45 to agriculture to dental medicine to shipping (anti-algal compounds to prevent algal fouling; anti-sulphate reducer compounds to prevent corrosion of steel) to construction (e.g. anti-sulphate reducer compounds to prevent corrosion of steel), etc., and to research tools (agonists/antagonists for all manner of selected life forms). [0075] The present invention provides a procedure to measure on an array, enzymatic activities of cells against many of the standard substrates and metabolites that characterize life, plus other substrates of interest. Because of the 50 chemical design of the substrates of the array, the array provides the identity of the reaction, the reaction products and, in a subsequent step, the enzyme itself. It therefore links a substrate/metabolite with its cognate enzyme. The reactome array thus forges a thus far missing link between metabolome and genome. Since many of the metabolites on the array are connected in pathways, it is also possible to reconstruct the metabolic network operating in any organism without any prior genomic information. 55 [0076] The use of the array to investigate the reactome of a microbial community is particularly interesting. The array represents a new possibility to have a metabolic overview of an entire community, without perturbing it in any way prior to preparation of the community lysate for analysis. And, in cases where it is difficult to obtain sufficient biomass for direct analysis, it is even possible to obtain a small molecule microarray analysis of a metagenomic library of a DNA

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sample of a community or enrichment and thereby obtain a detailed overview of the global metabolism of the sampled community. The reactome analysis described here uncovered new metabolic activities in organisms and communities (46 new functions in P. putida KT2440 and five in metagenomic communities, including a novel hydrogenase; cf. Table 4 and Figures 5 and 6), which not only provide interesting opportunities for new lines of investigation, but also revealed 5 new metabolic components of niche specificity and predominant microbial pathways shaping the overall metabolism of the individual habitats. Especially, the hydrogenase (cf. Table 4 and Fig. 6), which is a rather small enzyme, may be of interest in the field of energy production. [0077] Since a physico-chemical analysis of habitats is rather selective in terms of the parameters measured, detection limits defined by the instruments used, and usually does not discriminate between bioavailable and non-bioavailable 10 levels, whereas the array scores most of the metabolic potential of the cell or community in relation to the prevailing conditions and bioavailable fraction of compounds, another application of the array is the habitat characterization by metabolic profiling. This type of application can also be used in diagnosis of diseases/intake of drugs/toxic substances that influence metabolic activities of the microbial flora, through reactome analysis of faecal/skin biota, forensic analyses of diverse types (e.g. groundwater pollution), prospecting (e.g. for natural gas seeps that indicate underlying reservoirs), 15 detection of manufacturing sites of illegal substances, etc. Indeed, the design of custom arrays for use with particular organisms or communities will entrain a diverse spectrum of applications relating to enzyme activity profiles, including the phenotyping of organisms (microbes, plants, higher animals and humans), populations, mutant libraries and transgene libraries, the direct diagnosis of diseases and quality control in food industries, to cite some. [0078] The present invention will be illustrated in more detail in the following examples, but it is not restricted to the 20 special embodiments exemplified in these examples. [0079] Commonly used chemical and molecular-biological working methods are not described in detail here, but they can be referred to in, for example, Houben-Weyl, Methods of Organic Synthesis.

EXAMPLES 25 EXAMPLE 1:

General techniques.

30 [0080] Unless specified otherwise, reactions were carried out with dry solvents freshly purified by passage through a column of activated alumina (A-2) and supported copper redox catalyst (Q-5 reactant). All other reagents were purified according to standard literature methods or used as obtained from commercial sources. [0081] NMR spectra of all compounds were recorded at 600, 500, 400, or 300 MHz, using Varian I-600, Varian I-500, Varian M-400, Varian M-300, and Bruker Biospin 300 instruments. 1H NMR chemical shifts were reported relative to 35 13 residual CHCl3 (7.26 ppm). C NMR data were recorded at 125, 100, or 75 MHz, using Varian I-500, Varian M-400, or Bruker Biospin 300 MHz instruments, respectively. 13C NMR chemical shifts are reported relative to the central line of 59 CDCl3 (77.0 ppm). Co NMR measurements were carried out at room temperature with Bruker ASX-200 (B0=4.7 T, 59 Larmor frequency v0=48.1 and 52.9 MHz in Co resonance, Bruker MSL-300 (B0=7.1 T, v0=71.2 and 79.4 MHz), and Bruker Avance DSX-500 (B0=11.7 T, v0=120.4 and 132.3 MHz) spectrometers. Single-pulse MAS spectra were obtained 40 by using a Bruker MAS probe with a cylindrical 4-mm o.d. rotor. When necessary, continuous-wave proton decoupling with a radiofrequency (RF) field of 50 kHz was applied during acquisition. Spinning frequencies vr up to 17 kHz were utilized. A short pulse length of 1 ms corresponding to a nonselective n/12 pulse determined using an aqueous or DMSO solution of small molecules (SMs) was employed. Recycle times were 1 and 90 s in 59Co . The baseline distortions resulting from the spectrometer dead time (5-10 ms) were removed computationally using a polynomial baseline correction 45 routine. The dead-time problem was then overcome by Fourier transformation of the NMR signal, starting at the top of the first rotational echo. [0082] Molecular masses were analyzed at the SIDI Core Facility of the Autonomous University of Madrid. For each experiment, a magnetic high resolution mass spectrometer (8000 v acceleration) was used, with ionization source FAB (LSIMS - liquid secondary ion mass spectrometry with Cs ions) using m-nitrobenzoic alcohol (m-NBA) as matrix. The 50 samples (0.5-1.2 g) were dissolved in acetone, methanol or DMSO, depending of the solubility of the SMs-Cy3. Micro- analyses were performed by the SIDI Core Facility of the Autonomous University of Madrid, and are quoted to the nearest 0.1% for all elements, except for hydrogen, which is quoted to the nearest 0.05%. Reported atomic percentages are within the error limits of 6 0.3%. [0083] For N-terminal amino acid sequencing, polyacrylamide gel electrophoresis under denaturing conditions (SDS- 55 PAGE: 10%, v/v) was performed according to Laemmli (U.K. Laemmli, Nature 227, 680 (1970)), using a Mini-PROTEAN cell apparatus (Bio-Rad), and protein bands were blotted to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was stained with Coomassie Brilliant Blue R-250, after which the bands of the proteins were cut out and processed for N-terminal amino acid sequencing. The peptide sequences were initially scored against blastp nr to identify

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the best hits among full-length proteins, and then converted into coding sequences and screened for potential protein encoding genes (PEGs) via tblastn and tblastx search (F. Stephen et al., Nucleic Acids Res 25, 3389 (1997)) against the comprehensive non-redundant database sourced from the nucleotide (nr/nt) collection, reference genomic sequences (refseq_genomic), whole genome shotgun reads (wgs) and environmental samples (env_nt) databases. This was chosen 5 empirically to increase the number of matching potentially coding elements. Based on the best BLAST hits, degenerate oligos were designed and used to amplify full length ORFs from the metagenome libraries.

EXAMPLE 2:

10 Bacterial strains, culture, and growth conditions.

[0084] E. coli DH5F’ was used as a recipient for pGEMT plasmid (Promega) constructs containing cloned PCR fragments of P. putida KT2440 encoding hypothetical proteins and metagenomic proteins. E. coli TOP10 (Invitrogen) was used as a recipient for pCCFOS vector (EPICENTRE) constructs. E. coli cultures were grown in Luria-Bertani (LB) 15 medium and incubated at 37˚C on an orbital platform operating at 200 rpm. When required, cultures were supplemented with the following antibiotics: ampicillin (100 mg/ml), nalidixic acid (10 mg/ml) and chloramphenicol (12.5 mg/ml). P. putida KT2440 was grown in M9 minimal medium with 15 mM succinate as carbon source in 100-ml flasks shaken at 30 ˚C and 150 rpm from an initial turbidity at 600 nm of 0.02 to a final value of 0.760.05. Samples (3 ml) were removed, the cells harvested by centrifugation at 4˚C, and the cell pellets were washed with 20 mM Hepes pH 7.0 before storing at 20 -20˚C until use.

EXAMPLE 3:

General reactome strategy. 25 [0085] The general reactome strategy comprises five stages for array construction and protein-SMs transformation detection as follows (cf. Figure 1).

[1] Data searching and compound identification 30 [0086] Initially, an extensive data mining effort, focused mainly on the Kyoto Encyclopedia of Genes and Genomes (KEGG Database: http://www.genome.ad.jp/kegg/), the University of Minnesota Biocatalysis and Biodegradation Data- base (UM-BBD: http://umbbd.msi.umn.edu/) and PubMed (http://www.ncbi.nlm.nih.gov/sites/entrez), was undertaken to produce a list of compounds to be synthesized that are 35 substrates of one or more metabolic reactions and that collectively form most of the central metabolic networks of cellular systems. Additional metabolites characteristic of microbial metabolic activities were also identified for synthesis.

[2] Compound synthesis, modification and arraying

40 [0087] A library of 2483 identified SMs was synthesized using the strategies specified in Table 1. The purity of each SM was confirmed by NMR and molecular mass. Individual SMs were coupled to the Cy3 fluorescent dye and subsequently combined on specific positions to a nitrotriacetic-Co(II) complex containing a terminal poly A-tail (Table 2). Importantly, during synthesis, the Cy3 dye is attached to the molecule at specific positions that allow control of the reaction product formed, through creating for each molecule different Cy3-variants that collectively serve as substrates 45 for all possible reactions described in KEGG; the array thus assays multiple distinct reactions of the same metabolite. The resulting derivatives were dissolved at different concentrations from 0.5 to 100 nM (six concentrations) in dimethyl sulfoxide (DMSO) and stored at -70˚C until use in 384-well microtiter plates. Each well also contained cofactors, cations and supplements known or predicted to be required for efficient transformations. The 2483 SMs included in this study, together with the position of modification with Cy3 are given in Tables 1 and 2. These procedures were also used to 50 synthesize Cy3-modified X-Gal (obtained from Boehringer Mannheim), which was used as substrate for the β-galactosidase of E. coli (provided also by Boehringer Mannheim). Individual SMs were subsequently spotted onto Corning UltraGAPS glass slides in a spatially addressable manner, by means of a MicroGridII spotting device from Biorobotics operating at 20˚C and 50% relative humidity, and subsequently immobilized by standard UV cross-linking.

55 [3] Array analysis and cell extract

[0088] 60 ml quantities of cell lysates of microbial cultures, or libraries of metagenomic clones of microbial communities, diluted in PBS buffer to a final protein concentration of 0.1 mg/ml, were layered on the array, which was subsequently

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incubated at room temperature for 30 min.

[4] Data analysis and metabolic reconstruction

5 [0089] Arrays were scanned for fluorescence with a GenePix 4000B scanner (Axon Instruments) operating at 532 nm at 10 mm resolution with 100% laser power, and images (spot and background intensities were quantified in triplicate or more) were analyzed using the GenePix v5.1 software (Axon Instruments). Reconstruction of metabolic maps were carried out with GraphViz and SOI Linux software.

10 [5] Protein identification with gold beads

[0090] Proteins reacting with specific metabolites were identified by incubating a protein extract with metabolite-coated gold nano-particles, followed by protein sequencing of the captured proteins, reverse translation of the sequences into gene sequences, cloning of the corresponding genes, hyper-expression of the genes, and purification and characteri- 15 zation of the corresponding proteins.

EXAMPLE 4:

General procedure for the synthesis of probe compounds. 20 [0091] The general reaction strategy showing the successive steps for the construction of Cy3 modified metabolites and their integration into probe compounds is described below.

1. Preparation of nitrilotriacetic-Co(II) complexes. 25 [0092] The amino-nitrilotriacetic-Co(II) complex was formed by reaction of NR,NR-bis(carboxymethyl)-L-lysine hydrate (ANTA, Fluka) with an excess of cobalt(II) chloride (Sigma) in 20 mM HEPES in aqueous solution (36). Excess cobalt was precipitated by increasing the pH to 10, and the precipitate was removed by filtration through a 0.2 mm membrane (PTFE, Amicon). 30 2. Incorporation of poly(A) tails in nitrilotriacetic-Co(II) complexes (optional).

[0093] Activation of the phosphate groups of the poly(A) tails (Sigma Genosys, average molecular weight: 100 kDa) and subsequent amidation with the Co(II) complex was performed by overnight incubation with 3 mM N-hydroxysuccin- 35 imide (NHS, Fluka) and 3 mM 1-ethyl-3-[3 -(dimethylamino)propyl]carbodiimide (EDC, Sigma) in 20 mM HEPES buffer (pH 7.5).

3. Generation of double activated Cy3 dye.

40 [0094] Cyanine dye was linked via a histidine tag and a flexible linker through which the dye is linked to the Co(II) complex and the metabolite, respectively. Briefly, a histidine molecule was firstly incorporated to the Cy3 dye by enzymatic acidolysis of Cy3 NHS (a succinimidyl ester, GE Healthcare) with histidine and immobilized Lewatit lipase EL1 (37) from a cow rumen metagenome (37.39 mol% incorporation of histidine in 24 h, at a ratio 1 histidine:4 NHS ester). 3-Methyl- 2-butanol was used as solvent with a water content of up to 3.2%, and the reaction was carried out at 50-55 ˚C. High- 45 performance preparative liquid chromatography was used to analyze and purify the products of the acidolysis reaction. Purified Cy3-His was dissolved in DMSO at a concentration up to 4 M and stored at -70˚C until use [0095] In a second step, the Cy3-His was joined to a 4-amino-3-butyric acid linker. Briefly, the linker was dissolved in 0.1 M sodium borate buffer pH 8.5 and Cy3-His dissolved in a small amount of neutral water was added in aliquots until equimolar concentrations were reached. After incubation for 2 hours at room temperature, the labeled product was 50 purified by reverse phase HPLC on a Chemcobond 5C18 ODS column (4.6 x 150 mm). Elution was carried out with a linear gradient of 6% to 50% acetonitrile in 50 mM ammonium formate, pH 7.0, over a period of 30 min at a flow rate 1.0 mL/min, with monitoring of the eluate at 550 nm. The yield was 46%. HRMS (MALDI) calculated for C44H55N6O11S2 [M+] was 908.0906 and found was 908.0955. The Cy3-His-4-amino-3-butyric acid (N,N-dimethylamino) ethylester was synthesized by Lewatit lipase EL1-mediated esterification as described above. The final product was purified by reverse 55 phase HPLC, as described above, except that the gradient was 6% to 80% acetonitrile. The yield was 0.1%. HRMS + (MALDI) calculated for C53H64N9O13S2 [M ] was 1099.2815 and found was 1099.2841.

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4. His-tagged metabolite library synthesis.

[0096] The primary synthetic challenge involved finding a reaction path from a functionalized core with the highest yield (>26-90% overall). The purified metabolites were characterized by NMR and HPLC and were found to be 90% 5 pure; yields were >20%. A number of standard strategies were employed, and new ones developed for the synthesis of compound libraries synthesized on solid supports or by parallel synthesis using separate reaction vessels. The full spectrum of synthesis methods used in the present study (as well as NMR and high resolution mass spectra) will be available to the community on our web server (http://biology.bangor.ac.uk/people/staff/025123) and are briefly described in Table 1, together with metabolite synthesis 10 strategies. In some cases, the metabolites were directly purified from pure cultures using preparative HPLC (Waters 2795 XE). The purity of each SM was confirmed by NMR and mass determination (see Table 1). SMs were reconstituted and diluted in PBS buffer, DMSO or a mixture of both, accordingly to their solubility properties, and stored in 384-well microtiter plates at -70˚C until used. Metabolites were further functionalized via incorporation of a histidine tag at specific positions (SM-His; see Tables 1 to 3) using solid-solid and solid-liquid phase synthesis. 15 [0097] General synthetic methods to incorporate His-tags to different metabolites (SM) are listed in the following.

Metabolite Synthetic method to incorporate His tags characteristics

20 OH-containing metabolites Lipase (Thermomyces lanuginosus) esterification with histidine in 3-Methyl-2- butanol (Ferrer et al., Tetrahedron (2000) 56: 4053-4061)

NH2-containing metabolites Lipase (Candida antarctica) amidation with histidine in 3-methyl-2-butanol (Plou et al., Journal of Biotechnology (2002) 96: 55-66) Aliphatic metabolite (linear) Enzymatic incorporation of OH- groups via a wide spectrum dioxygenase followed 25 by esterification with lipase (Thermomyces lanuginosus) and histidine in 3-methyl- 2-butanol. Aliphatic metabolite (circular) The metabolite is dissolved in trifluoroacetic acid (20 ml) and refluxed for two hours under nitrogen. The solvent is evaporated and the residue is extracted with ethyl 30 acetate. The organic layer is washed with water, brine and dried over MgSO4. The hydroxylated compound is then esterified with histidine using Thermomyces lanuginosus lipase in 3-methyl-2-butanol. Aromatic The -OH group is incorporated as described by Callahan et al., Bioorganic and Medical Chemistry Letters 16, 3802 (2006) and the hydroxylated compound is then 35 esterified with histidine using Thermomyces lanuginosus lipase in 3-methyl-2- butanol. In this case a double bond is lost during the synthesis.

5. Generation of His-tagged metabolite-Cy3 library. 40 [0098] Incorporation of the fluorescence dye in the metabolite was achieved by reaction of the SM-His molecules with activated Cy3-His-4-amino-3-butyric acid (N,N-dimethylamino) ethylester, using solid-solid and solid-liquid phase synthesis. The resulting library is composed of individual metabolites coupled at different positions to Cy3 dye molecules, both of which carry histidine tags. The coupling of Cy3 at different positions was designed to create the full spectrum of 45 potential substrates needed to detect all possible catalytic reactions described in KEGG for a given core metabolite. Thus, each type of Cy3 substituent configuration determines the identity of the reaction and the reaction products. And, only when a reaction occurs at a specific position is the Cy3 dye released to give a fluorescent signal. [0099] General synthetic methods to incorporate different His-tagged metabolites (SM) to the activated Cy3 are listed in the following. 50 Metabolite Synthetic method to incorporate His tags characteristics Halogenated metabolites The activated Cy3 is reacted with SM-His halide in the presence of

55 a quaternary ammonium salt such as benzyltriethylammonium chloride using 50% aqueous sodium hydroxide as an acid-removing agent (Bull. Chem. Soc. Jpn., 54, 1879 (1981)).

19 EP 2 230 312 A1

(continued)

Metabolite Synthetic method to incorporate His tags characteristics 5 Non halogenated aliphatic metabolite (linear) An halogenated moiety is incorporated via treatment of the His-SM with CHCl3 + Fe(CO)5. Afterwards, the halogenated derivative is incorporated to the Cy3 as above Non halogenated aliphatic metabolite (circular) Enzymatic C-C bond formation via a wide spectrum aldolase. 10 Aromatic Aromatic nucleophilic substitution reaction between His-SM and activated Cy3 in anhydrous dimethylsulfoxide as described by Médebielle (Tetrahedron Letters, 37, 5119-5122 (1996))

15 6. Incorporation of His-tagged metabolite-Cy3 derivatives to the poly(A)-nitrilotriacetic-Co(II) complex.

[0100] The final step in the array development is the incubation of the histidine functionalized Cy3-SM with poly (A)-nitrilotriacetic-Co(II) complexes in 50 mM phosphate buffer, 50 mM NaCl, pH 7.5 for 1 hour at 25˚C. When required, DMSO was added to increase substrate solubility. The resulting complexes were separated from unbound enzyme 59 20 molecules by HPLC as described above. To ensure that each SM-Cy3 binds through both of its His residues, NMR was performed, and only derivatives incorporating single SM-Cy3 molecules were purified, and stored in 384 microtiter plates at -70˚C until used. These molecules were used directly for the construction of the array.

7. Binding of His-tagged metabolite-Cy3 derivatives to gold nanoparticles (optional).

25 [0101] Au-6,8-dithioctic acid (TA) clusters were synthesized as described by Abad et al. (Abad et al. in J. Am. Chem. Soc. 127, 5689 (2005)) and used to create Au-TA-ANTA-Co(II)-SMs-Cy3 clusters. Briefly, the Au-TA clusters were linked to the ANTA-Co(II)-SMs-Cy3 (prepared as described above) by overnight amidation in a single step in the presence of 3 mM N-hydroxysuccinimide (NHS, Fluka) and 3 mM 1-ethyl-3-[3 -(dimethylamino)propyl]carbodiimide (EDC, Sigma) 30 in 20 mM HEPES buffer (pH 7.5). Further purification was carried out by ultrafiltration through low-adsorption hydrophilic 30000 NMWL cutoff membranes (regenerated cellulose, Amicon).

SYNTHESIS EXAMPLE 1:

35 [0102] The following Synthesis Example 1 provides a complete synthesis of a probe compound comprising 5-bromo- 4-chloro-3-indoyl-beta-galactopyranoside (X-Gal) as test component (SM) and Cy3 as indicator component. Both the test component and the indicator component are linked to a transition metal complex comprising cobalt by one histidine linker moiety each. Test component and indicator component are linked by 4-amino-3-butyric acid as a linker moiety. The so-prepared probe compound is used for the analysis of the sensitivity of the reactome array. 40 1. Preparation of nitrilotriacetic-Co(II) complexes.

[0103] The amino-nitrilotriacetic-Co(II) complex was formed by reaction of NR,NR-bis(carboxymethyl)-L-lysine hydrate (ANTA, Fluka) with an excess of cobalt(II) chloride (Sigma) in 20 mM HEPES in aqueous solution (J. M. Abad et al., J 45 Am Chem Soc 127, 5689 (2005)). Excess cobalt was precipitated by increasing the pH to 10, and the precipitate was removed by filtration through a 0.2 mm membrane (PTFE, Amicon).

2. Incorporation of poly(A) tails in nitrilotriacetic-Co(II) complexes.

50 [0104] Activation of the phosphate groups of the poly(A) tails (Sigma Genosys, average molecular weight: 100 kDa) and subsequent amidation with the Co(II) complex was performed by overnight incubation with 3 mM N-hydroxysuccinimide (NHS, Fluka) and 3 mM 1-ethyl-3-[3 -(dimethylamino)propyl]carbodiimide (EDC, Sigma) in 20 mM HEPES buffer (pH 7.5).

55 3. Generation of double activated Cy3 dye.

[0105] Cyanine dye was linked via a histidine tag and a flexible linker through which the dye is linked to the Co(II)

20 EP 2 230 312 A1

complex and the metabolite, respectively. Briefly, a histidine molecule was firstly incorporated to the Cy3 dye by enzymatic acidolysis of Cy3 NHS (a succinimidyl ester, GE Healthcare) with histidine and immobilized Lewatit lipase EL1 (37) from a cow rumen metagenome (37.39 mol% incorporation of histidine in 24 h, at a ratio 1 histidine:4 NHS ester). 3-Methyl- 2-butanol was used as solvent with a water content of up to 3.2%, and the reaction was carried out at 50-55 ˚C. High- 5 performance preparative liquid chromatography was used to analyze and purify the products of the acidolysis reaction. Purified Cy3-His was dissolved in DMSO at a concentration up to 4 M and stored at -70˚C until use. [0106] In a second step, the Cy3-His was joined to a 4-amino-3-butyric acid linker. Briefly, the linker was dissolved in 0.1 M sodium borate buffer pH 8.5 and Cy3-His dissolved in a small amount of neutral water was added in aliquots until equimolar concentrations were reached. After incubation for 2 hours at room temperature, the labeled product was 10 purified by reverse phase HPLC on a Chemcobond 5C18 ODS column (4.6 x 150 mm). Elution was carried out with a linear gradient of 6% to 50% acetonitrile in 50 mM ammonium formate, pH 7.0, over a period of 30 min at a flow rate 1.0 mL/min, with monitoring of the eluate at 550 nm. The yield was 46%. HRMS (MALDI) calculated for C44H55N6O11S2 [M+] was 908.0906 and found was 908.0955. The Cy3-His-4-amino-3-butyric acid (N,N-dimethylamino) ethylester was synthesized by Lewatit lipase EL1-mediated esterification as described above. The final product was purified by reverse 15 phase HPLC, as described above, except that the gradient was 6% to 80% acetonitrile. The yield was 0.1%. HRMS + (MALDI) calculated for C53H64N9O13S2 [M ] was 1099.2815 and found was 1099.2841.

4. Preparation of His-tagged 5-bromo-4-chloro-3-indolyl-betagalactopyranoside (X-Gal).

20 [0107] A histidine molecule was firstly incorporated to the X-Gal by enzymatic esterification of histidine with X-Gal and immobilized Thermomyces lanuginosus lipase EL1 (11.2 mol% incorporation of histidine in 24 h, at a ratio 2 histidine : 1 X-Gal). 3-Methyl-2-butanol was used as solvent with a water content of up to 3.0%, and the reaction was carried out at 45˚C. High-performance preparative liquid chromatography using nucleosil C18 column using methanol:water as mobile phase was used to analyze and purify the products. Purified X-Gal-His was stored at -20˚C until use. This method 25 was used to link His molecules to carboxylate containing molecules.

5. Inorporation of X-Gal-His to Cy3-His-4-amino-3-butyric acid (N,N-dimethylamino) ethylester.

[0108] The activated Cy3 is reacted with X-Gal-His halide in the presence of a quaternary ammonium salt such as 30 benzyltriethylammonium chloride using 50% aqueous sodium hydroxide as an acid-removing agent (Bull. Chem. Soc. Jpn., 54, 1879 (1981)). By using this method, the Cy3 molecule is attached via the linker moiety to the halogenated moiety in the X-Gal molecule.

6. Incorporation of His-tagged X-Gal-Cy3 derivatives to the poly(A)-nitrilotriacetic-Co(II) complex. 35 [0109] The final step in the array development is the incubation of the histidine functionalized Cy3-X-Gal with poly (A)-nitrilotriacetic-Co(II) complexes in 50 mM phosphate buffer, 50 mM NaCl, pH 7.5 for 1 hour at 25˚C. When required, DMSO was added to increase substrate solubility. The resulting complexes were separated from unbound enzyme molecules by HPLC as described above. To ensure that each SM-Cy3 binds through both of its His residues, 59NMR 40 was performed, and only derivatives incorporating single SM-Cy3 molecules were purified, and stored in 384 microtiter plates at -70˚C until used. These molecules were used directly for the construction of the array.

EXAMPLE 5:

45 SM spotting and detection of protein-SM transformations.

[0110] SM-Cy3s (0.25 nL droplets of 3.5 pmol/l in DMSO/PBS = 1:1; spot size 400 mm diameter) were spotted by means of a MicroGrid II micro-arrayer (Biorobotics) onto a glass slide, followed by fixation by cross-linking through the poly A tail. Each SM was spotted in triplicate on each slide. Buffer controls were applied for comparison. Sixty ml of cell 50 lysate at a protein concentration of 0.1 mg/ml in PBS buffer was deposited on the slide and incubated at room temperature for 30 to 180 min. PBS buffer was used as a control. The slide was then washed with PBS and deionized water, dried by standard array slide centrifugation protocol and fluorescence intensities of the spots were measured with a microarray laser scanning system (Axon) set to a pmt of 500 and 100% laser power. Signals were analyzed and quantified using GenePix pro 4.1 software (Axon). Through the analysis of three micro-arrays of three biological replicates, average 55 values and standard deviations were calculated using the Microsoft Excel programme. The average deviation is given in the caption of Table 3. Each data point was normalized to the signal intensity obtained with X-Gal-Cy3 and pure β- galactosidase (150 pg/ml), and normalized signal intensities were compared to those produced by the control array incubated with buffer. Normalization with the Cy3 β-Gal-signal intensity eliminated errors of signal intensity variation

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between arrays. All values given in Table 3 are therefore Cy3-signal corrected signal intensities after lysate incubation compared to buffer-only incubation. On the average of four Cy3-signal intensity measurements, the signal intensity of SM-Cy3 on the buffer-incubated array was 0.2% lower than the signal intensity of lysate-incubated array, showing high reproducibility of the Cy3-signal intensity and thus the legitimacy of using it as standardization factor. 5 EXAMPLE 6:

[0111] Data Analysis. After background subtraction, signal intensities for each replica were normalized and manually analyzed using Excel program (Microsoft) and GenePix Pro 6 Demo Program (http://www.moleculardevices.com/product 10 literature/family links.php?familyid=14). MultiExperiment Viewer software (Sun Microsystems Inc) was used to visualize and compare differences in signal intensity.

EXAMPLE 7:

15 [0112] Construction of (meta)genomic libraries. DNA was extracted from three environments which differdiffering by in regard to the species composition and richness and main environmental constraints and the corresponding insert- libraries were constructed. Kolguev Island coastal seawater (KOL): A 200 ml sample of the coastal seawater of Kolguev Island (Barents Sea, Russia) was placed into a 1 L Erlenmeyer flask containing sterile crude oil (Arabian light, 0.5 % (vol/vol)) and nutrients 20 ([NH4]2PO4, 0.05% (w/vol)). After four weeks of incubation at 4 ˚C on a rotary shaker, total DNA was extracted the culture with G’NOME DNA Extraction Kit (Qbiogene; Carlsbad, California), and a metagenome expression library in the bacteriophage lambda-based ZAP phagemid vector (ZAP Express Kit, Stratagene), was constructed as described in the manufacturers’ protocols. A library of 8 x 106 phage particles, average insert size about 6 kbp, was thereby generated. Phage particles were used to infect E. coli XL1 Blue MRF’ and subsequent mass excision was performed by using E. 25 coli XLOLR cells, as recommended by the supplier. Vulcano Island (VUL): The fosmid library was established from the DNA isolated from the enrichment of microbial community from acidic pool of Porto Levante on Vulcano Island, Italy. Sulphfur and iron-containing sandy volcanic acidic (pH 1.5-4) hydrothermal pool sample was enriched with the medium 874 (pH 1.7) (DSMZ, http://www.dsmz.de) containing 0.1% (w/vol) yeast extract and was incubated for 4 weeks at 45˚C with shaking. The total amount of fosmid clones was 30 11520. The DNA was extracted using G’NOME DNA Extraction Kit (BIO101, Qbiogene) and was cloned using Fosmid Library Production Kit (Epicentre) as recommended by the suppliers. L’Atalante seawater-brine interface sample (L’A): The brine-seawater interface above hypersaline anoxic basin L’Ata- lante (Eastern Mediterranean Sea) was sampled during the MedBio2 oceanographic cruise in December 2006 from the depth of 3.431 meters. The 50 mL-samples were placed into sterile 100 ml Hungate bottles containing resazurine (anoxia 35 indicator), 1 g/L yeast extract and 2 mM 13C-glucose. The salinity measured immediately after the cast was 180 g/L (NaCl). After six months of incubation at 14˚C in the dark, the total DNA was extracted with G’NOME DNA Extraction Kit (BIO101, Qbiogene) and was further cloned using Fosmid Library Production Kit (Epicentre) as recommended by the suppliers. [0113] P. putida KT2440: the total DNA was extracted with G’NOME DNA Extraction Kit (BIO101, Qbiogene) from 40 100 ml culture of this bacterium grown as described above and was further cloned using Fosmid Library Production Kit (Epicentre) as recommended by the suppliers.

EXAPMLE 8:

45 [0114] Culture conditions and general procedure for the preparation of protein lysates. Cells of P. putida KT2440 were grown at 30˚C in 100 mL-flasks filled with 10 mL minimal medium (MM) prepared as follows. A solution with "Epure" water containing (NH4)2SO4 (2 g/L), Na2HPO412H2O (6 g/L), KH2PO4 (3 g/L) and NaCl (3 g/L) was adjusted to pH 7.0 6 0.2 and then autoclaved. The medium was supplemented with 20 mM MgSO4, 10 mM FeSO4 and 15 mM Na-succinate (from a stock solution sterilized by filtration through a 0.22 mm filter (Millipore)). Like in case of L’A and 50 Volcano libraries, the individual clones were incubated overnight without shaking at 37˚C in LB medium, 12.5 g/ml chloramphenicol, in 384 microtiter plates. An aliquot of 10 ml ach culture were pooled together in appropriate flasks filled with 1L medium and subsequently incubated 6 additional hours (OD600nm 1.5) at 37˚C. For lambda phage Kolguev library, mass excision in E. coli XLOLR was done following the protocol recommended by the supplier. The pool of clones (from phagemids of fosmids) was grown with shaking at 37˚C in LB medium, with 50 g/ml kanamycin until OD600nm 55 1.5. After cultivation the cells were harvested by centrifugation (5000 g) for 15 min to yield 2-3 g/L of pellet. The cell pellet was frozen at -80˚C overnight and then thawed. Cold PBS buffer was added directly to the frozen pellets (1.2 ml per 0.3 grs cell pellet). The mixture was vortexed to homogeneity and subsequently sonicated for 2 min (total time). The extract was centrifuged for 15 min at 15,000 g to separate cell debris and intact cells. The supernatant was carefully

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collected avoiding disturbing the pellet, and transferred to new tubes. Then, the extracts were immersed in liquid nitrogen and subjected to lyophilisation in order to avoid volatile contaminants. After that, extracts were resuspended in 1.2 mL PBS buffer and protein concentration was determined by a standard procedure (38) and further fixed to 0.1 mg/ml.

5 EXAMPLE 9:

[0115] Representative procedure for the synthesis of SMs-Cy3 gold nanoparticles for identification and N- terminal sequencing of SM-acting proteins. Au-6,8-dithioctic acid (TA) clusters were synthesized as described by Abad et al. (36) and used to create Au-TA-ANTA-Co(II)-SMs-Cy3 clusters. Briefly, the Au-TA clusters were incorporated 10 to the ANTA-Co(II)-SMs-Cy3 by overnight amidation in a single step in the presence of 3 mM N-hydroxysuccinimide (NHS, Fluka) and 3 mM 1-ethyl-3-[3 -(dimethylamino)propyl]carbodiimide (EDC, Sigma) in 20 mM HEPES buffer (pH 7.5).13 Further purification was carried out by ultrafiltration through low-adsorption hydrophilic 30 000 NMWL cutoff membranes (regenerated cellulose, Amicon). Enzymatic binding was carried out by incubation of the functionalized -3 15 nanoparticles (40 g cm ) in PBS buffer, pH 7.5 overnight at room temperature with protein solution (0.1 mg/ml in PBS). Au-TA-ANTA-Co2+-protein nanoparticles were separated from unbound enzyme molecules by ultrafiltration through low-adsorption hydrophilic 100 000 NMWL cutoff membranes (Amicon). Identification of bound proteins was performed by in situ trypsin digestion and ESI-Q-TOF MS/MS mapping (M. Ferrer et al., Nature 445, 91 (2007)) that provides improved mass measurement accuracy for peptide sequencing and enables unambiguous protein identification. ESI-

20 Q-TOF MS/MS analyses were performed at the Sequencing Core Facility of the Autonomous University of Madrid. For each experiment, up to three binding experiments were prepared and analyzed.

EXAMPLE 10:

25 PCR amplification of genes encoding hypothetical proteins from P. putida KT2440 and metagenomic libraries.

[0116] P. putida KT2440 and metagenomic hypothetical proteins were amplified by PCR from genomic and metagenomic DNA using the set of primers detailed in Table 4. Cycling parameters were 2 min at 95˚C followed by 25 cycles at 95˚C for 30 s, 66˚C for 30 s, and 72˚C for 20-140s (see specific elongation temperature for each protein in Table 4),

30 and ending with 10 min at 72˚C. KOL-1, -2 and -7 and VUL-9 proteins were amplified by PCR from the corresponding metagenomic DNA library using the set of primers detailed in Table 4. Cycling parameters were 5 min at 95˚C followed by 25 cycles at 95˚C for 1 min, 55˚C for 1 min, and 72˚C for 1 min, and ending with 7 min at 72˚C. PCR products were ligated in pGEMT plasmid (Promega).

35 EXAMPLE 11:

[0117] Gene cloning, expression and purification. All proteins characterized in this study were cloned into pET- 41 Ek/LIC vector (Novagen) and expressed with an N-terminal fusion to 6xHis tag, according to manufacturer’s instruc- tions and plasmids were subsequently isolated and introduced into E. coli ORIGAMI(DE3)pLysS expression host. Full-

40 length of gene coding for proteins were amplified from the P. putida genome by polymerase chain reaction (PCR), whereas full-length of proteins from libraries were amplified with degenerated primers based on the Q-TOF peptide obtained (Table 4). Proteins were expressed in Escherichia coli strain ORIGAMI(DE3)pLysS (Novagen). Cultures were grown overnight in Luria-Bertani medium containing 100 mg/ml ampicillin and 50 mg/ml kanamycin, then diluted 1:100 in fresh medium. Cells were grown at 37˚C to a final OD600 of 0.5, induced with 1 mM isopropyl--D-thiogalactopyranoside 45 (IPTG) and incubated at 37˚C for an additional 4 h. Cell pellets were collected by centrifugation at 4 1C for 20 min at 8,000g. Pellets were then suspended in 25 ml prechilled lysis buffer (Complete EDTA-free protease inhibitor tablet (Roche), 150 mM NaCl, 1 mM dithiothreitol, 50 mM Hepes, pH 7.0, 5 mM imidazole) and lysed by sonication on ice for 3 min with 30 s intervals. Cell lysates were centrifuged once more at 4˚C for 20 min at 30,000 g, and the soluble fractions were retained. Proteins were purified using a 1-ml HisTag (Novagen) according to the manufacturer’s protocols and

50 eluted in 250 mM imidazole and 50 mM HEPES, pH 7.0. Elutions of 500 1 were collected, pooled and concentrated 100-fold using Microcon YM-3 spin columns (Millipore). The molarities of all purified proteins were determined by using the corresponding extinction coefficient. Purified proteins were stored at -80 ˚C until further use.

EXAMPLE 12:

55 Receiver Operating Characteristic (ROC) analysis.

[0118] The "receiver operating characteristic" (ROC) analysis is used to evaluate the performance of a binary classifier

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separating two populations (positive and negative cases) and it is becoming a standard in evaluating and comparing prediction methods (T. Fawcett, Pattern Recogn Lett 27, 861 (2006)). This analysis can be applied to classifiers (prediction methods) which associate a numerical score to each case. Ideally the classifier would tend to associate high scores to the positive cases and low scores to the negative ones (or the other way around). The analysis is performed by sorting 5 all the cases (positives and negatives) by their associated scores. This sorted list is then cut at different score thresholds and for each cut the true and false positive rates (TPR and FPR) are calculated as

10

15 [0119] Where, TP: "true positives" - cases predicted as positives (above the threshold for that particular cut) which are in fact positives (correct predictions); FN: "false negatives" - cases predicted as negatives (below the threshold) which are actually positives (wrong predictions); TN: "true negatives"; FP: "false positives". "TP/(TP+FN) is also known as "sensitivity" and gives an idea of the fraction of positives recovered at this particular cut of the list (note that TP+FN 20 is the total number of positive cases). "TN/(TN+FP)" is also known as "specificity". [0120] The ROC plot is constructed by plotting TPR against FPR for the different cuts of the list. In such a representation, a random method (a classifier without discriminative power which spreads positives and negatives uniformly across the sorted list of scores) will produce a diagonal line from (0,0) to (1,1). Methods with some discriminative power will generate curves above this line. The higher the curve (closer to the (0,1) corner) the better the method. A method with a perfect 25 discriminative power (which puts all the positives together at the top of the list, associated to the highest scores) would be represented by a single point at (0,1). A parameter based in this representation which quantifies the global performance of a method in the AUC ("area under curve"). The random method commented above will produce an AUC value of 0.50, while discriminative methods will produce AUC values between 0.50 and 1.00 (perfect discrimination). [0121] In this study case, we used this analysis for evaluating the ability of our array to discriminate compounds which 30 are actually metabolized by P. putida from those which are not. The score is the fluorescence intensity, under the idea that there will be a positive relationship between the intensity in the array and the compound being metabolized in P putida. We assume that compounds metabolized by P. putida are those which act as substrates in one or more chemical reactions in the metabolism of P. putida as reconstructed from KEGG (cf. above) for that organism. We took as P. putida reactions those for which the EC code or the KEGG orthologs code (ko) are associated to a P. putida K2440 gene 35 ("PPU" in KEGG nomenclature).

EXAPMLE 13:

[0122] Calculation of Z-scores for comparing samples in terms of functional classes. The z-score (Zi) for a 40 given intensity value (Ii) is calculated as:

45

Where I and σ are the average and standard deviation of the all the intensity values in the array respectively. A Zi value of 0 means that the intensity is right in the average. A positive value means that the intensity is higher than the average, 50 and the other way around for negative values. For each KEGG functional class for which more than 1676 compounds are in the array, we calculate the average Zi value of all the compounds belonging to that class (Zi). A high Zi value indicates that this class of compounds is highly metabolized in that particular sample (array). Comparing the Zi values for a given class in two arrays (samples) it is possible to known which metabolic activities are "emphasized" or "repressed" from one condition to the other. To quantify 55 that, for each class we calculate the difference of its Zi values in the two samples (∆Zi = Zi2 - Zi1). It is important to note that the results in terms of which classes are over/under expressed are exactly the same using Zi or Ii values since, by definition, they are correlative (see equation above). We used Zi values simply because they are easier to interpret in terms of relative intensities.

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EXAMPLE 14:

[0123] Hydrogenase activity and IR measurements. The oxidation of H2 (H2 uptake) was followed spectrophoto- metrically due to the reduction of methyl viologen (MV) as described by De Lacey et al., J Biol Anorg Chem 9, 636 (2004): 5 - + 2+ .+ H2 2e + 2H +2BV 2BV

[0124] Buffers: The buffers used for the activity assay were: 1 mM MV in Tris-HCl 20 mM buffer pH 8.1 and sodium dithionite 10 or 100 mM in Tris-HCl 20 mM buffer pH 8.1. 10 Sample preparation: 0.2 mg/mL L’A62 protein in buffer Tris-HCl 20 mM pH 8.1. To this solution 1 mL of dithionite 10 mM solution is added. The IR spectra was measured as described by De Lacey et al. using a protein solution of 12 mM in Tris-HCl 50 mM pH 8.1.

EXAMPLE 15: 15 [0125] Construction rRNA gene clone libraries and clone sequencing. PCR amplification was performed with 0.1 ng DNA template. 16S rRNA genes were amplified using the Eubacteria-specific forward primer F27 (5’- AGAGTTT- GATCMTGGCTCAG-3’) in case of KOL and L’A and a universal F530 primer (5’-TCCGTGCCAGCAGCCGCCG-3’) for VUL library, in all cases in the combination with the universal reverse primer R1492 (5’-CGGYTACCTTGTTACGACTT- 20 3’). Amplification was done in 20 ml reaction volume with recombinant Taq DNA Polymerase (Invitrogen, Gemany) and original reagents, according to the basic PCR protocol, with an annealing temperature of 45˚C (VUL) and 50˚C C (L’A and KOL), for 30 cycles. PCR amplicons were purified by electrophoresis on 0.8% agarose gels, followed by isolation from excised bands using a QIAEX II Gel Extraction Kit (Qiagen, Germany). The purified PCR products were ligated into plasmid vector pCRII-TOPO (TOPO TA Cloning kit, Invitrogen, Germany) with subsequent transformation into 25 electrocompetent cells of E. coli (TOP 10) (Invitrogen, Germany). After blue/white screening, randomly picked clones were resuspended in PCR-lysis solution A without proteinase K (67 mM Tris-Cl (pH 8.8); 16 mM NH4SO4; 5 M - mer- captoethanol; 6.7 mM MgCl 2; 6.7 M EDTA (pH 8.0) (Sambrook and Russel, 2002) and heated at 95C C for 5 min. The lysate (1ml) was used as DNA template for PCR amplification using primers M13F (5’-GACGTTGTAAAACGACGGCCAG- 3’) and M13R (5’-GAGGAAACAGCTATGACCATG-3’). After verification on the agarose gel, PCR products were purified 30 with MinElute 96 UF PCR purification kit (Qiagen, Germany) and sequenced with M13 and M13R primers according to the protocol for BigDye Terminator v1.1 Cycle Sequencing Kit from Applied Biosystems (USA).

EXAMPLE 16:

35 Dose-response curves determined with E. coli β-galactosidase (β-Gal).

[0126] To prove that the present array can discern active and non-active proteins and in parallel to determine the sensitivity of the system, we determined the molecule dose-response behaviour of active and inactive -Gal and vice versa. 5-Bromo-4-chloro-3-indolyl-D-galactopyranoside (X-Gal) modified with Cy3 as described in SYNTHESIS EXAM- 40 PLE 1, was used as substrate. Figure 3 shows the X-Gal-associated pixel intensity from the scanned slides plotted against the amount of X-Gal. As shown, 30 min incubation with a solution of only 5 ng pure β-Gal ml-1 at 20˚C was -1 sufficient to ensure 50% of maximum fluorescence (F50) when 0.25 nl of a solution containing 0.12 pmol ml substrate was spotted (signal to noise (S/N) ratio above 71), while inactive protein was unable to release the Cy3 dye. Further, using 2.52 pmol X-Gal-Cy3 ml-1 50% of maximum fluorescence was reached above 1.86 ng β-Gal ml-1 (signal saturation 45 above 95 ng ml-1). According to this data, micro-array analysis were further performed by spotting 0.25 nl of substrate solutions at concentration of 2.52 pmol ml-1 and by arraying the slides with at least 0.10 mg protein lysate ml-1 to ensure detection of all proteins in the extract (it should be noted that enzyme concentrations as low as 1.5 ng ml-1 were sufficient to ensure appropriate detection with a S/N 10).

50 EXAMPLE 17:

Characteristics of microbial communities examined.

[0127] In order to assess the utility of the array for the analysis of complex microbial communities as represented in 55 metagenomic libraries, we obtained samples from three habitats with distinct physico-chemical characteristics and therefore distinct microbial communities and metabolic characteristics. The general characteristics of the three habitats are as follows: (i) acidic (pH 1.0-3) sulfur- and iron-rich (from 3 to more than 500 mg/l) sediments of a hydrothermal pool (25-75˚C) of Porto Di Levante, Vulcano Island (Italy) (ii) oil-polluted, cold (1˚C) coastal seawater sampled near Kolguev

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Island, Barents Sea, Russia, and (iii) the seawater-brine interface of the deep hypersaline anoxic brine lake of the L’Atalante Basin, Eastern Mediterranean Sea (14˚C). All samples were used to produce enrichments cultures to obtain higher biomass levels for the subsequent analyses. The Vulcano Island sediment was introduced into an acidic (pH 1.7) ferrous iron- and sulfate-rich liquid medium and incubated for 4 weeks at 45˚C to produce enrichment VOL; the Koluev 5 Island coastal water was enriched with crude oil and incubated for 4 weeks at 4˚C to produce enrichment KOL; and the seawater:brine interface sample from the L’Atalante Basin was supplemented with glucose and yeast extract and incubated anaerobically for 6 months at 14˚C, to produce enrichment L’A. Thus: the microbial communities obtained for analysis represent communities from very distinct, rather extreme habitats: a low energy, low nutrient, heavy metal-rich habitat (VOL), a nutrient and energy-rich, organic pollutant-contaminated habitat (KOL), and a hypersaline, anaerobic 10 environment, inoculated with a sample taken also from a high pressure habitat but which was subsequently maintained at atmospheric pressure, so should contain facultative barophiles (L’A).

EXAMPLE 18:

15 General pan-reactome considerations.

[0128] As shown in Figure 5, the VUL reactome consisted of 807 compounds, the KOL reactome consisted of 1493 compounds, and the L’A reactome 2386. The restricted metabolic activity of the VUL sample was not unexpected, since the diversity of the community is low, the biomass concentration is low, and the prevailing physico-chemical conditions 20 highly selective of a restricted metabolism. Similarly, the reactome of KOL was also not unexpected, since the excess carbon in the hydrocarbon-based enrichment leads to high cell densities and much recycling of cellular carbon in all its diverse forms. At first sight, it might seem that the extremely diverse metabolic profile of the L’A metagenome library is surprising, given the highly restricted diversity of the original community. However, it has been shown that a wide range of physico-chemical conditions prevail in the extremely steep chemoclines of the seawater:hypersaline brine interfaces 25 of the brine lakes and this might select for organisms expressing a broader range of metabolic activities. In addition, it should be kept in mind that, on one hand, the oligotrophic environment selects for organisms expressing a wide range of nutrient scavenging systems constitutively at low levels, and in addition, anaerobic metabolism and salt and pressure tolerance systems not specified or expressed by the other two communities, and, on the other, the E. coli cellular environment may result in expression of a range of aerobic metabolism systems encoded, but not necessarily expressed 30 under natural conditions, by the community organisms. Nevertheless, the exceptional richness of the metabolic profile of the L’A library is impressive.

EXAMPLE 19:

35 The micro-array served as experimental platform to identify gene functions.

[0129] The methodology presented here provides a new window to study the functional composition of single cells and microbial communities without apparent need of sequence information as well as to identify many uncharacterized gene-coding enzymes. This has been shown by combining the array concept with high-throughput mass spectrometry 40 peptide sequencing using metabolite-containing nanoparticles. To further prove this hypothesis we extent this analysis not only for P. putida extracts but also for the metagenome-derived extracts to analyse the possibility of mining protein diversity. [0130] To test this, we randomly select nine SMs exhibiting positive signals (fluorescence values up to 35512) in the micro-array: four for P. putida lysates (cis-2-hydroxypenta-2,4-dienoate, γ-carboxymuconolactone, 3-hydroxyanthrani- 45 late and dimethylallyl diphosphate) and five for KOL, VUL and L’A lysates (undecane, (S)-4,5-dihydroxypentan-2,3- dione, 2-bromo-1-chloropropane, phosphatidylinositol-4,5-bisphosphate and methyl viologen). To identify the proteins responsible for their transformation the corresponding Cy3 derivatives were synthesized, immobilized in a gold-magnetic nano-particle, and further incubated with P. putida or library lysates (Figure 1, step 5). After 30 min at 20˚C incubation, the gold particles were separated by either magnetic attachment or centrifugation (5000 g x 15 min) and the attached 50 proteins were identified by trypsin digestion followed by Q-TOF sequencing. Using peptide sequence fragments, degenerated oligonucleotides were designed, the corresponding genes were amplified using (meta)genomic DNA, cloned into the pET30 Ek/LIC expression vector and the proteins purified with a 6 x His tag. Analysis of fluorescence emission when different amounts of pure protein were incubated with different concentration of substrates and vice versa revealed that the obtained results matched the reactome data (Table 3): Cy3 fluorescence emission increased when increasing 55 the amount of both protein and substrate while inactive proteins do not (Figure 6). [0131] P. putida proteins: Sequence analysis revealed the identity of proteins acting against cis-2-hydroxypenta-2,4- dienoate, γ-carboxymuconolactone, 3-hydroxyanthranilate and dimethylallyl diphosphate - all of them correspond to the hypothetical proteins with no assigned function annotated PP_1394, PP_1752, PP_2949 and PP_1642 (Table 4). How-

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ever, the experimental analyses provided here suggest that these enzymes are accordingly new 2-keto-4-pentenoate hydratase, 3-carboxy-cis,cis-muconate cycloisomerase, 3-hydroxyanthranilate 3,4-dioxygenase and isopentenyldiphos- phate delta-isomerase (see Table 4). This has also been shown by examining the activity of the pure proteins against standard substrates (data not shown). The correlation of this identity with genomic context is as follows: here we observed 5 that surrounding compounds are also metabolized by P. putida lysates, thus suggesting the presence of new metabolic pathways. A case of interest is the ability of protein PP_1394 to transform the conversion of cis-2-hydroxypenta-2,4- dienoate to cis-2-hydroxypenta-2,4-dienoate as a part of the biphenyl degradation pathway. This pathway is not annotated in KEGG for P. putida KT2440 strain (cf. above), in spite of the fact that strains F1, GB1 and W619 clearly possess some genes responsible for p-cymene and 4-chlorobiphenyl degradation (see http://www.genome.jp/kegg/). In contrast, 10 array hits were found for the entire set of metabolites ranging from p-cymene to 2-hydroxy-6-oxo-7-methylocta-2,4- dienoate as well as from 4-chlorobiphenyl to cis-2,3-dihydro-2,3-dihydroxy-4’-chlorobiphenyl (fluorescence values up to 6931) (Figure 6) and further analysis of nanoparticle proteome analysis have also revealed the identity of proteins doing those transformations. These data suggest that the lysate of KT2440 contains enzymes that are able to metabolize those intermediates, even though genome information per se does not provide any evidence for that. This suggests in 15 turn that many proteins may potentially enable, yet unknown, important catabolic activities expanding our knowledge on microbial metabolism of this bacterium. [0132] Metagenomic proteins: Sequence analysis revealed that all of metagenomic proteins acting against undecane, (S)-4,5-dihydroxypentan-2,3-dione, 2-bromo-1-chloropropane, phosphatidylinositol-4,5-bisphosphate and methyl viologen exhibited a high degree of similarity to predicted hypothetical proteins with no function assigned (Table 4), except 20 L’A62, a 162-amino acid polypeptide with a predicted molecular mass of 18,068 Da along with estimated pI of 4.55, that exhibited high similarity to a predicted [NiFe] hydrogenase from Carboxythermus hydrogenoformans (7e-54). Data suggest these enzymes are new alkane hydroxylase, S-ribosylhomocysteine lyase, haloalkane dehalogenase, phosphatidylinositol-bisphosphatase and hydrogenase. To prove that assigned function are correct we select and preliminary characterized one of the most promising enzyme candidates, the L’A62 protein and reaffirmed this was indeed the case. 25 A FTIR (Fourier Transform Infrared) spectrum of protein 62 was recorded at a final concentration of 8 M (Figure 6). The bands that appear in the 2150-1900 cm-1 region are typical of the 1 carbonyl and 2 cyanide ligands of the active site of standard NiFe-hydrogenases (J.C. Fontecilla-Camps et al., Chem Rev 107, 4273 (2007)). The band of frequency value of 1947 cm-1 and the group of bands between 2080 and 2095 cm-1 correspond to the carbonyl ligand and the cyanide ligands respectively of this type of hydrogenases in the "unready" and "ready" oxidized states (A. De Lacey et al., 30 Biochem Biophys Acta 832, 69 (1985)). The band at 1936 cm-1 can be assigned to the carbonyl ligand of irreversibly inactivated enzyme, whereas the bands at 2060 and 2073 cm-1 can be assigned to the cyanide ligands of the same state. Further, the H2-uptake activity of protein 62 was measured in a spectrophotometer using methyl viologen as electron acceptor (Figure 6, inset). The protein isolated under aerobic conditions had the typical lag-phase of several minutes in the activity profile of standard NiFe-hydrogenases in the "unready" oxidized state (V. M. Fernandez et al., 35 Biochim Biophys Acta 832, 69 (1985)). Incubation under H2 of the protein during several hours led to disappearing of the lag-phase and an increase of the maximum activity. This activation process is also a functional characteristic signature of standard NiFe-hydrogenases. Remarkably, the structural and functional data measured suggest an active site very similar to that of standard NiFe-hydrogenases, although the overall protein structure is unique up to date for this type enzymes (just 1 subunit of very small size, an amino acid sequence with no motifs for iron-sulfur clusters nor with the 40 typical L1 or L2 signatures) (P.M. Vignais, B. Billoud, Chem Rev 107, 4206 (2007)).

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227 EP 2 230 312 A1

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229 EP 2 230 312 A1

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230 EP 2 230 312 A1

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231 EP 2 230 312 A1

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232 EP 2 230 312 A1

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233 EP 2 230 312 A1

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234 EP 2 230 312 A1

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235 EP 2 230 312 A1

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236 EP 2 230 312 A1

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237 EP 2 230 312 A1

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238 EP 2 230 312 A1 0 0 135 686 1905 2083 2800 5 (- L’A background) 491 774 2149 1993 2171 2888 10 1467 104,75 16,75 4790,5 2776,5 4562,5 4474,5 417,25 329,25 1529,25 1441,25 10099,3 10011,3 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 15 0 (- VUL background)

20 6 35 74 -46 -21 -17 7292 7219 2505 2429 4852,25 4764,25 4450 4384 5975,75 5887,75 (raw data) VUL 0 0 0 0 0 0 25 0 89 29 19157 19091 1641,75 1553,75 (- KOL background)

30 -6 95 72 34 -58 -16 155 2988 2922 -67 6662 6596 -117 4487 4421 -83 (raw data) KOL 0 0 0 0 11724 11633 -29 0 0 0 35 0 45 40 80 73 2721 2564 10 168 520 454 216 -61 2440 (- KT succinate background) Table 3. Extensive description of microarray data

40 73 83 -86 -63 -53 169 109 119 164 204 292 197 340 -118 9281 9157 -60 5225 5101 2795 2729 -16 2440 (raw data) KT succinate

45

50

55 v-Triazole 1,2,3-Triazine 1,2,3,5-Oxatriazole 1,2,3-Oxadiazole 5-Chloro-3-methylcatechol Compound gamma-Nonalactoneepsilon-Caprolactone10-Oxodecanoate1-Bromodecane2-Bromodecane10-Formyltetrahydrofolate4-Hydroxy-5-phenyltetrahydro-1,3-oxazin- 2-one 86 -89 532 0 480 -290 011-Hydroxyundecanoate 20 408 2329 356 0 81 2488 2263 0 3216 -59 0 2383 65 2900 3150 4951 0 38 2834 0 7 4846 -47 -21 0 1332,5 219,25 0 0 0 1244,5 291 131,25 549 512,5 399,25 203 461 311,25 424,5 3-Methylcrotonyl-CoA 2-Chloromaleylacetate 1,3-Dichloropentane 11-Oxoundecanoate Oxatriazole (R)-1,2,4-Butanetriol 1,4,2-Dioxazole 1,3,4-Oxadiazine 2-Methyl-1,3,4-oxadiazole

239 EP 2 230 312 A1 0 282 156 305 392 435 5 (- L’A background) 370 244 393 480 523

10 1960 660,25 572,25 334,25 246,25 3201,5 1187,5 936,25 848,25 3102,5 3014,5 615,75 527,75 7111,25 7023,25 6554,75 6466,75 5598,25 5510,25 1415,25 1327,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 (- VUL background) 3 20 5 46 -90 -64 -54 -46 -85 -115 2621 2555 5414,5 5326,5 (raw data) VUL 0 0 0 0 0 25 0 29 76 (- KOL background)

30 0 95 -32 -39 -34 142 -121 1838 1772 37 5729 5663 -96 6500 6396 21 4394 4238 10773 10617 3737,5 3649,5 5477 5411 (continued) (raw data) KOL 0 1 0 0 0 0 0 0 0 35 0 75 62 7106 7033 -10 38 7243 7155 22 1671 1605 3061 2995 181 128 -71 116 491 340 -142 472 3064 2998 -11 2440 (- KT succinate background)

40 -7 51 24 82 71 -72 -74 199 305 252 162 125 186 240 146 596 -181 -285 2440 (raw data) KT succinate

45

50

55 1,2-Dihydroxynaphthalene 1-H-pyrazole-3-carboxamide Compound 1,2-Benzoquinone 1,4-Dichloro-2-butene 1,2-Didecanoyl-3-beta-D-galactosyl-sn- glycerol Decane-1,2-diol 1,1-Dibromopentane Dodecane-1,2-diol 1,2-Dihexanoyl-3-beta-D-galactosyl-sn- glycerol 1,2,4-Triazole 1,2-Dipropionyl-3-beta-D-galactosyl-sn- glycerol 1,2-Dibutyryl-3-beta-D-galactosyl-sn- glycerol cis-1,2-Dihydroxy-1,2-dihydro-8- methylnaphthale 1,2-Butanediol 1,2,5-Oxadiazole 2,4,6-Tribromophenol 1,3,5-Triazine(1R,2S)-1,2-Dihydronaphthalene-1,2-diolketone methyl isobutyl Oxathiazine 3010 2886 41 252 0 128 4988 -67 4922 0 -23 3071,5 0 1057,5 9135,5 7121,5

240 EP 2 230 312 A1 291 1509 4145 477,5 5 (- L’A background) 379 1597 10 4233 565,5 687,75 599,75 240,75 152,75 351,75 263,75 6489,75 6401,75 2863,25 2775,25 3974,75 3886,75 L’A (raw data) 0 0 0 0 0 0 0 0 15 0 18 (- VUL background)

20 3 84 (raw data) VUL 0 25 0 34 -107 (- KOL background)

30 51 100 3803 3737 -118 2112 1985 -47 1526 1336 -149 2800 2734 -37 1046 817 -10 4962 4740 4173 3951 360,75 272,75 (continued) (raw data) KOL 0 0 0 0 0 0 35 0 25 2403 2213 53 177 4802 4736 31 104 2440 (- KT succinate background)

40 50 -62 -18 -46 -83 -51 -19 149 301 238-45 114 228 2440 (raw data) KT succinate

45

50

55 1,2-Dipalmitoyl-beta-D-galactosyl-sn- glycerol 1,2-Oxazine 1,2-Dioctanoyl-3-beta-D-galactosyl-sn- glycerol 6-Deoxyerythronolide 1,2-Dipalmitoyl-3-O-[alpha-D- glucopyranosyl(1->2)-O-alpha-D- glucopyranosyl]-sn-glycerol Compound glucopyranosyl)-sn-glycerol 1,2-Didodecanoyl-sn-glycerol 1,2-Dihydroxydibenzothiophene cis-1,2-Dihydroxy-1,2- dihydrodibenzothiophene 1,2-Didodecanoyl-3-O-(alpha-D- glucopyranosyl(1->2)-O-alpha-D- glucopyranosyl)sn-glycerol 1,2-Didodecanoyl-3-O-(alpha-D- 1,2-Dichlorobenzene1,2-Dichloropentane1,1-Dichloropentane3-Hydroxy-1-indanone1,2-Diaminopentane(1S,2S)-1,2-Dihydronaphthalene-1,2-diol1,2-Didodecanoyl-3-beta-D-galactosyl-sn- glycerol 88 149 88 340 62 25 0 368 0 216 2889 0 3514 244 8558 68 2823 3448 3304 3271 8492 2 -80 106 3238 3205 -29 2927 40 41 0 97 0 2861 608,25 539,25 0 31 968,5 264 520,25 451,25 2380,75 374 880,5 2292,75 176 286

241 EP 2 230 312 A1 4 3905 5 (- L’A background) 92

10 3993 10105 10017 2487,5 2399,5 2315,25 2227,25 L’A (raw data) 0 0 0 0 15 0 (- VUL background)

20 15 56 64 (raw data) VUL 0 25 0 23 (- KOL background)

30 89 55 -41 4222 4103 59 (continued) (raw data) KOL 0 0 35 0 90 1002 868 2489 2355 7077,5 5063,5 161 1713 1580 -42 141 2440 (- KT succinate background)

40 8 0 -109 0 92 26 135,25 47,25 26 18 -95 214 285 265 2440 (raw data) KT succinate

45

50

55 Compound 1,2-Epoxycyclohexane1,2-Heptadecanediol1,7-HeptanediolHexacosane-1,2-diol1,2-Hexadecanediol1,2-Hexanediol1,2-Nonanediol 431,2-Nonadecanediol3-Isopropylbut-3-enoyl-CoA 51,2-Octadecanediol 01,2-Octanediol 82 -31propionateD-()-Solketal 97 01,3-Oxazole -781,2-Pentanediol 0 886 0 261,2-Dibutyryl-sn-glycerol -45 3971 85 01-phosphate Propane-1,2-diol 0 1497 762 104 -92 3853 0 3338 0 10 0 241 1431 -153 -52 38 39 2343 0 3252 1084 188 -17 0 929 153 -29 117 0 162 2277 0 72 0 62 960 122 1880 863 0 4667 29 110 245,75 0 96 4 0 -43 136 0 164 1750 4601 214,75 1884 3339 157,75 44 420,25 51 0 70 33 4029 0 126,75 415 -55 1812 76 3188 332,25 1278 0 3963 498,75 6402,5 -128 2802 537,5 0 0 327 1190 410,75 540 -20 6314,5 2651 449,5 0 6693,5 400 7103,5 0 452 9752,25 6605,5 312 7015,5 9664,25 10291,3 10203,3 1,2-Dipaimitoyl-3-O-alpha-D- glucopyranosyl-sn-glycerol Propane-1,2-diol (R)-Propane-1,2-diol (S)-Propane-1,2-diol 1,2-bis-O-Sinapoyl-beta-D-glucoside 1,2-Tetracosanediol

242 EP 2 230 312 A1

5 (- L’A background)

10 L’A (raw data)

15 (- VUL background)

20 (raw data) VUL

25 (- KOL background)

30 (continued) (raw data) KOL

35 2440 (- KT succinate background)

40 2440 (raw data) KT succinate

45

50

55 Compound 1,2-Thiazole1,2-Undecanediol2-Chloro-4-hydroxy-6-amino-1,3,5-triazine1,3,4-Oxadiazole1,3,4-Thiadiazole 867Dithiazine1,2,5-Oxadiazine 7431,3,5-OxadithianeThiadiazine 224 2443-isopropylmalate -151,2-biphosphateD-myo-inositol 1,3,8-Trihydroxynaphthalene 46 100 120 2012,3-bisphospho-D-glyceratecyclic 01,3-Butanediol 2602,4-Dichloroaniline 154 4551 0 1290 -16 561 771,3-Diazole 721,3-Dibromobenzene 48 236 4485 1248 136 88 11661,2-Dihexanoyl-sn-glycerol 216 -7 437 24 0Deoxyuridine 0 4614 1121,3-Dichloropentane 1124 5453 4885 0 -1 25251,3-Dichlorobenzene 92 0 0 611,3-Didecanoyl-sn-glycerol 4548 1618,75 3254 5387 207 243 4819 1151,3-Dimethylbenzene -103 2455 1691 3070 0 39 1530,75 105 1767,25 3184 11 0 49 42 231 49 119 83 -55 0 1567 3004 7041,75 39 316 -89 0 0 2616 -52 0 2525 6204 72 2421 107 0 0 97 2963 68 0 5027,75 113 192 48 2376 0 6138 2492 606 112 2296 2897 10711 0 6721,25 0 6 5366 0 3497,25 10 0 5526 2784,25 606 89 -48 1638,75 4707,25 647,75 0 518 0 8697 3409,25 -18 5278 0 4437 121 120,25 770,25 5460 2 447 0 0 0 -114 1984 0 4371 0 32,25 194 -73 33 8282 0 1822 4179,25 770,75 0 -26 128 -9 8123 0 4091,25 -52 5702,5 682,75 0 270 467,5 779 0 5614,5 654 0 132,5 379,5 182 691 436,5 566 60 44,5 348,5 0

243 EP 2 230 312 A1 970 257,5 5 (- L’A background)

10 1058 345,5 4823,25 4735,25 L’A (raw data) 0 0 15 0 31 6256,25 6168,25 (- VUL background)

20 2 39 97 (raw data) VUL 0 25 0 (- KOL background)

30 45 (continued) (raw data) KOL

35 0 14 5359 5293 2440 (- KT succinate background)

40 64 138 2508 2384 3899 3731 55 2177 2053 24 2440 (raw data) KT succinate

45

50

55 Compound methyl 1,7-dimethyluric acid1,3-Diolein1,3,2-Dioxazole(R)-2-Methylmalate1,3,5-Trithiane1,2-Dipalmitoyl-3-myristoylglycerol 541,3-Dioctadecanoyl-sn-glycerol1,3-Dithiane1,2-Dioleyl-sn-glycerol 03-Hydroxy-3-methyl-2-oxobutanoate 2Pentadecane-1,2-diol 117 87 0 55Propane-1,3-diol 44712,6-Dihydroxycyclohexane-1-carboxyl-CoA 1411 0 -801,3-Oxathiole 0 11116 4405 0 01,3-Oxazole 0 1287 331,3-Pentanediol 106 0 10992dTTP 2661 -86 6858 -7 -142 -426-Hydroxycyclohex-1-ene-1-carboxyl-CoA 5927 3 12201,4-Butanediol 0 0 2595 147 6666 4179 2851-Butanoyl-sn-glycerol3-phosphate 0 5861 0 0 0 1154 0 379,25 3038 18554 -51 81 4055 2040 106 161 -86 -101 345 -50 18360 31 2972 291,25 55 92 42 -148 1974 24 0 3813 0 3264,75 0 0 279 -142 0 0 26 2755,5 0 2327 126 3711 0 0 5721,25 3176,75 0 -147 485,75 193 236,25 0 12140 2667,5 2496 161,75 2203 5633,25 2436 482 -79 -43 2 348,25 397,75 12074 0 148,25 127 2394 335,5 4407 73,75 2329 3105 394 0 0 260,25 329,75 3753,5 -101 11491,5 4169 3398 247,5 3039 241,75 2104,75 -82 11403,5 3665,5 0 3291 -26 2016,75 0 10192,8 0 8466,5 0 10104,8 0 5192,5 8378,5 1052,25 295,25 5104,5 964,25 207,25 1,4-Dibromo-2-butene 4-phosphateD-Erythrose 1,4-Dichlorobutane

244 EP 2 230 312 A1 69 789 335,5 5 (- L’A background) 877 10 157 423,5 214,75 126,75 3116,75 3028,75 1530,75 1442,75 2984,25 2896,25 L’A (raw data) 0 0 0 0 0 15 0 (- VUL background)

20 65 -72 -20 -74 951 739 5144,75 5056,75 (raw data) VUL 0 0 2 0 25 0 (- KOL background)

30 38 68 28 -78 744 678 174 108 (continued) (raw data) KOL 0 0 0 35 0 23 378 2464 2206 -134 2440 (- KT succinate background)

40 51 79 -13 502 147 119 4325 4201 55 1780 1656 4223 4157 -77 2440 (raw data) KT succinate

45 cis-13-trans- 50

55 Compound 1-Heptadecanoyl-sn-glycerophosphocholine (15S)-15-Hydroxy-5,8,11- icosatetraenoate 1,3-Dichloropropane1,4-Dimethylbenzene1,4-Dioxane1,4-DithianeMaltoseMaltotrioseMaltotetraoseMaltopentaose 20Maltohexaose 53Maltoheptaose1,5-Anhydro-D-fructose 01,5-Anhydro-D-mannitol 108 01,5-Anhydro-D-glucitol 471,2-Dihydroxycyclohexa-3,5-diene-1- 27carboxylate 3578 0 94 511,5-Dichloropentane 71 294 0 3512 41 0 21 -141 59 0 97 0 0 4721 170 50 -801,2-Pentanediol 14 0 0 02-Pentadecanol -29 4552 682 0 7533 4718 01,1-Dichlorohexane 0 0 0 810 1439 63 -164 7467 4640 616 0 5321 26922 6575 1358 57 657 7655 9755 26856 0 5255 0 -11 184 -94 12858 7589 6487 1510 9677 0 0 -16 336,75 4838,25 0 96 12777 0 1357 677,75 506,75 4750,25 248,75 278 0 0 770 465 318,5 2738,75 589,75 418,75 80 4796,25 220,25 2650,75 230,5 682 377 4708,25 132,25 320 232 1,6-Naphthyridine

245 EP 2 230 312 A1 0 0 57 90 155 4780 5 (- L’A background) -6 145 178 243

10 4868 347,75 259,75 965,25 877,25 500,25 412,25 1840,25 1752,25 L’A (raw data) 0 0 4 0 5 4 0 15 0 11 (- VUL background)

20 -6 70 11 71 (raw data) VUL

25 0 (- KOL background)

30 -32 919 853 319 253 2569 2465 -94 2894 2819 79 3770 3704 -48 4520 4435 96 9662 9528 22 (continued) (raw data) KOL 0 0 0 0 0 0 0 35 0 137 889 823 168 876 810 8222 8156 100,25 12,25 520 2549 2483 5632 5566 45,75 2440 (- KT succinate background)

40 2 39 21 83 -30 261 292 103 644 -193 0 3479 3413 -3 0 1457 1369 -205 2440 (raw data) KT succinate

45

50

55 Compound 1-Butanoyl-sn-glycerol-3-phosphocholine -99 1-Chlorobutane 1-Chlorodecane 1-Bromobutane 1-Bromodecane 1,4-Dichlorobenzene beta-D-Fructose 6-phosphatebeta-D-Fructose Dicarbomethoxynaphthalene 2-Chloro-4-methylphenol 3-Chloropropanoic acid 3-Chloropropanoic 6-MethyluracilviologenMethyl 1,7-Naphthyridine1,8-Dichlorooctane1,8-Naphthyridine1-Aminocyclopropane-1-carboxylate1-Acetyl-sn-glycerol3-phosphate 1-Acetyl-5-Hydroxy-1,5-dihydro-pyrrol-2- one 3284 2361-Butyrylglycerol 621-Octanoyl-sn-glycerol-3-phosphate 92 88 953,5-Dibromo-4-hydroxybenzoate 3160 112acetate 4-Bromophenyl 26 0 8193 0 140 0 0 568 161 0 8069 25 74 209 2868 502 4558 9 37 531 50 7282 2713 143 0 4485 -126 0 465 -18 7216 36 0 -16 -45 -37 0 3907,75 -61 -22 0 -66 0 0 0 0 325,5 3819,75 0 0 105 7873,25 0 5948,5 168 237,5 141 2733,75 7785,25 16 0 5860,5 6222,5 80 2645,75 53 6134,5 0 84,5 151,75 0 63,75

246 EP 2 230 312 A1 50 77 27 84,5 76,5 5019 5442 3802 6210 736,5 172,5 5 (- L’A background) 138 165 115 5107 5530 3890 10 6298 172,5 164,5 824,5 260,5 422,75 334,75 703,25 615,25 304,25 216,25 5315,5 5227,5 176,25 88,25 3296,5 3208,5 350,25 262,25 5545,75 5457,75 5144,25 5056,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 25 31 (- VUL background)

20 3 91 33 28 -11 -10 -22 -49 161 -198 -167 7501 7435 2278,75 2190,75 (raw data) VUL 0 0 0 0 0 0 0 0 25 0 67 33 (- KOL background)

30 -2 61 99 99 24 -23 -37 -88 -62 133 111 4491 4425 -29 7517 7451 6321 6255 -12 2759 2693 97 1601 1403 41 (continued) (raw data) KOL 0 0 0 30534 30341 -61 5 0 0 0 0 0 0 0 1 0 0 35 0 19 67 50 3484 3418 23 223 1238 1019 920 701 497,25 409,25 925 1198 1132 -82 623 4643 4506 63 2440 (- KT succinate background)

40 2 39 19 95 90 77 -44 -11 143 118 129 191 347 174 747 125 -117 -102 1774 1650 1678 1612 -85 1049 2440 (raw data) KT succinate

45

50

55 Compound 1-Dodecanoyl-sn-glycero-phosphocholine 13 1-Decanoyl-sn-glycerol3-phosphate 1-Dodecanol Chlorotoluene1-Deoxy-D-altro-heptulose7-phosphate 114 21 0 3928 3739 -25 0 423,5 335,5 trans-1-Decalone 2,6-Dichlorophenol 1H-2-Benzopyran-1-one 1-Decanol 1-Decanoyl-sn-glycero-phosphocholine 1,5-Naphthyridine 1-Hydroxybutane-1,2,4-tricarboxylate 1-Heptadecanol 1-Dodecanoyl-sn-glycerol3-phosphate 3,4-Dichlorophenol 2-Heptanol 1-Heptanol trans-Cyclohexane-1,2-diol 1-Palmitoylglycerophosphocholine 1-Methylnicotinamide 4-Imidazolone-5-propanoatemethyl beta-D-Fructose 1,6-bisphosphatebeta-D-Fructose 1-Hexanoyl-sn-glycero-phosphocholine

247 EP 2 230 312 A1 422 1331 437,5 309,5 5 (- L’A background) 510

10 1419 525,5 397,5 1239,5 1151,5 994,75 906,75 6149,5 6061,5 2925,5 2837,5 9375,5 9287,5 5818,5 5730,5 3581,5 3493,5 6607,5 6519,5 235,75 147,75 11424,3 11336,3 16604,3 16516,3 5873,75 5785,75 L’A (raw data) 0 0 0 0 0 0 0 01 10615,5 0527,5 0 0 0 0 0 0 0 0 15 0 14 1141,25 1053,25 (- VUL background)

20 7 66 83 32 16 -46 -51 -71 -89 -51 -65 246 180 2205,5 2117,5 -125 (raw data) VUL 0 0 0 0 0 0 0 0 0 0 25 0 (- KOL background) 2 5 30 1 46 26 34 36 70 -32 210 144 2151 2085 -87 5110 5001 -4 5326 5099 905 678 9948,75 9860,75 (continued) (raw data) KOL 3 0 0 0 0 0 0 0 0 0 35 0 20603 20466 -29 21 98 537 2440 (- KT succinate background)

40 41 64 87 -54 -28 -25 661 127 102 105 101 145 222 1936 1812 2880 2781 -12 4187 4063 -50 1140 1016 6076 5945 105 2906 2782 15631 15565 80 15919 15795 6610 6515 2440 (raw data) KT succinate

45

50

55 Compound 1-Methoxynaphthalene 1-Monododecanoylglycerol 2-Carboxy-2,3-dihydro-5,6-dihydroxyindole 1620 1496 -35 1-Indanone 1R-Indenol 1-Monotetradecanoylglycerol Methyl pyridine-3-carboxylateMethyl 2-Hydroxymethylnaphthalene 1-Hexanol 1-Monohexanoylglycerol Indan-1-ol 1-Nitrosonaphthalene 1-Nitronaphthalene 1-Nitronaphthalene-7,8-oxide 1-Nonadecanol 1-Nonanoyl-sn-glycero-3-phosphocholine 107 1-Monooctanoylglycerol 1-Monodecanoylglycerol 1-Methylnaphthalene5-Hydroxyindoleacetaldehydenucleoside 2,5-Diaminopyrimidine triphosphate 243 180 119 56 54 7308 0 7176 3891 571-Nitronaphthalene-5,6-oxide 3759 0 5554,25 673,25 5466,25 585,25

248 EP 2 230 312 A1 0 623 7868 6153 2093 1265 2973 5 (- L’A background) 711 7956 6241 2181 3279 10 3061 5336,5 5248,5 667,25 579,25 7994,5 7906,5 3077,5 2989,5 1749,75 1661,75 1047,25 1530,75 1442,75 6340,75 6252,75 L’A (raw data) 0 0 0 0 0 8 0 0 0 0 0 15 0 16 (- VUL background)

20 -8 80 74 24 82 -56 -86 -51 -36 -14 3244 3178 713,25 625,25 (raw data) VUL 0 0 0 0 0 0 0 0 0 0 25 0 15526 15460 5345,5 5257,5 (- KOL background) 1

30 -2 16 45 19 20 20 -68 100 211 145 -218 7410 7344 3646 3580 9173,25 9085,25 3053 2987 17695 17629 6858,75 6770,75 (continued) (raw data) KOL 1 0 0 4 0 0 35 0 13 68 72 68 4061 3995 -2 83 3310 3244 22 45 4321 4255 186 120 198 266 412 3361 3295 -47 134 2440 (- KT succinate background)

40 67 53 81 52 322 125 390 196 169 207 258 128 192 2157 2033 -43 11899 11775 2553 2208 2963 2618 7928,25 7840,25 2440 (raw data) KT succinate

45

50

55 Compound 1-Hexadecanoyl-sn-glycerol3-phosphate 536 Phenylacetate 1-O-Hexadecanoyl-glycerol 1-Propionyl-sn-glycero-3-phosphocholine 81 12-Pyren-1-yldodecanoicacid 1-Nonanol1-Oleyl-sn-glycerol3-phosphate6-Oxo-2-hydroxycyclohexane-1-carboxyl- CoA 2-Octanol1-Octadecanoyl-sn-glycerophosphocholine 351-Octanol 0 79 72 0 264 0 -95 140 -6 0 -95 0 -70 0 1222 121 0 1134 0 917 195,25 829 107,25 1-Palmitoyl-3-stearoyl-rac-glycerol 1-Pentanol 1-Octadecanol 1-Octanoyl-sn-glycero-3-phosphocholine 192 2,3,4-Trichlorobiphenyl 1-Pyrroline 1-Tetracosanol 1-Tetradecanoyl-sn-glycerophosphocholine2,2’,5-Trichlorobiphenyl 137 1-Undecanol 1-O-Sinapoylbeta-D-glucoside

249 EP 2 230 312 A1 0 0 0 272 312 539 355 4335 2866 191,5 326,5 111,5 773,5 5 (- L’A background) 360 400 443 -7,5 6349 2553 10 2954 279,5 199,5 861,5 5260,5 5172,5 1840,5 1224,5 1136,5 166,75 78,75 19140,8 19052,8 5431,75 5343,75 1408,75 L’A (raw data) 0 0 0 0 7 0 0 0 0 0 0 0 0 15 0 71 (- VUL background)

20 41 13 -54 -41 -15 -32 -46 137 6931 6865 414,5 4305 4239 (raw data) VUL 0 0 0 0 0 0 0 25 0 13 17 (- KOL background) 7

30 -6 79 21 83 -66 -46 -66 3488 3366 -44 2323 2182 -61 4364 4298 -13 2789 2610 137 3570 3504 3396 3330 4075,75 3987,75 3526 3407 126 2575 2509 -49 2490 2424 7968 7902 2862 2796 -81 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 0 0 0 0 35 0 17 652 -36 195 188 2440 (- KT succinate background)

40 98 10 67 21 54 96 82 15 -21 -20 -40 -63 141 776 319 118 312 -145 2440 (raw data) KT succinate

45

50

55 Compound 2,3,5,6-Tetrachlorobiphenyl 2,1-Benzisoxazole1-Tetradecanol1-Bromo-2-(2-bromoethyl)benzene1-Chloro-2-(chlorophenylmethyl)benzeneacid 2,2-Dichloropropanoic -411-Pyrroline-5-carboxylate 3 112-(2,6-Dibromophenyl)-1H-pyrrole 0 2665 0 0 9629 2541 2275 9563 39 62 2183 6 0 73 0 0 8768 0 6 8658 872,25 252,75 0 294,25 784,25 164,75 1799,25 206,25 0 1,3-Thiazole (2,3-Dihydroxybenzoyl)adenylate 2,3,4-Trichlorophenol 2,4,5-Trichlorophenol 2,3,6-Trichlorophenol 2,3-Butanediol 2,4,4’-Trichlorobiphenyl 2,3-Dihydroxybenzoate 1-Phenyl-1,3-butanedion 2,3-Dihydro-2,3-dihydroxybenzoate 2,3-Dihydroxy-3-methylpentanoate 1-Chloro-3-(2-chlorophenyl)benzene cis-2,3-Dihydroxy-2,3-dihydro-p-cumate2,3-Dihydro-2,3-dihydroxybiphenyl 7055 6931 -4 3-(4-Chlorophenyl)benzene-1,2-diol

250 EP 2 230 312 A1 0 0 0 0 0 23 33 31 239 1179 410,5 333,5 5 (- L’A background) 327 111 121 1885 3193 10 2045 498,5 995,5 441,25 353,25 632,75 364,75 276,75 487,25 399,25 1432,5 456,75 368,75 252,25 164,25 382,75 294,75 7286,75 7198,75 2123,75 109,75 3514,75 3426,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 (- VUL background) 0 20 3 21 10 -40 105 -124 3619 3420 1570,5 8296 8192 (raw data) VUL 0 0 0 0 0 25 0 10 43 9912 9846 421,5 39 -214 53 (- KOL background)

30 -79 -48 -77 123 109 105 119 3087 3021 10 1863 1797 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 35 0 22 5671 5585 -29 62 3613 3494 46 188 -36 138 2676 2610 24 117 7126 6905 83 205 11709 11643 353 287 2440 (- KT succinate background)

40 3 73 98 35 32 50 -11 -16 312 109 262 146 241 329 186 9198 9074 2223 2157 -137 1443 1319 -33 1698 1574 5753 5631 87 3515 3391 3628 3475 -11 4796 4672 12 2565 2441 1954 1841 103 2440 (raw data) KT succinate

45

50

55 Compound 2,3-Diketo-L-gulonate 2,3-Dihydroxy-isovalerate 2,4,5-Trichlorobiphenyl 1,2-Dimethylnaphthalene 2,3-Dihydroxy-p-cumate cis-2,3-Dihydro-2,3-dihydroxy-4’- chlorobiphenyl 2,4’,5-Trichlorobiphenyl 4-Hydroxyphenylpyruvate 2,3-Dihydrodipicolinate 1-Chloro-4-(2,2-dichloro-1-phenylethyl) benzene 2-(Formamido)-N1-(5-phospho-D-ribosyl) acetamidine 2,3-Dihydrofuran (R)-2,3-Dihydroxy-3-methylbutanoate methyl trans-2,3-Dihydroxycinnamate methyl 2,4,6-Trichlorobiphenyl 2,2’-Dimethoxybiphenyl 2,3-Dihydroxy-3-methylpentanoate methyl 2,3-Dihydroxyphenylpropanoate 2,4,6-Trichlorophenol 2’,2,4-Trichlorobiphenyl 2,4-Dichlorobenzoate

251 EP 2 230 312 A1 0 88 19 286 335 502 2723 2341 330,5 5 (- L’A background) 374 176 423 107 590 2811 10 2429 418,5 130,25 42,25 216,25 128,25 5783,5 5695,5 338,75 250,75 205,75 117,75 316,75 228,75 6856,25 6768,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 15 0 47 (- VUL background)

20 1 57 16 12 11 90 28 -33 -37 3650 3584 63,75 7053 6926 5249 5183 3992,25 3904,25 4746 4632 417,75 329,75 (raw data) VUL 3 0 0 0 0 0 0 0 0 0 25 7 52 26 (- KOL background)

30 69 55 26 92 -99 -43 -29 -91 -69 126 121 -181 -150 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 0 0 35 0 57 2098 2032 27 27 4692 4626 113 33 1770 1704 35 37 2440 (- KT succinate background)

40 43 37 49 96 25 13 19 -26 -10 100 181 151 157 161 -105 1304 1180 2573 2507 35 2255 2131 623 494 -87 2440 (raw data) KT succinate

45

50

55 Compound 2,5-Diamino-6-(5’-phosphoribosylamino)-4- pyrimidineone 2,5-Diamino-6-hydroxy-4-(5’- phosphoribosylamino)-pyrimidine methyl-D-gluconate 2,5-Didehydro-D-gluconate 2-Bromo-1,3-dichlorobenzene2,4-Dichlorophenoxyacetategamma-Glutamyl-gamma- aminobutyraldehyde 2,4-Dichlorophenol 2,4-Dinitroaniline -76 522,5-Dichloro-2,5-cyclohexadiene-1,4-diol 01,4-Dichloro-2-phenylbenzene 2,5-Dichlorohydroquinone 0 61 4276 2676 4210 2583 -26 26 0 0 1339,75 1251,75 360 272 N-acetyl-LL-2,6-Diaminoheptanedioate 2,3-Dihydroxyphenyl acetate 2,3-Dihydroxyphenyl LL-2,6-Diaminoheptanedioate 2-cis,6-trans-Farnesol (2Z,6E)-3,7,11-Trimethyldodeca-2,6,10- trienal 2,5-Dichlorophenol 3,5-dichlorophenol 2-propyl-glycidyl

252 EP 2 230 312 A1 0 0 0 98,5 4212 267,5 134,5 5 (- L’A background) 35 67,5 79,5

10 4300 355,5 2738,5 2650,5 3282,5 3194,5 6038,5 5950,5 292,25 204,25 5148,5 5060,5 1416,25 1328,25 3385,75 3297,75 L’A (raw data) 0 3 0 0 0 0 0 0 0 0 15 0 89 (- VUL background)

20 41 92 -37 6261 6044 379,75 291,75 (raw data) VUL 0 0 0 13586 13520 478,25 390,25 0 25 0 (- KOL background)

30 64 43 -188 4532 4466 -61 1195 1040 -18 7757 7691 69 6884 6647 -37 3729 3582 116 3508 3442 155 4080 4014 9109 9043 3504,75 3416,75 (continued) (raw data) KOL 0 0 7 0 0 0 0 35 0 70 2387 2321 -99 86 42 3909 3843 -132 470 -129 130 759 14476 14410 31981 31915 186,5 2440 (- KT succinate background)

40 21 76 43 39 113 594 194 131 254 210 883 166 -127 2001 1877 2761 2694 -9 12742406 1150 2282 37 4038 3807 12863 12632 222,5 2440 (raw data) KT succinate

45

50

55 Compound cis-2,3-Dihydroxy-2,3-dihydroethylbenzene 89 2-Amino-3-oxobutenoate 2-Arachidonoylglycerol 2,3-Dihydroxyethylbenzene 2,3-Bisphospho-D-glycerate Bromobenzene-3,4-dihydrodiol Methyl 3-hydroxydecanoate Methyl 2-Amino-4-hydroxy-6-hydroxymethyl-7,8- dihydropteridine diphosphate 2-Amino-4-hydroxy-6-hydroxymethyl-7,8- dihydropteridine 2,2-dimethyl-1,3-dioxane-4-propyl carboxylate 2-Aceto-2-hydroxybutanoate (S)-2-Aceto-2-hydroxybutanoate 2-Aminobenzenesulfonate 2-Aminobenzoate 2-Hydroxymuconate 2,6-Dimethylnaphthalene2-trans,6-trans-Farnesol2-trans,6-trans-FarnesalL-2-amino-3-oxobutanoatesulfonate benzene 2-Amino 133 30 21 -36 9 0 0 0 8 73 94 -34 0 7 0 0 -89 52 99 56 0 0 0 0 4239,25 3968 4151,25 338,25 456 3880 250,25 368 4-Fluorobenzoate

253 EP 2 230 312 A1 28 441 973 1890 578,5 187,5 5 (- L’A background) 116 529 1978 10 1061 275,5 235,25 147,25 2496,25 2408,25 L’A (raw data) 0 0 0 0 0 15 0 (- VUL background)

20 75 (raw data) VUL

25 0 13809 13743 666,5 89 (- KOL background)

30 175 109 3754 3688 1378,25 1290,25 -103 1752 1686 -37 (continued) (raw data) KOL 0 0 0 10355 10289 7769 7703 7234,25 7146,25 35 0 67 5194 5026 11182 11014 4859,75 4771,75 621 3083 3014 -143 169 2246 2180 1957 1891 833 2504 2415 -43 2440 (- KT succinate background)

40 1 -26 -88 745 293 957 191 -105 1664 1540 281 3954 3830 2336 2237 -74 3516 3392 2640 2574 -96 2440 (raw data) KT succinate

45

50 D-arabino-heptonate 7-

55 Compound 2-Butyne-1,4-diolBromobenzene2-Bromobutane2-Bromoethylbenzeneacid2-Bromopropanoic 1-Bromo-2-ethenylbenzeneButan-2-ol(E)-2-Chlorobut-2-ene2-Chlorobiphenyl 192-Chlorobutane 190 462-Chlorodecane 303 -76 201-(2-Carboxyphenylamino)-1’-deoxy-D- 05’-phosphate ribulose 66 179 0 0 82 0 116 4361 179 2633 54 -38 7740 24 0 0 4295 -25 2567 -12 55 7614 0 0 138 1517 114 0 71 0 120 28 0 3210 1098 1433 72 0 -45 -115 5 0 3035 1995 0 942 41 196,5 273,5 0 0 1918 3259 4555 6285,5 232 -119 0 108,5 185,5 3371 467,5 -103 6197,5 3171 4467 76 0 1815,5 3294 379,5 0 2096,25 539,25 1727,5 5847,75 2008,25 341,5 451,25 5759,75 253,5 2-Dehydro-3-deoxy- phosphate acid 2-Chloropropanoic cis-2-Hydroxypenta-2,4-dienoate 1-Chloro-2-methylbenzene 2-Chlorophenol 2-Phenylpropanenitrile 3-(p-Chlorophenyl)coumarin phenyl propionate 2-Dehydro-3-deoxy-L-arabinonate 2-Dehydro-3-deoxy-D-gluconate

254 EP 2 230 312 A1 1 2895 4518 11,75 5 (- L’A background) 89 2983 10 4606 99,75 4263,5 4175,5 909,75 821,75 2553,75 2465,75 1632,75 1544,75 5347,25 5259,25 L’A (raw data) 0 0 0 0 0 15 0 24 66 13 (- VUL background)

20 79 -36 -67 5095 5012 482,25 394,25 (raw data) VUL 0 0 0 0 25 0 34344 34243 1900,25 1812,25 (- KOL background)

30 9 37 10 94 261 172 155 488 422 -38 -118 3076 2969 4200 4093 5036,75 4948,75 8788 8722 6810 6744 6437,25 6349,25 4648 4551 93 5591 5524 24 (continued) (raw data) KOL 0 6 0 0 0 0 0 0 0 35 0 128 2440 (- KT succinate background)

40 27 97 55 -47 -21 130 115 108 252 -103 -111 4771 4647 2092 2026 -2 2607 2483 3633 3567 90 2440 (raw data) KT succinate

45

50

55 Compound Dodecan-2-ol 2-Dehydropantolactone Demethylmenaquinone 2-Deoxy-D-ribose1-phosphate 2-Ethylhexan-1-ol 2-Demethylmenaquinone 2-Deoxy-D-ribose5-phosphate 2-Keto-L-gulonate 2H-1,2,4-Benzoxadiazine 2-Dehydropantoate 2H-1,2-Oxazine 2-Deoxy-D-gluconate2-Dehydro-D-glucose2-Dehydro-3-deoxy-D-xylonate5-Dehydro-2-deoxy-D-gluconate2-Dehydro-D-gluconate2-Deoxy-D-glucose2-Dodecanoylglycerol 209 1502-Decanoylglycerol 1552-Dehydro-3-deoxy-D-galactonate 962-Dehydro-3-deoxy-D-galactonate6- 85phosphate 26 13355 31Decan-2-ol 0 382 13231 236 -93 702 118 -52 -44 188 258 0 170 628 0 0 0 0 10981 64 90 47 0 2261 12416 2105 10915 10770 5602 24 12311 2195 0 2039 -63 675,75 10704 5464 1130 63 425 84 587,75 312,5 0 4505,5 47 1064 0 337 4417,5 18 224,5 541,25 0 4659 113,25 5012,5 453,25 4571 382,75 4924,5 25,25 294,75

255 EP 2 230 312 A1 156 2972 6241 1214 5319 6052 824,5 428,5 5 (- L’A background) 244 3060 1302 6329 5407 10 6140 912,5 516,5 6353,5 6265,5 282,25 194,25 694,25 606,25 187,25 99,25 771,75 683,75 164,25 76,25 7052,75 6964,75 3624,25 3536,25 1957,75 1869,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 39 (- VUL background)

20 6 81 53 -40 -20 -10 158 222 156 105 4248 4180 589,25 501,25 (raw data) VUL 0 0 0 0 0 0 0 25 0 43 (- KOL background)

30 14 41 70 33 17 103 109 1744 1636 -113 6372 6276 -19 9783 9717 -52 1916 1850 32 3627 3507 43 6063 5997 183 117 1265,25 1177,25 (continued) (raw data) KOL 0 2 0 0 0 0 0 0 0 10993 10796 117 0 0 0 35 0 40 2950 2726 89 104 -64 2440 (- KT succinate background)

40 71 71 84 70 85 18 15 -17 -15 -15 -14 126 100 164 228 1563 1439 1928 1862 -29 1739 1615 16465 16399 -89 1211 1087 -29 2440 (raw data) KT succinate

45

50

55 2-Hydroxy-6-oxo-6-phenylhexa-2,4- dienoate Compound 2-Hydroxybutane-1,2,4-tricarboxylate 2-Hexadecanoyl-glycerol 2-Hydroxy-6-keto-2,4-heptadienoate 2-Hydroxy-6-oxo-7-methylocta-2,4- dienoate methyl (S)-2-Hydroxy-2-methyl-3-methyl oxobutanoate 2-Phenyl-4-propyl-tetrahydro-1,4-oxazine2-Hydroxy-3-oxopropanoate 2-Hydroxy-3-oxosuccinate 213 2-Hydroxy-5-methyl-cis,cis-muconic semialdehyde 2-Hydroxyadipate 2H-1-Benzopyran 3H-1,2,4-dioxazole 2-Hydroxy-2-hydropyrone-4,6- dicarboxylate 2H-1,2-Benzoxazine 2-Hydroxy-3-carboxy-6-oxo-7-methylocta- 2,4-dienoate 4-hydroxybenzoate propyl 4-Methyl-1,3-oxazinane-2-thione 3-Methyl-2H-1,4-benzoxazine

256 EP 2 230 312 A1 3219 339,5 5 (- L’A background)

10 3307 427,5 452,25 364,25 335,25 247,25 4566,5 4478,5 474,25 386,25 5712,25 5624,25 1761,25 1673,25 1842,25 1754,25 L’A (raw data) 0 0 0 0 0 0 15 0 28 79 3848,25 3760,25 (- VUL background)

20 (raw data) VUL

25 11 -101 (- KOL background)

30 559 493 -86 140 3392 3326 -77 4221 4155 287 221 365,25 277,25 1982 1916 94 2794 2728 -37 2325 2198 206 6032 5966 -70 2560 2494 58 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 35 0 61 2075 2002 3105 3032 122 885 819 -14 2440 (- KT succinate background)

40 6 13 53 -81 -93 105 116 112 246 185 100 2440 (raw data) KT succinate

45

50

55 Compound Pyran-2-one 2H-Pyran p-Tolualdehyde 2-Hydroxybiphenyl 2’-Hydroxybiphenyl-2-sulfinate 2-Isothiocyanatoethylbenzene 1-chloro-2-ethenylbenzene N-Methylimidazolidine-2,4-dione 2-Hydroxycyclohexan-1-oneHeptadec-2-nol2-HeptadecanoylglycerolHexacosan-1-olL-ArgininosuccinateHexadecan-2-ol -392-HexanoylglycerolHexan-2-ol(R)-2-Hydroxyglutarate 15 02,4-Dichlorobenzoyl-CoA -752H-Imidazole2-Hydroxymalonate 1194 2571 24 0semialdehyde 2-Hydroxymuconate 02-Hydroxyhexadecanal 110 -134 2379 1070 -131 0 85 621 5111 122 48 0 0 -63 0 4421 5045 497 0 107 56 -24 7339 0 -2 0 4298 7553 -67 -71 -12 7115 0 37 0 7487 701,5 27 164 0 0 0 0 -61 81 1414,5 0 1738 613,5 97 0 -154 5388,25 23 -27 0 1326,5 1672 7183,25 0 5300,25 133 0 258 0 7095,25 -91 5644,5 0 40 66 359,75 170 5556,5 1092,5 0 336 3798,25 271,75 0 1004,5 3710,25 298,25 248 1859,75 210,25 1771,75 2-Bromo-1-chloropropane

257 EP 2 230 312 A1 777 4272 498,5 5 (- L’A background) 865

10 4360 586,5 533,75 445,75 993,75 905,75 5160,5 5072,5 6893,75 6805,75 1622,75 1534,75 9120,75 9032,75 L’A (raw data) 0 0 0 0 0 0 0 15 0 28 3856,75 3768,75 16 3182,75 3094,75 (- VUL background)

20 -6 54 100 116 (raw data) VUL 0 0 25 0 24 4815 4749 5109,5 5021,5 67 (- KOL background)

30 -9 37 139 247 169 -124 4256 4144 -34 4059 3993 -37 6130 5956 63 5146 5080 1185 1119 9250,75 9162,75 3396 3191 596 391 (continued) (raw data) KOL 0 0 0 0 0 0 0 35 0 15 293 4069 3772 88 144 2572 2414 291 133 1705,5 1617,5 2440 (- KT succinate background)

40 -2 79 22 19 -18 104 417 139 119 268 -107 1478 1354 2153 2087 -30 2618 2494 90 2440 (raw data) KT succinate

45

50

55 Compound cis-2-Methylaconitate 2-Methylcitrate 2-C-Methyl-D-erythritol4-phosphate 2-Methyl-1-propanol 2-Methyl-1,3-dioxolan 2-Methylbenzyl alcohol 2-Methylbenzyl 4-Methylbutanoyl-CoA S-Glutaryldihydrolipoamide 2-Indanone2-Isopropylmaleate(R,S)-piperazine-2-tert-butylcarboxamide2H-Pyrrole5-Dehydro-2-deoxy-D-gluconate 462-Keto-D-gluconicacid 2-amino-3-oxobutanoatemethyl L-2-amino-3-oxobutanoatemethyl 04-Methylthio-2-oxobutanoate 34 32-Oxopent-4-enoicacid 53 5431 99 26 161 0 0 42 0 5322 -16 0 0 37 116 -105 4694 3772 0 -72 125 -64 4585 50 0 3464 -147-7 0 0 961,5 59 -18 63 0 66 0 873,5 187 35 0 0 10 0 4492 121 0 12772,3 7551,5 0 4426 458,5 12684,3 796,5 6809,75 7463,5 2029,75 1289,75 370,5 6721,75 708,5 1941,75 1201,75 2-Methyl-1,3-cyclopentane 2-Methyl-4-amino-5- hydroxymethylpyrimidine diphosphate 2-Maleylacetate 2-Methylbenzo-1,3-dithiole

258 EP 2 230 312 A1 2890 4563 5047 3443 4497 5 (- L’A background) 2978 4651 5135 3531 10 4585 1822,5 1734,5 4579,25 4491,25 8822,25 8734,25 4485,25 4397,25 7215,25 7127,25 12543,8 12455,8 8474,25 8386,25 4429,25 4341,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 15 0 45 (- VUL background)

20 2 50 71 -72 -81 111 336 246 4574,75 4486,75 895 829 2407,25 2319,25 -155 -166 (raw data) VUL 0 0 0 0 0 0 0 0 0 25 0 43 -110 (- KOL background)

30 65 98 16 37 70 31 54 -49 109 0 -6 0 7771 7699 1220 1148 13038,5 12950,5 (continued) (raw data) KOL 0 0 0 0 0 35 0 53 4077 4011 -33 18 3428 3362 448 382 5350,25 5262,25 25 26 3907 3764 1552 1409 1164,75 1076,75 20 170 2520 2381 -7 267 218 4115 3958 411 254 5427,25 5339,25 110 -141 145 2426 2360 363 297 2149,5 2061,5 458 2323 2216 47 287 181 4958 4820 183 2440 (- KT succinate background)

40 64 74 15 -31 -64 -88 294 391 177 342 234 142 149 150 269 582 411 305 144 -169 2440 (raw data) KT succinate

45

50

55 1-Nonadecanoyl-sn-glycerophosphocholine 2-Nonanoylglycerol 2-Nonadecanoylglycerol 2-Octaprenyl-3-methyl-5-hydroxy-6- methoxy-1,4-benzoquinone 2-Octaprenyl-6-methoxyphenol Nonadecan-2-ol 2-Methylserine Compound 1-Methyl-2-nitrobenzene 1-Hydroxymethylnaphthalene 2-C-Methyl-D-erythritol2,4- cyclodiphosphate 1-Methoxy-2-phenylbenzene 3,4-dimethylphenol 2-Nitronaphthalene 2-Naphthoic acid 2-Naphthoic 2-Methylnaphthalene 2-Hexaprenyl-3-methyl-6-methoxy-1,4- benzoquinone 2-Methylpyridine [(2S)-Oxiran-2-yl]methylacetate 2-Nitroaniline 2’-O-Methyllicodione

259 EP 2 230 312 A1 1601 5 (- L’A background)

10 1689 745,75 657,75 2777,5 2689,5 857,25 769,25 7896,75 7808,75 4846,25 4758,25 L’A (raw data) 0 0 0 0 0 15 0 (- VUL background)

20 -38 -88 -69 -99 -63 (raw data) VUL 0 0 0 0 25 0 (- KOL background)

30 66 98 -42 -132 2244 2178 34 3658 3592 2805 2739 5156,25 5068,25 2558 2492 209 143 3365,75 3277,75 2912 2642 3754 3484 1513,75 1425,75 (continued) (raw data) KOL 0 0 0 0 0 0 0 35 0 2440 (- KT succinate background)

40 87 34 41 39 -60 121 104 111 1691 1567 -33 2440 (raw data) KT succinate

45

50

55 Compound Nonan-2-ol2-Nonaprenyl-6-hydroxyphenol2-Nonaprenylphenol2-Oxobutanoate2-OctadecanoylglycerolOctadecan-2-ol -302-Octanoylglycerol2-Octanol3-aminobenzoate 0Thiocyanoethylbenzene 263 3981-Monobutanoyl-glycerol 5812-Octaprenyl-6-methoxy-1,4-benzoquinol 467 47 1392-Octaprenyl-3-methyl-5-hydroxy-6- 274methoxy-1,4-benzoquinol 457 202 81 7 343 -57 29 0 -107 806 135 269 78 143 85 0 0 0 -84 0 0 563 11 -146 145 3654 19 0 -4 -11 5078 0 144 34 189 0 3588 9 -2 -76 5012 0 0 8654,25 0 4938 123 0 2849 0 1288,75 0 0 8566,25 1652 4161,75 4872 -111 2698 5786 -21 1200,75 -65 4073,75 1402,25 1974 1586 12363,8 1559,75 0 5698 0 1314,25 1898 12275,8 0 1471,75 4088 1718,75 1593,25 864 5400,5 4000 1630,75 1505,25 5312,5 776 2-Octaprenyl-3-methyl-6-methoxy-1,4- benzoquinol 2-hydroxybenzoate propyl acid 2-Oxooctadecanoic 2-Phospho-4-(cytidine5’-diphospho)-2-C- methyl-D-erythritol 2-Octaprenylphenol 3-Phenylbutan-1-ol Pentan-2-ol 2-phosphateD-Glycerate

260 EP 2 230 312 A1 3310 2926 5 (- L’A background) 3398 10 3014 305,75 217,75 995,75 907,75 5560,75 5472,75 2552,75 538,75 5624,75 5536,75 5601,75 5513,75 L’A (raw data) 0 0 0 0 0 0 0 15 0 (- VUL background)

20 76 59 -73 -55 -20 -108 3493 3427 8320,75 8232,75 (raw data) VUL 0 0 0 0 0 25 0 47 (- KOL background)

30 3 96 26 66 113 -111 2814 2680 -13 4067 4001 1280 1214 3887,75 1873,75 (continued) (raw data) KOL 0 0 30505 30439 -1 0 0 0 35 0 29 41 211 -29 865 2440 (- KT succinate background)

40 6 87 98 77 47 153 335 989 165 -126 2440 (raw data) KT succinate

45

50

55 Compound 2-S-isocapryloyl-3R-hydroxymethyl- gamma-butyrolactone 2-Octaprenyl-6-hydroxyphenol Tetradecan-2-ol 2-Propylglycerol2-Phosphoglycolate2-Pyrone-4,6-dicarboxylate2-Octaprenyl-6-methoxy-1,4-benzoquinonePropionaldehyde 22882-Propen-1-ol4,5-Dihydro-1H-pyrazolePyran-2-one 2164 5254 103752,3-Dihydro-1H-pyrrole 51(2S)-Flavanone -122 5130(2S)-Flavan-4-ol 10251(1R,6R)-2-Succinyl-6-hydroxy-2,4- 0cyclohexadiene-1-carboxylate 79 113 0 4877 -77 143 1228 4811 27 0 -17 0 0 51Tetracosan-2-ol 1112 19 -118 -26 0 9495 4255 0 90 6 -31 1293 0 0 9429 41741-Chloro-2-[1-(4-chlorophenyl)ethenyl] 0 887benzene 1351 0 1178 0 0 1980 1051,5 -4 20 4870 776 -103 1263 323 1751 1910,5 4789,75 963,5 4804 0 0 -2 25590 0 4701,75 257 1822,5 -156 25361 4906,25 2532,5 0 6983,5 48 0 4818,25 2974,5 2444,5 6895,5 2242 0 2886,5 134,75 2154 273,75 46,75 185,75 2-Tetradecanoylglycerol Ethylcysteine 2-Methyl-4,5-dihydro-1,3-thiazole Undecan-2-ol

261 EP 2 230 312 A1 185 54,5 3567 393,5 203,5 5 (- L’A background) 273

10 3655 481,5 291,5 142,5 7303,5 7215,5 228,75 140,75 7416,25 7328,25 5125,25 5037,25 3721,75 3633,75 L’A (raw data) 0 5 0 0 0 0 0 15 0 50 27 (- VUL background)

20 93 -49 4458 4263 795,75 707,75 (raw data) VUL 0 25 0 (- KOL background)

30 72 -59 8541 8475 -4 6153 6087 116 2472 2406 20 5094 5028 -74 1030 964 5184 5118 50 (continued) (raw data) KOL 0 0 0 0 0 0 35 0 30 1515 1449 71 660 4419 4353 -57 112 2440 (- KT succinate background)

40 76 50 96 -77 -19 236 784 154 -112 -213 4234 4110 1418 1232 78 2440 (raw data) KT succinate

45

50

55 Compound 2-Fluorobenzoate 3,5-Dihydroxybenzoic acid 3,5-Dihydroxybenzoic 3-Amino-1,2,4-triazole 3-Amino-5-deoxyfructose6-phosphate 3-Fluorobenzoate 3,4,5-Trichlorophenol3’,4’,5-Trihydroxy-3,7-dimethoxyflavone3,4-Dichloroaniline1,3-Dichloro-2-phenylbenzene -563,4-Dihydroxymandelate3,4-Dihydroxyphenylacetaldehyde3,4-Dihydroxy-trans-cinnamate -144 03,4-Dihydroxybenzoate -65Cystathionine -1033,4-Dihydroxyphenylacetate -110 4399 03,4-Dichlorobut-1-ene 24 -73,4-Dihydro-1naphthalene (2H) 0 0 4333 0 -43methylphenylpyruvate 1501 0 -31 0 108 235 -1321,3-Dichloro-5-phenylbenzene 4507 0 1377 394 1455 -189 0 2068 42 111 0 4441 49 22 328 1331 2002 2263 0 5805,5 -33Monochlorobenzene 17 -233-carboxy-2-hydroxy-4- -173 0 -129ethylpentanoatecarboxamide 0 -6 2157 3791,5 3734 0 0 0 0 0 -55 17 7922,5 3635 0 2704 410,75 6458,75 5908,5 381,75 -78 69 0 0 5734 6370,75 322,75 2616 293,75 0 2715,5 3 602,75 5646 2627,5 514,75 5325,25 631 5237,25 543 3-(3,4-Dihydroxyphenyl)lactate

262 EP 2 230 312 A1 44,5 3582 126,5 333,5 5 (- L’A background)

10 3670 214,5 132,5 421,5 158,75 70,75 4012,5 3924,5 609,25 521,25 588,25 500,25 3947,25 3859,25 5509,75 5421,75 6717,75 6629,75 2241,75 2153,75 L’A (raw data) 0 8 0 0 0 0 0 0 0 15 0 84 23 (- VUL background) 3 1

20 -3 89 15 15 -74 (raw data) VUL 0 0 0 25 0 50 47 (- KOL background)

30 -42 -12 -18 130 113 1966 1900 150 1791 1638 -24 1751 1685 89 (continued) (raw data) KOL 0 0 0 0 0 0 0 35 0 69 6700 6634 74 17 3177 3111 586 3714 3565 76 2440 (- KT succinate background)

40 2 0 1134 1068 -39 0 2891,5 2803,5 -3 78 17 23 53 37 193 710 141 -151 -150 4315 4191 -72 2440 (raw data) KT succinate

45

50

55 Compound 4-Chlorobenzoate1-Chloro-3-phenylbenzene1-Chloro-3-ethenylbenzene3-Chlorotoluene3-Carbamoyloxymethylcephemlactatemethyl 3-Dehydrocarnitine 323-Dehydro-L-gulonate6-phosphate 78 559 444 0 435 0 130 320 110 3227 22 4196 -2 2246 6 3123 44 4130 2133 0 0 -49 -37 279 -82 -80 1587 6322 0 0 213 0 0 1366 6256 7852 3090,75 1709 414,75 481 2380,5 70 7723 3002,75 1621 326,75 2292,5 260 143,5 4 4572,25 4724,75 55,5 4484,25 4636,75 2-isopropylmalate3-Chloro-4-hydroxy-phenylacetatemethyl3-Carboxy-4-methyl-2- oxopentanoate 164 177 40 -23 53 5029 0 4963 -89 65 0 0 3805,75 3717,75 324 236 3-Dehydroquinate 3-Dehydro-L-gulonate 1,4,2-Oxathiazole 3-Demethylubiquinone-9 3-propylcatechol 2,3-Dichloropentane 3-Dehydro-L-threonate 3H-Dioxazole 3H-1,2-Dithiole 3-Dehydroshikimate 2H-1,3-Dithiole

263 EP 2 230 312 A1 105 810 3518 8665 9765 473,5 207,5 635,5 5 (- L’A background) 193 898 3606 8753 10 9853 295,5 723,5 6485,5 6397,5 333,25 245,25 622,25 534,25 579,25 491,25 354,75 266,75 639,75 551,75 4412,75 4324,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 15 0 52 (- VUL background)

20 -60 107 118 (raw data) VUL 0 0 25 0 (- KOL background)

30 -90 -10 -13 4972 4659 -67 2593 2527 -36 7376 7310 -101 2015 1949 -21 6226 6160 10230 10164 561,5 3041 2904 29 2130 2064 9565 9499 9451 9385 1611 1545 3906,25 3818,25 (continued) (raw data) KOL 0 0 3 0 0 0 0 20225 20159 -63 0 0 0 0 35 0 29 2349 2283 -29 98 4325 4206 35 237 2242 2176 7617 7551 295 2890 2773 49 2440 (- KT succinate background)

40 3 23 55 49 27 -26 -84 -67 153 114 127 361 419 222 -179 2440 (raw data) KT succinate

45

50 hydroxy-4-oxobutanoic

55 Compound (3S)-3-Hydroxydecanoyl-CoA 3-Hydroxy-2-benzopyronemethylglycine methylester3-Hydroxy-2-methylpropanoate3-Hydroxyanthranilate3-Hydroxy-4-methylanthranilate3-Hydroxyoxolan-2-one -8(2S)-2,4-Diamino-3- -21 31 283-Hydroxybenzaldehyde 0 6643alcohol 2-Hydroxybenzyl 0 0amine 3-Hydroxybenzyl 154 0 6519 -53 13913 5651 30 13847 -72 1271 0 5451 85 1205 -39 0 9 -31 -119 19 0 3614 0 0 0 8383,75 3531 51 403,75 55,75 8295,75 2385,75 1642,75 0 315,75 2297,75 1554,75 0 545,75 457,75 acid 3-Hydroxybenzoate (3S)-3-Hydroxyhexacosyl-CoA 3H-Indole ceph-3-em-4-carboxylate3-Hydroxymethyl 3-Hydroxy-cis,cis-muconate 12 (3S)-3-Hydroxyoctadecanoyl-CoA 3-Hydroxycinnamate (S)-acetoin (3R)-3-Hydroxytetradecanoicacid (3S)-3-Hydroxydodecanoyl-CoA (3S)-3-Hydroxyhexadecanoyl-CoA

264 EP 2 230 312 A1 262 6629 6978 578,5 711,5 579,5 5 (- L’A background) 350 6717 10 7066 666,5 799,5 433,75 345,75 3858,5 3770,5 306,75 218,75 579,25 491,25 802,75 714,75 7457,5 7369,5 3792,5 3704,5 4133,25 4045,25 5369,75 5281,75 3911,75 3823,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 (- VUL background) 8 -2 20 -5 22 49 -69 -34 -183 1674 1461 1835,25 1747,25 (raw data) VUL 0 0 7 5 0 0 0 25 0 47 8447 8381 (- KOL background)

30 -6 61 13 -72 194 152 113 4068 3961 -80 3942 3683 -28 5699 5544 -61 6468 6401 290 223 5326,75 5238,75 1465 1354 35059 34948 309,75 221,75 6500 6355 100 5473 5407 26 7211 6927 4593 4309 10158,5 10070,5 3357 3291 6541 6475 667,5 (continued) (raw data) KOL 0 0 0 0 0 0 3 0 0 0 2 0 0 35 0 18 54 136 4286 4220 207 4570 4487 3732 3649 1473,25 1385,25 2440 (- KT succinate background)

40 3 16 54 26 65 70 -59 -37 121 142 178 127 260 331 101 126 -184 -108 2631 2507 4546 4292 -10 1711 1587 73 3377 3253 71 2440 (raw data) KT succinate

45

50

55 Compound 3-beta-Hydroxysterolhexadecanoate 3-Hexaprenyl-4-hydroxy-5- methoxybenzoate 3-beta-Hydroxysteroldodecanoate 3-beta-Hydroxysterolhexanoate 3-(3-Hydroxy-phenyl)-propanoicacid (3R)-3-Hydroxypalmitoyl-CoAacid 3-Hydroxyhexadecanoic 3-Hexaprenyl-4,5-dihydroxybenzoate 3-Hydroxypimeloyl-CoA -503H-Pyrrole 0 24293-Hydroxypropanoate 21923-beta-Hydroxysterololeate -1353-beta-Hydroxysteroloctanoate 03-(Imidazol-5-yl)pyruvate 385,75 297,75 3-Hydroxypropanal (3S)-3-Hydroxytetradecanoyl-CoA 3-beta-Hydroxysteroltetradecanoate 3-beta-Hydroxysteroldecanoate (S)-3-(Imidazol-5-yl)lactate 3-Methylbutanol 3-Methylbutan-2-ol 3-Indoleacetonitrile

265 EP 2 230 312 A1 649 287 6258 5294 8122 1299 487,5 344,5 554,5 233,5 5 (- L’A background) 737 375 6346 5382 10 1387 575,5 432,5 642,5 321,5 3151,25 3063,25 7472,75 7384,75 1561,25 1473,25 7706,25 7618,25 5089,25 5001,25 3569,25 3481,25 1360,25 1272,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 15 0 32 74 (- VUL background)

20 9 98 44 65 -13 -72 -55 585 509 5348,25 5260,25 (raw data) VUL 0 0 0 0 0 0 39402 39226 8210 25 0 57 -103 10 -115 90 (- KOL background)

30 39 89 -11 -32 -73 553 487 -13 166 194 128 -51 362 242 (continued) (raw data) KOL 0 0 0 0 35 0 24 -106 36 88 61 3344 3271 147 18 18281 18215 1328 1262 1615,25 1527,25 101 193 4425 4344 -87 129 2440 (- KT succinate background)

40 73 -46 -15 225 317 148 160 253 109 212 121 185 1798 1674 -8 4483 4359 123 2491 2367 4480 4414 -111 1616 1492 23 2980 2856 173 107 4138 4072 1780 1656 172 101 518 447 2440 (raw data) KT succinate

45

50

55 )methyl propionate )methyl 6 Compound 3-Hydroxytoluene 4-Nitroanilide ethylferulateGlutaconyl-CoAalcohol3-Methylbenzyl 3-Methyl-1-benzothiopheneCl-(3-Indolyl)-glycerol3-phosphate 3-(Methoxycarbonyl)pent-2-enedioate 3-Methylcatechol 283 167 6164 70 159 43 6040 0 7138 4751 6975 -48 7072 4657 6755 0 3018 -33 -39 2924 163 0 0 2159,5 97 1728,5 543 2071,5 12623,3 1640,5 455 12535,3 4-Methylbutanoyl-CoA 3-Oxobutyryl-CoA phenylpropionate 3-Oxododecanoyl-CoA trans-2-(4-Nitrophenyl)-3-phenyloxirane3-Methyl-2-oxobutanoate 3-Methylquinoline 142 (S)-3-Methyl-2-oxopentanoate ( 3-Oxodecanoyl-CoA acid 3-Nitrobenzoic 3-Oxohexanoyl-CoA (3-Nitrophenyl)methanol 3-Oxohexadecanoyl-CoA

266 EP 2 230 312 A1 162 75,5 7873 2153 2649 746,5 781,5 5 (- L’A background) 250 9887 2737 10 2241 869,5 163,5 5421,5 5333,5 375,25 287,25 335,75 247,75 2444,25 2356,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 15 0 (- VUL background)

20 72 17 -17 -21 -82 -106 2063 1997 834,5 1899 1666 813,75 725,75 2748 2382 1502,75 1414,75 (raw data) VUL 0 0 0 0 0 0 25 0 48 56 (- KOL background)

30 -35 -55 -34 865 686 -32 114 134 3771 3689 -104 3416 3241 6756 6581 4400,5 4312,5 (continued) (raw data) KOL 0 0 0 0 35 0 25 10 66 242 -77 140 104 144 146 2440 (- KT succinate background)

40 14 68 32 -56 -32 149 134 366 264 268 190 1182 1058 232 1523 1399 3302 3236 -35 2440 (raw data) KT succinate

45

50

55 Compound (R)-3-Hydroxy-butanoyl-CoA 3-Sulfocatechol 2,5-dihydro-1H-pyrrole 3-Thiaoctanoyl-CoA 3-Oxooctadecanoyl-CoA3-Oxohexacosyl-CoA3-Oxooctanoyl-CoAethyl-3-oxobutanoate2-ethyl-1,3-hexanediol3-Octaprenyl-4-hydroxybenzoate3-Oxotetradecanoyl-CoA 2403-Oxoadipyl-CoA 1063-Oxoadipate 116enol-lactone3-Oxoadipate 2495 5134 91 79 0 2371 5010 60 1263-Phenyl-propionicacid 05-O-(1-Carboxyvinyl)-3-phosphoshikimate 38 -16 0 68 0 25 2 1368 3787 200 -25 0 0 0 128 -19 0 3664 12444,4’-Dichlorobiphenyl 22056 76 -45 0 120 62 3887 -18 4794 0 21990 32 4463 0 4877,75 3821 0 4728 4737,25 0 -56 4189 4789,75 -97 4502 0 5121,5 4649,25 4563,5 0 793 611,75 4436 5033,5 4475,5 3430 1153,75 523,75 705 752,25 3364 1065,75 664,25 7640,75 7552,75 4-Acetamidobutanoate 3-Phenylpropan-1-ol 2-Phospho-D-glycerate 3-Phosphohydroxypyruvate 2-Phosphonooxypyruvate (3S)-3-Hydroxy-butanoyl-CoA

267 EP 2 230 312 A1 36 59 273 151 5 (- L’A background) 361 124 239 10 147 142,75 54,75 409,25 321,25 534,25 446,25 352,25 264,25 960,75 872,75 266,25 178,25 3325,75 3237,75 3406,25 3318,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 15 0 10 21 (- VUL background)

20 9 87 21 37 -53 -37 (raw data) VUL 0 0 0 0 25 0 (- KOL background)

30 4 73 -18 0 -6 0 3888 3822 -108 7231 7165 -89 2898 2832 55 4614 4548 (continued) (raw data) KOL 0 0 0 0 35 0 75 -118 45 33 3667 3548 98 18 2440 (- KT succinate background)

40 14 -15 -29 -78 -34 199 169 157 142 18331620 1709 1496 3056 2614 2943 2545 41 79 2023 1899 2420 2317 19485 19382 3338,5 3250,5 2440 (raw data) KT succinate

45

50

55 Compound 4-Bromobenzoic acid 4-Bromobenzoic 4-Bromocatechol (4S,5S)-4,5-Dihydroxy-2,6-dioxohexanoate(4S)-4,6-Dihydroxy-2,5-dioxohexanoate 90Adenine4-Aminobutanoate -314-Aminobutanal4-Aminobenzoate 04-Amino-4-deoxychorismate 04-Amino-5-hydroxymethyl-2- methylpyrimidine 9444-Amino-2-methyl-5- phosphomethylpyrimidine 70 1400 721 -22 1427 694 1304 4 1276 789 1303 0 570 1180 91 4625 566 2541 3758 1967 157 4559 25 4345,5 2445 3674 1901 91 981,25 4257,5 8140 -3 -113 27 893,25 8 8044 0 0 0 -38,5 0 207,25 451,75 97,5 0 306,25 119,25 363,75 9,5 218,25 4-Bromobutan-1-ol 4-Bromophenol 1-Bromo-4-methylbenzene Bromobenzene-2,3-dihydrodiol 4-Dimethylaminophenol 5’-diphospho)-2-C-methyl-D-4-(Cytidine erythritol 4-Carboxy-2-hydroxymuconate semialdehyde 4-Carboxy-2-hydroxyhexa-2,4-dienedioate -24 4-Carboxy-2-hydroxy-cis,cis-muconate

268 EP 2 230 312 A1 95 375 278 3576 174,5 385,5 112,5 5 (- L’A background) 183 463 366

10 3664 262,5 473,5 200,5 418,75 330,75 151,25 63,25 1585,25 1497,25 1677,75 1589,75 3698,25 3610,25 1103,25 1015,25 L’A (raw data) 0 0 8 0 0 0 0 0 0 0 0 15 0 (- VUL background)

20 8 -87 -20 -33 -151 2501 2435 1801,5 1713,5 (raw data) VUL 0 0 0 3 25 0 42 (- KOL background)

30 41 69 119 108 -112 5692 5556 9487 9351 4517 4451 13 4037 3906 53 3329 3250 87 1457 1320 5891 5754 2147,5 2059,5 4995 4849 100 (continued) (raw data) KOL 0 3 0 0 0 0 0 2 0 35 0 29 17 2422 2335 -100 158 3826 3737 -25 133 3006 2940 -31 876 -62 2440 (- KT succinate background)

40 60 44 63 18 -43 -34 127 282 153 141 257 126 -132 1000 2440 (raw data) KT succinate

45

50

55 Compound 4-Carboxy-2-oxo-4-pentenoate4-Carboxy-4-hydroxy-2-oxoadipate5,7,4’-Trihydroxy-3’-methoxyflavone4-Chlorobenzoyl-CoA1-Chloro-4-ethenylbenzene -97 244-Dehydropantoate 4904-Chlorotoluene2,5-diamino-6-ribitylamino-4 (3H)-pyrimidinone5’-phosphate 0 0 366 -36 -64 4625 3058 3135 2186 0 4559 2927 0 3053 47 2062 -120 5618 1735 192 4010 5602 0 5487 1637 0 3944 110 5436 2967,75 2519 97 -12 2500,25 208,5 4447 2879,75 2396 2412,25 0 0 120,5 4281 -55 2938,5 458,25 1824 0 2850,5 370,25 1736 2479,75 2391,75 4-propyl phenol 4-propyl 1-Fluoro-4-methylbenzene Diazepine 4-Hydroxy-2-oxopentanoate 4-Hydroxy-2-oxoglutarate 3-Methyl-1,3-oxazinane methyl 4-Hydroxy-3-methoxybenzoatemethyl 4-Hydroxy-3-methoxycinnamicacid 6-Methyl-3,4-dihydro-2H-1,4-benzoxazine -46 4-Hydroxymandelate Biphenyl 4-Phenyl-1,3-oxazinane-2-thione 1,3-Benzoxazine-2,4-dione 4-Hydroxy-2-butynal

269 EP 2 230 312 A1 254 589 2968 1474 590,5 475,5 201,5 5 (- L’A background) 342 677 3056 10 1562 678,5 563,5 289,5 5950,5 5862,5 840,75 752,75 588,75 500,75 361,75 273,75 1595,75 1507,75 2610,75 2522,75 3757,25 3669,25 1349,25 1261,25 2804,75 2716,75 2028,25 1940,25 2919,75 2831,75 7249,25 7161,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 75 (- VUL background) 5 4 2

20 -1 86 15 26 88 -18 141 1257 1191 5832,25 5744,25 5386 5320 6961,5 6873,5 (raw data) VUL 0 0 0 0 4 0 0 25 0 59 (- KOL background)

30 4 -67 521 371 116 125 -117 -105 5638 5396 -64 3901 3798 97 1133 1042 -13 2382 1971 1300 1234 -111 1966 1900 40 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 0 0 35 0 50 1251 996 -82 74 3563 3497 273 -41 817 251 -95 196 139 2440 (- KT succinate background)

40 1 55 46 15 89 10 58 63 92 37 -36 198 174 397 941 124 375 320 1674 1550 2940 2874 2582 2516 517,75 429,75 1986 1862 3194 3128 -33 2212 2088 3596 3509 39 1520 1396 3711 3645 -123 2440 (raw data) KT succinate

45

50

55 Compound methyl4-Hydroxycinnamate 4-Hydroxy-L-threonine 4-Hydroxybenzoate D-4-Hydroxyphenylglycine alcohol 3-Hydroxybenzyl 3-Hydroxy-2-formylbenzothiophene 4-Hydroxybenzaldehyde 4-(Aminomethyl)phenol 3-Methoxy-4-hydroxymandelate 4-Hydroxybutanoate 3-Hydroxy-3-methyl-2-oxopentanoate L-4-Hydroxyglutamatesemialdehyde 4-Methyl-2-oxopentanoate alpha-tolualdehyde (1S,4S)-4-Hydroxy-3-oxocyclohexane-1- carboxylate 4-Hydroxy-4-methyl-2-oxoglutarate 4-(Hydroxymethyl)phenol 4-Hydroxybenzoyl-CoA 4-Hydroxybenzoylformate 4’-Methoxy-5,7-dihydroxyflavone 4-Methyl benzyl alcohol benzyl 4-Methyl 3-Methylbutanal

270 EP 2 230 312 A1 472 1848 1522 5 (- L’A background) 560 1936 10 1610 1379,5 1291,5 477,75 389,75 915,75 827,75 6596,25 6508,25 4118,75 4030,75 4473,25 4385,25 3144,25 3056,25 1818,25 1730,25 L’A (raw data) 0 0 0 0 0 0 0 0 15 0 (- VUL background) 4 20 3 -19 307 161 8406,5 8318,5 -130 4468 4402 (raw data) VUL 0 0 0 0 25 0 (- KOL background)

30 79 -50 -54 -36 2430 2364 1914 1848 3861 3702 -171 1211 1081 4955 4825 5430,5 5342,5 5707 5641 23 (continued) (raw data) KOL 0 0 0 0 35 0 75 63 31 2094 1889 56 85 612 3725 3659 -68 468 7958 7892 2440 (- KT succinate background)

40 86 47 50 77 19 199 187 736 155 209 592 2028 1904 34 2440 (raw data) KT succinate

45

50

55 Compound 4-MethylcatecholMethyl-cyclohexane4-Methylcyclohexan-1-ol4-Methylcyclohexan-1-one4-Methylcycloheptan-1-ol4-chloro-2-Methylphenol5-(2-Hydroxyethyl)-4-methylthiazole4’-O-Methylisoflavone -162-Hydroxy-6-oxo-octa-2,4-dienoate 401 111 1914-Methyl-5-(2-phosphoethyl)-thiazole 119glucuronide 4-Methylumbelliferyl 45 277 0 163 0 67 58 964 0 0 -33 5 6396 52 39 2397 840 0 520 4173 6306 2138 0 0 29 0 18 5028 375 4020 4626 2729 -15 -54 424 0 4772 260 0 115 4536 2470 307 55 0 0 143 6376,75 115 2026,75 3078 0 6288,75 651,5 1938,75 1461,75 77 6808,25 0 3012 8 8451,75 1373,75 6720,25 563,5 8188,75 8363,75 1072 9376 0 8100,75 984 9288 7323,75 7235,75 Pyran-4-one 4-Nitrotoluene 4-Oxalocrotonate 4-Nitroaniline 4-Nitrophenol 4-Oxalomesaconate 4-Oxoproline 4-Phospho-L-aspartate 4-Phospho-D-erythronate D-4’-Phosphopantothenate (R)-4’-Phosphopantothenoyl-L-cysteine5-O-Methyl-myo-inositol 2245 2121 5514 5448 45

271 EP 2 230 312 A1 0 0 101 599 1538 1686 488,5 230,5 428,5 5 (- L’A background) 189 687

10 1774 576,5 23,75 26,25 373,75 285,75 107,25 19,25 4068,5 3980,5 507,25 419,25 4605,75 4517,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 15 0 37 3766,25 3678,25 (- VUL background)

20 31 103 6429 6363 516,5 (raw data) VUL 0 25 0 10206 10127 1626 77 9465 9399 318,5 84 (- KOL background)

30 40 -56 4365 4055 12 1117 1051 -75 1203 1074 14 7576 7510 199 133 8421,5 8333,5 4953 4887 61 2391 2293 3029 2931 1302,75 1214,75 2302 2164 39 8235 8169 5978 5912 248,75 160,75 (continued) (raw data) KOL 0 0 0 0 9 0 0 0 0 35 0 53 3058 2933 4251 4126 300,75 212,75 108 514 430 645 3956 3890 -158 436 7500 7434 -57 234 143 302 3576 3429 70 786 1255 1189 766 700 604 4116 3892 73 2440 (- KT succinate background)

40 -2 74 49 35 69 69 -19 -45 232 177 358 133 560 426 910 728 2440 (raw data) KT succinate

45

50

55 Compound 5-Benzamido-2-benzyl-4-oxopentanoate5-Bromo-4-chloro-3-indoylphosphate 5-carboxyamino-1-(5-phospho-D-ribosyl) -120 imidazole 5-Dehydro-D-fructose 2-Dehydro-D-glucono-1,5-lactone 4-(1-D-Ribitylamino)-5-aminouracil4-Chlorobiphenyl5,7,4’-Trihydroxyflavoneacid 2-Aminodecanoic 2090carboxylate 4-imidazole 2,2’-Dihydroxybiphenyl methyl-5-amino-4-oxopentanoate 19665-Amino-6-(5’-phosphoribitylamino)uracil -40 4801 3195 769 4583 3071 0 170 -10 -71 0 0 0 2256,75 -33 3809 2168,75 3743 0 5781,5 1628,75 3767,5 0 5-Amino-6-(5’-phosphoribosylamino)uracil5-Amino-4-oxooctanoate 1134acetyl-CoA Benzoyl 1010 150 5-Dehydro-D-gluconate 5’-Fluoro-5’-deoxyadenosine 4-amino-5-imidazolecarboxamide 2-Aminohexadecanoic acid 2-Aminohexadecanoic 5-Dehydro-4-deoxy-D-glucarate 5H-1,2,5-Oxathiazole

272 EP 2 230 312 A1 0 0 327 4367 6916 6050 5 (- L’A background) 415 8,75 4455 7004 10 6138 925,25 837,25 1682,5 1594,5 618,75 530,75 1346,25 1258,25 5060,75 4972,75 8021,758789,25 7933,75 8701,25 12175,8 12087,8 6190,75 6102,75 1790,75 1702,75 4643,25 4555,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 15 0 22 13 (- VUL background)

20 1 48 41 74 79 30 -32 -87 622 416 8014,25 7926,25 -137 3626 3560 8419,25 8331,25 (raw data) VUL 0 0 0 0 0 0 0 25 0 66 99 (- KOL background) 4

30 -7 -56 -14 165 6390 6235 -23 5509 5216 18 6316 6228 -85 2530 2357 49 (continued) (raw data) KOL 0 0 0 0 0 0 0 35 0 65 2124 2058 54 31 -134 153 132 141 -31 501 3436 3248 8729 8541 74,75 2440 (- KT succinate background)

40 82 55 56 13 27 84 -25 277 189 265 178 110 625 155 7983 7859 4270 4056 -7 2985 2861 -276 4177 4053 6920 6854 88 2790 2666 101 2047 1923 2177 2083 617 523 2440 (raw data) KT succinate

45

50

55 Compound 5-Methylthioadenosine S-Methyl-5-thio-D-ribose1-phosphate S-Methyl-5-thio-D-ribulose1-phosphate 5-HydroxyfuranocoumarinS-Methyl-5-thio-5-deoxy-D-ribose1- sulphate 2,5-Diamino-6-(5’-triphosphoryl-3’,4’- trihydroxy-2’-oxopentyl)-amino-4- oxopyrimidine 118-Methoxyfuranocoumarin 5-Methoxyfuranocoumarin 0 16 0 -32 0 335,75 247,75 2(3H)-oxazolone 5-Nitro-1,2,4-triazol-3-one 5-Methyltetrahydrofolate 6-Hydroxyhexanoate N1-(5-Phospho-alpha-D-ribosyl)-5,6- dimethylbenzimidazole 6-Acetyl-beta-D-galactoside 1,2-Ditetradecanoyl-sn-glycerol 4-Pyridoxolactone 6-Chloro-3-indolyl-beta-D-galactoside 6-Chloro-3-indolyl-beta-D-glucoside 6-S-Acetyldihydrolipoamide Melibiose

273 EP 2 230 312 A1 966 950 2200 6456 5084 511,5 5 (- L’A background) 2288 6544 1038 1054 10 5172 599,5 362,25 274,25 188,75 100,75 413,75 325,75 1213,75 1125,75 2105,75 2017,75 3359,25 3271,25 2667,75 2579,75 1225,25 1137,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 15 0 (- VUL background)

20 0 33 26 -54 -33 -86 -29 (raw data) VUL 0 0 22772 22560 5946,25 5858,25 0 0 0 0 25 0 13 61 5628 5562 (- KOL background)

30 26 -72 -33 -30 -23 127 9215 9149 -54 2864 2782 -33 4684 4554 -153 6091 5997 7947 7853 2192,5 2104,5 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 35 0 82 2429 2294 34 73 168 131 166 -27 2440 (- KT succinate background)

40 42 99 49 57 -70 -42 -83 292 290 101 206 197 -115 1692 1568 -132 1228 1104 3766 3700 29 2485 2361 5546 5480 -10 1773 1649 1397 1304 2929 2836 6010,25 5922,25 2819 2695 185 119 169 103 7212,75 7124,75 13118 12994 2864 2713 506 355 6746,5 6658,5 2440 (raw data) KT succinate

45

50

55 Compound 1,2-Didodecanoyl-sn-glycerol2,3,6-trimethyl-1,3-oxazinane5-Methylthio-D-ribose7,8-dihydropterin6-Hydroxymethyl 11325 135 11201 36 11 -171 -154 0 0 0 -75 328 19 262 0 37 0 2674,5 2586,5 574,75 0 486,75 327,5 239,5 6-Hydroxymethyl 7,8-dihydropterin6-Hydroxymethyl pyrophosphate 6-Oxohexanoate Acyclic-7-deazapurine 6-Phospho-2-dehydro-D-gluconate 2’-Aminobiphenyl-2,3-diol 9,10-Dihydrophenanthrene 9-Oxononanoic acid 9-Oxononanoic 6-Pyruvoyltetrahydropterin 6-Phospho-D-glucono-1,5-lactone Xanthen-9-one 8-Hydroxyfuranocoumarin Uridine 5’-triphosphate 1,2-Anthracenediol 5-Hydroxy-L-tryptophan 1-Deaza-5-azapurine 1-Deazapurine 6-Phospho-D-gluconate 7H-pyrrolo[2,3-d]pyrimidine

274 EP 2 230 312 A1 0 0 23 612 2,25 5833 3495 374,5 176,5 5 (- L’A background) 80 700 111 5921 10 3583 462,5 264,5 90,25 253,25 165,25 4424,5 4336,5 4170,5 4082,5 4849,25 4761,25 3413,25 3325,25 3605,25 3517,25 L’A (raw data) 0 0 0 0 0 0 0 0 15 0 92 4693,25 4605,25 (- VUL background)

20 11 -100 2278 2139 4539 4338 (raw data) VUL 0 0 0 0 25 0 47646 47541 3522,75 3434,75 (- KOL background)

30 37 3424 3330 -81 3075 2983 28 3642 3561 173 7082 7016 26 3286 3213 39 5179 5013 44 (continued) (raw data) KOL 0 4 0 0 0 35 0 30 182 1951 1826 6158 6033 -0,75 640 6663 6597 6908 6842 138 4713 4520 -23 270 5232 5166 10708 10642 4178,75 4090,75 931 3716 3650 3491 3425 5341,5 5253,5 145 2661 2494 73 771 -55 2440 (- KT succinate background)

40 4 40 13 10 12 764 128 306 154 262 394 269 895 1399 1275 6736 6670 6207 6141 5203,5 5115,5 1401 1277 2894 2797 2716 2619 1393 1269 -9 1055 2769 2645 24 1197 1073 -74 2635 2511 4415 4349 5346 5280 2440 (raw data) KT succinate

45

50

55 Compound 8-Amino-7-oxononanoateimidodiphosphate 5’-adenylyl 1588 1464 13 0 -42 0 514,75 0 Acetate 3-Acetoacetyl-CoA Tri-L-Alanine 2-Hydroxy-3-oxoadipate Aminoacetaldehyde (S)-5-Oxo-2,5-dihydrofuran-2-butyrate Acenaphthalen-1-ol N-Acetyl-D-mannosamine6-phosphate Naphthyl-2-methyl-succinyl-CoA 1133 1009 1563 1497 2599 2533 7887,5 7799,5 Acetaldehyde O-Acetylcarnitine (R)-acetoin N-Acetyl-L-glutamyl5-phosphate N-Acetyl-L-glutamate5-semialdehyde Acetoacetate 5’-phosphate Adenosylcobalamin N-Acetyl-D-glucosamine 2,2-Azino-bis-3-ethylbenzthiazoline-6- sulfonic acid Acetyl-CoA 1-Phenylethanone

275 EP 2 230 312 A1 0 104 255 5 (- L’A background) 192 10 343 189,25 101,25 235,25 147,25 6178,5 6090,5 539,75 451,75 3777,75 3689,75 L’A (raw data) 0 0 0 0 0 15 0 24 (- VUL background)

20 11 84 -87 (raw data) VUL 0 0 25 0 (- KOL background)

30 18 3231 3165 15423 15357 3853,75 3765,75 30021671 2886 1605 9225 9109 90 13,75 3987 3921 47 (continued) (raw data) KOL 0 0 2 35 0 44 728 4218 4085 -12 2440 (- KT succinate background)

40 16 111 107 126 168 852 7392 7268 87 3575 3451 28 5971 5847 3778 3703 39 2440 (raw data) KT succinate

45

50

55 Compound Adenosyl cobinamide Adenosyl cobinamide phosphate Adenosylcobalamin N-Acetyl-D-glucosamine6-phosphate N-Acetyl-L-glutamate2-Amino-3-oxoadipate 2763AcenaphthaleneN-Acetylneuraminate3-oxo-4-methylpentanoate 2639cis-AconitateN2-Acetyl-L-ornithine 4323 107N5-Propyl-L-ornithineester 5482-butenoyl-CoAAcridine 4199 -121 17Acrolein 114 66 424O-Acetyl-L-serine 8773Adenosine 22 0 47 11670 303N-Acetyl-D-glucosamine1-phosphate 0 0 1622 8707 11604 2797 0 179 0 112 1498 63 34 79 5422 2476 2697 3604,5 4117 46 -7 0 69 5356 13 0 0 -79 93 2623 3516,5 4000 1355 4461 537 0 3315 -93,5 0 -124 0 4 0 0 -5 4395 1231 3249 9383 2566 449 2934 0 0 1283 5258 209,25 1373 0 0 2604,75 9281 2413 2868 6419 1217 5089 121,25 2516,75 1830,75 1285 6180 125 4186 -12 6303 5731 7803 1742,75 6027 4098 5665 0 7634 14,5 37 2653,75 202,75 4011,5 48,5 0 2565,75 114,75 3923,5 0 2-Aminopurine 4-Aminobiphenyl N-Acetyl-D-mannosamine Acetyl phosphate Agmatine

276 EP 2 230 312 A1 0 6,5 5446 6979 5 (- L’A background) 8,5 5534 10 7067 5653,5 5565,5 433,25 345,25 3786,75 3698,75 3782,75 3694,75 L’A (raw data) 0 0 0 1 15 0 11 27 (- VUL background)

20 24 77 (raw data) VUL

25 16 15 (- KOL background)

30 133 2037 1920 -60 1348 1266 83 3147 3081 11841 11775 94,5 3088 3022 24 (continued) (raw data) KOL 0 0 0 0 35 0 78 1775 1631 -162 42 1704 1638 93 2440 (- KT succinate background)

40 -9 56 57 -57 202 166 -120 1657 1533 81 2440 (raw data) KT succinate

45

50 phospho-D-ribosyl)imidazole-

55 Compound ADPADP-L-glycero-D-manno-heptoseADP-D-glycero-D-manno-heptoseAlginate2-amino-4,6-dimethoxypyrimidineADP-ribose -12 120HexanedinitrileAdenosine-GDP-cobinamideUndecane -74 03,4-Dihydroxymandelaldehyde 01-Acetyl-sn-glycero-3- 1199 1200phosphoethanolamine 0 3123S-Adenosyl-L-homocysteine 133 2132-Amino-4-hydroxy-6-(erythro-1,2,3- 1075 1134 2352trihydroxypropyl)dihydropteridine 1819 2916 -116triphosphate 70 9 1419 265 89 2228 1695 -29 3 0 2031 456 1353 0 3903 199 3124 1689 2142 1907 948 0 0 -89 332 3837 3029 6832,75 1623 3 2076 2629 812 6420 6744,75 10297 5217,5 -42 37 0 -33 22 0 2519 10202 2730,5 6345 5129,5 0 2773,75 0 0 0 76 -27 220,25 2642,5 144 2685,75 281,25 62 2929,25 132,25 1692,5 0 0 69 2841,25 193,25 0 1604,5 4001,75 2199 -68 3913,75 27,5 2111 0 0 1-Acetyl-sn-glycero-3-phosphocholine 1-Acetyl-sn-glycerol3-phosphate D-Alanine Acetylhistone 4-carboxamide 1-aminoimidazole 5-Amino-1-(5- D-Alanyl-D-alanine

277 EP 2 230 312 A1 143 5 (- L’A background)

10 231 L’A (raw data)

15 0 (- VUL background)

20 (raw data) VUL

25 (- KOL background)

30 4539 4473 56 (continued) (raw data) KOL 0 35 0 11079 11013 169 103 1426,25 1338,25 2440 (- KT succinate background)

40 4 75 2440 (raw data) KT succinate

45

50

55 Compound (S)-2-Acetolactate4-hydroxy-4-ethyl-2-oxoglutarateN-Acetyl-beta-alanineL-AlanineAlanyllactateN-Methyl-L-alanine 7485-AminolevulinateAlditol 8872methyl gluconate 624 -142D-AldoseADP-glucose 8748 70652-buten-1-ol 0D-Allose 2151 2884 923 6999 164Allophanate -48S-Adenosyl-L-methionine 6454 2816 20275-Amino-4-imidazolecarboxylate 50 799 2083 40(S)-Allantoin 6388 0L-2-Aminoadipate6-semialdehyde -87 2704 72Altrose 133 1959 0 4598 1624 2334(R)-Allantoin 2579 -37 2638 2848 13236-phosphateAminofructose 0 36 4924 4490 0 0 556-semialdehyde2-Aminomuconate 1500 2210 2455 241 115 2257 -56L-2-Aminoadipate 0 61 1199 4142,25 4471 4512 1378 0 1769 3532 722 2906 1361 0 49 3364 153 4054,25 1058 0 10686 3612 4404 65 0 1312 1593 3419 2781 1664 580 770 3291 1889 10562 176,5 934 267,25 415 3524 -115 129 24 2561 -4 0 1571 93 -17 3399,25 1568,75 1772 4526 -6 179,25 88,5 62 2495 1566 291 63 0 3311,25 1480,75 0 134,5 0 0 -120 0 4460 0 -119 1473 0 922 2814,75 241,25 -108 -102 46,5 0 81,75 639 258 1862,5 2726,75 0 285 856 4871 153,25 0 0 1848,75 486 0 1774,5 170 4805 15 -46 197 1760,75 2607,25 41 2318 89 2519,25 0 0 2230 0 3764,25 1 4632,25 3676,25 4544,25

278 EP 2 230 312 A1 0 193 291 80,5 1155 122,5 351,5 723,5 5 (- L’A background) 281 379

10 1243 210,5 439,5 168,5 42,75 811,5 4877,5 4789,5 288,75 200,75 3526,5 3438,5 2079,5 1991,5 3824,25 3736,25 5592,25 5504,25 6826,75 6738,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 15 0 46 (- VUL background) 8 0 8 20 0 31 -26 239 173 152,75 64,75 1081 959 (raw data) VUL 0 0 0 5 25 0 (- KOL background)

30 4 10 20 1813 1747 -58 7583 7517 -59 4091 4025 -48 5823 5504 2169 1850 9183,5 9095,5 6168 6102 2801 2625 71 5272 5206 (continued) (raw data) KOL 0 0 0 0 0 0 35 0 89 3757 3375 902 1309 1243 -147 157 -157 295 151 3366 3300 -73 690 2440 (- KT succinate background)

40 55 74 10 53 -37 419 281 213 275 -203 -132 1026 2071 1947 1685 1561 5772 5706 -29 1399 1275 71 2440 (raw data) KT succinate

45

50

55 Compound 5-Amiriovaleric acid 5-Amiriovaleric Aminoacetone 2-Aminophenol S-Adenosyl-4-methylthio-2-oxobutanoate 814 D-AltronateAcetyl-maltoseS-Adenosyl-L-threonineS-AdenosylmethioninamineAllantoateD-glucoside Methyl Aminoimidazole ribotide 1400 843 1618 -119 1276Naphthyl-2-methylene-succinyl-CoA 719 1494Naphthyl-2-hydroxymethyl-succinylCoA 0 2785 202 23 36 2234 148 2719 78 2110 0 0 5142 82 1588 939 6716 5076 47 -28 1512 873 6547 988 0 0 113 112 -24,5 900 48,5 37 99,25 0 616,75 11,25 0 528,75 Naphthalen-1-ylphosphate hydrogen 2-Aminomuconate Amylose Amylopectin 5-Carboxymethyl-2-oxohex-3-ene-1,6- dioate AMP 1,3-Cyclohexanedione 3-Hydroxycyclohexanone Naphthyl-2-oxomethyl-succinyl-CoA

279 EP 2 230 312 A1 0 373 360 3831 259,5 5 (- L’A background) -2 461 448 47,5

10 3919 5805,75 5717,75 6384,25 6296,25 L’A (raw data) 0 0 0 15 0 (- VUL background)

20 9 48 -71 4698 4594 (raw data) VUL 0 0 1 11680 11614 25 0 (- KOL background)

30 26 -102 5838 5772 1250 1184 293 227 143,25 55,25 4179 4113 12141 12075 189,25 101,25 (continued) (raw data) KOL 0 0 0 35 0 15 41 1855 1655 5719 5519 484 1880 1814 -107 790 6233 6167 4694 4628 329,25 241,25 2440 (- KT succinate background)

40 17 45 139 165 123 608 107 914 1425 1301 -1 7129 7005 67 2440 (raw data) KT succinate

45

50

55 Compound Arbutin 6-phosphate Biphenyl-2,3-diol AnisoleNaphthalen-1-ylacetate Aniline2-aminosuccinateAnserineAnthraceneD-Arabinono-1,4-lactoneP1,P5-Bis(5’-adenosyl)pentaphosphate 1973-O-L-Alanyl-1-O-phosphatidylglycerolN-Acetylputrescine 2313-Aminopropanal -118 73 557 -127alpha-Aminopropiononitrilealpha-Pinene 468 285 107 3135sulfate 5’-Adenylyl 0 433 0 -97sulfate 2’-Phosphoadenylyl 218 344L-Arabinono-1,5-lactone -36 2926 1615-phosphate D-Arabinose 2430 72 2209 3178 1098D-Arabinose 0 94 -162 3760 -58 192 0 2364 2085 1032 3054 6 1973 3694 -70 0 0 5034 68 -18 -5 1943 276 5 1907 -80 49 0 4910 379,5 4073 -64 1877 0 56 0 152 0 0 4650 0 -238 4007 291,5 56 0 -263 373,25 181,25 86 4584 75 326 23 0 324,5 285,25 0 93,25 0 1094 -46 266 236,5 0 238 0 193 192,25 1006 819 0 178 3585,5 104,25 105 731 4468 3497,5 4380 L-Arginine L-Arabinose Pl,P4-Bis(5’-adenosyl)tetraphosphate

280 EP 2 230 312 A1 0 494 628 305 144 5032 278,5 5 (- L’A background) 582 716 393 10 232 366,5 3817,5 3729,5 3193,5 3105,5 352,75 264,75 5685,5 5597,5 3419,5 3331,5 185,25 97,25 187,75 99,75 5319,5 3305,5 1533,75 1445,75 3129,75 3041,75 1549,75 1461,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 43 1988,25 1900,25 32 56 (- VUL background) -2 20 -2 98 -86 -11 -78 122 6239 6173 -64,5 (raw data) VUL 0 0 0 0 0 14761 14411 5120 0 25 0 28 (- KOL background) 4

30 -3 94 -18 -32 187 121 -20 1747 1654 70 (continued) (raw data) KOL 0 0 0 0 0 35 0 23 4019 3953 -118 66 5583 5401 35 248 3853 3542 -8 161 242 120 170 392 2473 2407 -64 502 2476 2410 -52 852 2080 2014 33 165 -41 359 320 254 109 120 5180 5114 63 2440 (- KT succinate background)

40 90 66 18 12 -36 -11 372 285 147 294 516 190 626 976 289 483 244 4473 4349 -10 2064 1940 5101 5035 6505 6439 1408,25 1320,25 19136 19012 -180 2440 (raw data) KT succinate

45

50

55 Compound N-ethylaniline L-Aspartate L-Asparagine octanoate Ascorbyl Atropine 2-Hydroxylminostilbene Arene oxideArachidic acidester methyl L-Arginine ester methyl acid L-Arginosuccinic 591 72 467 0 4120 13532 4054 13466 51 125 0 59 398,5 2764 310,5 2676 Ascorbyl acetate Ascorbyl hexanoate 4-Hydroxymethylsalicylate Aziridine ATP Azetidine Arginine p-nitroanilineArginine Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly- Leu-Met-NH2 4-semialdehydeL-Aspartate butyrate Ascorbyl propionate Ascorbyl Atrazine

281 EP 2 230 312 A1 0 0 154 738 46,5 20,5 1589 5245 5 (- L’A background) 242 826 1677 10 5333 134,5 108,5 140,75 52,75 202,25 114,25 443,75 355,75 167,75 79,75 4889,25 4801,25 L’A (raw data) 0 0 0 0 0 0 0 15 0 86 19 3613,25 3525,25 21 38 -115,25 56 (- VUL background)

20 8 87 39 -19 122 -144 5477 5382 391,75 303,75 3410 3344 3277,25 3189,25 (raw data) VUL 0 0 0 0 0 25 0 (- KOL background)

30 16 16 -86 2915 2849 8593 8527 -14,5 5391 5325 85 4683 4617 1803 1736 -77 1493 1427 3216 3150 234,75 146,75 (continued) (raw data) KOL 0 0 0 0 0 35 0 16 4611 4511 16 132 -106 347 3064 2991 -117 363 2027 1953 160 280 -111 177 144 1373 1293 -68 354 2440 (- KT succinate background)

40 6 40 43 81 256 471 487 404 301 118 478 268 140 -139 1438 1314 810 744 1411 1287 3809 3743 104 1837 1713 29 2440 (raw data) KT succinate

45

50

55 Compound Phenylmethyl hexanoate Phenylmethyl 1,2-Benzophenanthrene Benzamide Phenylmethyl acetate Phenylmethyl propanoate Phenylmethyl Benzoyl-CoA (R,S)-Tetrahydrobenzylisoquinoline Benzimidazole tryptophan methylestergamma-Butyrobetainyl-CoACycloheptaamyloseButanoyldolichol2-methyl-3-oxobutanoateBenzyl (2R,3S)-2-methyl-3-Benzyl hydroxybutanoate 242 324-Phenylbutan-2-one Phenylmethyl benzoate -84 118 -22 0 61 0 -75 0 2641 3794 0Benzyl alcohol 0 2575 3661 3728 3419 61 3595 74 153 3353 -56 0 82-Benzothiophene 87 23 0 130,75 3540 414,25 0 42,75 150,5 3452 326,25 551,25 62,5 463,25 2,1-Benzoxazole Poly-beta-Hydroxybutyrate Benzene 1-Benzothiophene

282 EP 2 230 312 A1

5 (- L’A background)

10 L’A (raw data)

15 (- VUL background)

20 (raw data) VUL

25 (- KOL background)

30 (continued) (raw data) KOL

35 2440 (- KT succinate background)

40 2440 (raw data) KT succinate

45

50

55 Compound Betaine aldehydeBenzocyclobuteneBenzofuran2,6-DichlorobenzonitrileBenzoate2-Methylthiobenzothiazole2H-BenzotriazoleBenzoxazole 1174Benzocycloheptene 50Betaine 586Benzonitrile 1050 201N-Benzoylanthranilate -39 0CDP-1,2-Dihexadecanoylglycerol 462 5981Myrcene 1324 77 59Bromobenzene-2,3-oxide 0 4230 5859 164Nicofuranose 35 -170 1967Bromobenzene-3,4-oxide 1200 -12 41182-Bromomaleylacetate 9947 -194 0 98 50m-Bromobenzotrifluoride 2510 1843 0 0butenoyl-CoA 301 -69 9825 0 7 704 24034-Aminophenol 0 237 2444 4612epsilon-N-Biotinyl-L-lysine 0 103 -36 3505,5 177Butylamine -77 2249 0 25 580 0 184 132 171 4546 74 203 335 -37 136 3417,5 29 0 40 172 0 5248 -70 0 -25 66 257,5 60 132 8 79 211 -87 0 142 12 18 5182 -38 0 84 2381 0 3294,75 169,5 221,25 59 0 -16 160,25 2849 -62 44 68 -25 751 0 7 4632,5 2315 3206,75 0 -46 133,25 0 1693,75 17 13 2727 0 72,25 0 0 685 4544,5 219,5 21 0 6 1605,75 0 -42,75 0 7621 2138 0 1559 42 0 6350,5 89 131,5 11091 0 7499 1933 148,75 0 0 6688 1493 10811 2655 6262,5 230 0 23 2686,25 2597,5 31 60,75 -107 6600 9 429,75 2567 2759,25 2598,25 142 2509,5 57 0 0 341,75 -150 2671,25 0 4053,5 3084 0 0 203 3965,5 5405,25 2996 115 5317,25

283 EP 2 230 312 A1 0 67 24 233 49,5 52,5 4723 357,5 273,5 208,5 5 (- L’A background) 155 112 321

10 4811 445,5 361,5 140,5 666,75 578,75 773,25 685,25 2053,5 1965,5 2393,75 2305,75 1495,75 1407,75 1784,75 1696,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 15 0 (- VUL background)

20 0 12 22 11 -31 5360 5256 137,5 1255 1189 3341,25 3253,25 3368 3284 5059,25 4971,25 (raw data) VUL 0 0 0 0 0 0 0 25 0 (- KOL background)

30 32 50 11 -38 140 1902 1756 -59 1800 1734 4425 4359 -3,25 3172 2947 -29 1085 1019 -17 1409 1343 -10 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 35 0 81 -113 745 2804 2660 111 918 3025 2959 11275 11209 296,5 217 3437 3124 8645 8332 2440 (- KT succinate background)

40 6 19 96 89 -18 -97 -10 -76 -33 869 114 205 341 2068 1944 -104 1327 1203 15 1191 1067 2406 2328 11255 11177 245,75 157,75 2126 2002 2949 2669 -93 2008 1884 4206 4140 46 1042 2440 (raw data) KT succinate

45

50

55 Compound ButyrateButyl butyrateButyryl-CoAUridinecarboxylate 4-imidazole methyl Cyclohexa-1,5-diene-1-carbonyl-CoA Caffeate 432 2515 -49 2391 308 2211 0 -11 5345 2087 5591 5279 0 13715 5450 6701 13647 -197 6635 7787 63 0 7719 2541,5 0 3225 2453,5 -78,5 2281,25 3137 2193,25 0 Cellobiose-1,5-lactone Cadaverine Caffeoyl-CoA Butyl paraben Butyl 3’,5’-CyclicAMP Hexadec-1-ene epsilon-Caprolactam Hexanoyl-CoA N-Carbamyl-L-glutamate caffeate Methyl 5-Carboxy-2-pentenoyl-CoA Carbazole 8-Hydroxy-2-methyl-alpha-carboline N-Butyl-beta-carboline-3-carboxylate (-)-trans-Carveol Catechol

284 EP 2 230 312 A1 0 0 0 4,25 257,5 5 (- L’A background) 65 26

10 68,5 92,25 345,5 446,75 358,75 405,75 317,75 410,75 322,75 7394,5 7306,5 3734,5 3646,5 L’A (raw data) 0 0 0 0 0 0 9 15 0 10 (- VUL background)

20 26 75 78 19 -60 4745 4619 484,25 396,25 (raw data) VUL 2 0 0 25 0 89 35 (- KOL background)

30 68 -99 -35 101 8980 8914 9999 9933 4793,75 4705,75 1088 1022 40 2189 2123 10 5444 5367 54 6172 6106 4337 4271 349,75 261,75 (continued) (raw data) KOL 0 0 0 0 0 35 0 10 75 93 190 1649 1521 881 753 2440 (- KT succinate background)

40 -7 77 -59 -36 134 107 199 112 217 314 1421 1297 2429 2310 -102 1640 1516 155 1143 1019 -123 2440 (raw data) KT succinate

45

50

55 Compound CDP-Hexadecanoylglycerol Capryldihydroxyacetonephosphate 2-hydroxy-4-phenyl-butyricmethyl acid ester CDP-1,2-Dinonadecanoylglycerol Benzyl-L-cysteinamide CDP-butyrylglycerol 8-Hydroxy-2-methyl-beta-carboline8-Hydroxy-2-methyl-gamma-carbolineCarnosineCarvone 5152-(3-Carboxy-3-aminopropyl)-L-histidine 219CatechinN-Carbamoyl-L-aspartate 205 391 953-IsopropylmalateCobinamidephosphate Carbamoyl 130 81 50m-Aminophenol methyl4-Hydroxyphenylglyoxylate 573 1277 643’,5’-CyclicCMP 1197 0 234 1153 449 1128 -92 270 7cis-1,2-Dihydrobenzene-1,2-diol 110 235 2457 3655 -105 50 146 0 0 122 11 2361 3586 0 247 0 6026 3962,5 3903 3985,75 -80 0 10179 181 5960 2280 3897,75 3874,5 3815 1241 0 9995 5618 2212 78 1141 3669 5552 26 140 12 330,75 3581 181,5 72 3086,25 0 242,75 93,5 2998,25 7175,75 7087,75 3’,5’-CyclicdAMP trans-Hex-2-enoyl-CoA

285 EP 2 230 312 A1

5 (- L’A background)

10 L’A (raw data)

15 (- VUL background)

20 (raw data) VUL

25 (- KOL background)

30 (continued) (raw data) KOL

35 2440 (- KT succinate background)

40 2440 (raw data) KT succinate

45

50

55 Compound CDP-1,2-DibutyrylglycerolCDP-cholineCDP-DodecanoylglycerolCDP-1,2-DinonanoylglycerolCDP-TetradecanoylglycerolCDP-1,2-Ditetradecanoylglycerol 1490CDP-1,2-DihexanoylglycerolCDP-1,2-DioleylglycerolCDP-1,2-Dioctanoylglycerol 33 1366 -15CDP-Octanoylglycerol 4 83Cytidine diphosphate 15 640 10245CDP-1,2-Didodecanoylglycerol 0 0CDP-1,2-Dioctadecanoylglycerol 0 0CDP-1,2-Dipropionylglycerol 10121 574 209 0 341CDP-Decanoylglycerol 2999 -16CDP-ethanolamine 2841 1559 2040 3857 2153 163-Hydroxyaminophenol 4031 217 85 -23 2933 520CDP-glycerol 0 2775 1803 3791 2087CDP-hexanoylglycerol 1435 3965 83 5315 2565CDP-Nonanoylglycerol 16 0 0 396CDP-Nonadecanoylglycerol -65 3325,25 2582 33 -26 22 152 5095CDP-propionylglycerol -8 2499 0 26904-Hydroxybiphenyl 0 3466 2853 3237,25 530 -64 2516 0 0 0 109 6378 0 28 0 2446 3400 455 2787 22 401,5 35 406 -107 -52 0 6312 3592,25 51 3282,25 2757,25 0 8318 10 187 8862 5651 304 709,5 -59 4835 313,5 3504,25 0 3194,25 0 2669,25 0 502 -81 8074 0 8796 5585 0 573,5 0 555 -86 621,5 99 4697 0 8378 28 234,25 157,5 3648,5 4743,5 0 414 -12 2477 485,5 4815 431 79 0 8192 146,25 2652 3560,5 4655,5 69,5 0 2411 136 4677 0 2 0 601,75 2641 2586 3503 3547,75 -83 38 -120 513,75 2455 383,25 0 20 3437 3459,75 4483,25 0 0 367 295,25 -169 30,75 0 4395,25 682,25 417,25 279 0 399,25 0 594,25 329,25 311,25 4110,5 4022,5

286 EP 2 230 312 A1 17 207 5975 2455 5 (- L’A background) 105 295

10 6063 651,75 563,75 558,75 470,75 224,25 136,25 2143,5 2055,5 1668,25 1580,25 4445,25 4357,25 11096,3 11008,3 1735,25 1647,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 15 0 46 3359,25 3271,25 55 1654,25 1566,25 (- VUL background)

20 5 85 94 26 28 22 -41 121 (raw data) VUL 0 0 0 0 0 0 10443 10304 2543 0 25 0 57 (- KOL background)

30 26 37 -70 123 2237 2171 -104 1949 1883 23 4263 4153 6951 6841 2192,75 2104,75 3929 3863 9472 9406 5912,75 5824,75 (continued) (raw data) KOL 0 0 0 0 0 0 0 35 0 278 296 230 170 104 1814,5 1726,5 316 4292 4091 94 418 3521 3455 -126 135 2151 2085 6068 6002 3695,25 3607,25 458 -90 2440 (- KT succinate background)

40 93 96 45 81 -22 113 402 105 440 542 259 102 582 1405 1281 -83 2588 2464 25 4175 4051 -27 1428 1304 4983 4917 112 1885 1761 -54 2440 (raw data) KT succinate

45

50

55 Compound Choline 2,6-dinitrotoluene CellobioseCellotrioseCellotetraoseCellulosecis-3-(3-Carboxyethenyl)-3,5- cyclohexadiene-1,2-diol CDP-1,2-Didecanoylglycerol L-Cysteinylglycine 44 91 -65 200 0 0 0 76 786 105 159 682 -7 39 61 99 0 71 -6 0 -30 5 0 7,75 0 3682 740,25 0 3387 652,25 3594 3299 Cetyl alcohol 4-Chlorocatechol Cyclohexan-1,2-dione Chitobiose acetate Ethyl Cyclohexanone Chitin Chitosan 3’,5’-CyclicGMP Tolylacetate Cyclohex-2-enone 6-Carboxyhexenoyl-CoA ethylhexanoate

287 EP 2 230 312 A1 146 861 45,5 4753 2017 378,5 5 (- L’A background) 234 949 4841 10 2105 466,5 133,5 385,25 297,25 472,25 384,25 8151,5 8063,5 4029,5 3941,5 366,75 278,75 318,75 230,75 1827,25 1739,25 3719,75 3631,75 2551,25 2463,25 4264,75 4176,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 10 2934,25 2846,25 (- VUL background)

20 58 72 72 -85 -66 -73 -32 158 -121 8673 8607 289,75 201,75 6537 6471 4106,75 4018,75 7095 7029 161,25 73,25 (raw data) VUL 0 0 0 0 0 0 0 0 0 0 0 25 0 (- KOL background)

30 6 45 19 -73 -21 390 211 792 613 3062,25 2974,25 -156 3771 3623 -60 3425 3231 -62 2192 2116 -52 5893 5827 -51 (continued) (raw data) KOL 0 0 0 0 0 0 0 35 0 67 5118 5052 -80 34 -122 54 -102 96 348 262 1342 1276 25 196 2584 2518 3606 3540 5411,75 5323,75 527 -60 2440 (- KT succinate background)

40 2 96 12 52 -94 -13 472 386 320 651 191 158 178 220 -126 2077 1953 2941 2875 -49 1916 1792 1039 973 498 432 1995,25 1907,25 4440 4316 -27 2932 2808 -35 2252 2128 43 2096 1972 31536 31412 4924 4820 95 2440 (raw data) KT succinate

45

50

55 Compound 2-Hydroxy-2,4-pentadienoate 1-Bromopentane 1-Chloropentane Cinnamaldehyde acetate Cinnamyl N-Acetyl-D-glucosamine1,6-bisphosphate 4-Chlorophenol 119 Chorismate 5,7-Dihydroxychromone Styrene oxide 2,3-Dibromopentane Cinnamoyl-CoA L-Citrulline Cinnoline 4-Chloro-m-cresol 1,2,3-Trimethylbenzene CMP Citrate trans-Aconitate (R)-(+)-Citronellal (-)-Citronellol CMP-3-deoxy-D-manno-octulosonate propyl acetate

288 EP 2 230 312 A1 4028 5 (- L’A background)

10 4116 367,75 279,75 8319,5 8231,5 309,75 221,75 9265,5 9177,5 6432,5 6344,5 4003,75 3915,75 4602,25 4514,25 L’A (raw data) 0 0 0 0 0 0 0 15 3 (- VUL background)

20 69 -37 109 336 270 293,75 205,75 (raw data) VUL 0 25 0 73 41 (- KOL background)

30 62 139 107 4562 4496 41 (continued) (raw data) KOL 0 0 19726 19660 7 9 35 0 93 -141 16 46 2586 2520 29 168 23902 23784 -45 2440 (- KT succinate background)

40 70 55 -46 292 217 140 170 133 4000 3876 1534 1375 111 2440 (raw data) KT succinate

45

50

55 Compound 4-Coumarate p-Coumaroyl-CoA delta-aminovalerate2-ChlorobenzoateCardiolipin3-Carboxy-cis,cis-muconate4-Chlorobutan-1-olChloroxylenolStyreneChlorogenate 1652-Amino-3-carboxymuconate semialdehyde 1788 -177gamma-Carboxymuconolactone 107Coenzyme A 41 1664Coniferyl alcohol 2450 -101 0Coniferaldehyde 03-Chloro-cis,cis-muconate 2070 -80 1808 3369 2326acid Coronamic 2977 0 1974 1684 74 0 16 3245 220 138 2879 75 8 -15 1326 5272 1931 283 -82Coumaran 0 96 135 0 0Coumarin 224 9 1236 5148 0 0 1807 2508 159 37 0 4521 25 95 100 25 -117 186 2434 2442 2170,75 2257 4455 58 229,25 486,75 0 0 2153 2368 2082,75 0 120 8055 0 2065 -129 141,25 398,75 0 1972 229,25 4913 7989 20 103 43 332,25 1703,5 0 128 124,75 141,25 277,75 4825 244,25 1615,5 0 0 0 2667,75 36,75 0 189,75 2579,75 574,25 6038,75 2915 5848 5950,75 486,25 2827 5760 3-Ethyltoluene Crotonoyl-CoA p-coumarate Methyl CDP-Octadecanoylglycerol

289 EP 2 230 312 A1 125 388 354,5 557,5 5 (- L’A background) 213 10 476 264,75 176,75 6256,5 6168,5 6008,25 5920,25 3318,25 3230,25 2952,75 2864,75 L’A (raw data) 0 0 0 0 0 15 0 (- VUL background)

20 -57 (raw data) VUL

25 0 81 10193 10092 645,5 (- KOL background)

30 1617 1465 -114 3815 3749 60 (continued) (raw data) KOL 0 35 0 92 5530 5453 14357 14280 458,75 370,75 29 1546 1431 20468 20353 442,5 479 2409 2311 -96 2440 (- KT succinate background)

40 -64 603 113 216 153 2041 1917 3237 3135 9973 9871 1904 17806007 3983 5883 3917 2009 9683 911 9617 4757 4058,5 3659 3970,5 2509,25 2421,25 2013 1889 3541 3465 -45 1186 1062 182 3143 3019 100 2810 2686 1766 1700 60 2440 (raw data) KT succinate

45

50

55 Compound L-pipecolate 2-CoumarinateCreatineEthylglycocyamine3-ChlorocatecholCTP2-Chloro-cis,cis-muconateCytosineCoronafacic acidCrotono-betaine -173,5-Dichlorocatechol -50Coronatine 36 750 138aldehyde p-Cumic 0Cumene 0 626 14N6-(1,2-Dicarboxyethyl)-AMP 0 165 3576 245 335 1063Cyclobutane 129 116 5 1276 4755Cycloheptane 3452 47 121Cycloheptadecane 182 939 1173Cyclohexanol 4669 0 5 -10 3602 0Cyclohexylamine -30 2123 5274 94 -18 81 3522 2597 -41 2057 3337 0 0 5129 0 3239 0 2531 0 3196 0 24 2239 -81 352,25 3159 114 118,75 24 129 3763,75 2173 0 264,25 485 0 2820 30,75 48 3675,75 -54 0 41 344,25 397 510 2732 6066,75 0 4147,75 256,25 5978,75 422 4059,75 130,75 42,75 p-Cumic alcohol p-Cumic Cyclohexane Cyclohexadecane

290 EP 2 230 312 A1 37 135 940 619 6464 2587 569,5 327,5 5 (- L’A background) 125 223 707 1028 6552 10 2675 657,5 415,5 528,25 440,25 194,25 106,25 3650,75 3562,75 1104,25 1016,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 15 0 21 (- VUL background) 7 1

20 -5 13 -12 100 -101 (raw data) VUL 0 0 0 25 0 (- KOL background)

30 84 -25 873 807 5067 5001 6453 6387 2865 2690 127 3498 3432 87 5086 5020 9674 9608 (continued) (raw data) KOL 0 0 0 0 0 0 35 0 25 23 8047 7981 2148 2082 9568,75 9480,75 490 4483 4380 -49 206 -42 2440 (- KT succinate background)

40 -1 15 39 28 39 -10 -27 614 330 149 2497 2373 -33 3180 3056 8276 8129 127 2440 (raw data) KT succinate

45

50

55 Compound Cyclohexene oxideCyclononaneCyclopentaneUreaCyclopropanedADPD-CycloserineCycloundecane 1837L-Cysteinetrans-Cyclohexane-1,2-diol 1713 209acid Decanoic 3298 3371 2310 85 3174 2630 3195 201 -42 2186 -72 2232 1970 787 2506 20820 1397 3593 77 2003 0 1846 662 24067 0 20644 3505 1273 -36 6248 23816 235 2309 -2 250 5692 1969 14 6182 -50 2243 0 18 5626 0 162 1903 -98 4713 0 15325 0 39 184 -63 339,25 4647 15108 0 2746,25 716,25 0 251,25 96 3030,25 0 499,25 2658,25 6950,75 628,25 2942,25 4154,75 411,25 2140,25 6862,75 4066,75 2052,25 L-Cystine Cyclopentanone Caproyldihydroxyacetoneposphate 2,4-Dihydrofuran Acetylcysteine Cysteine p-nitroaniline Cytidine 2-methyl-3-oxosuccinateDiethyl L-Cystathionine Cystinyl p-nitroalinineCystinyl Methyl 2-phenylacetate Methyl D-4-Hydroxyphenyl-glycineestermethyl 147

291 EP 2 230 312 A1 0 990 4497 5251 283,5 5 (- L’A background) 40,5 4585 5339 10 1078 371,5 491,75 403,75 317,75 229,75 5314,5 5226,5 287,25 199,25 394,75 306,75 281,25 193,25 3075,75 2987,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 15 0 98 (- VUL background)

20 9 15 -66 -45 -167 7014 6948 (raw data) VUL 0 0 0 25 0 26 77 7547 7481 388,333 300,333 (- KOL background)

30 92 17 -84 143 4617 4551 164 1826 1732 4756 4662 370,25 282,25 3154 3088 -54 (continued) (raw data) KOL 0 0 0 0 0 35 0 30 4454 4294 -75 59 18 2451 2307 17460 17316 177,75 89,75 29 2065 1994 -37 373 4559 4493 9099 9033 491,75 403,75 103 3813 3747 152 4282 4190 -57 2440 (- KT succinate background)

40 32 19 15 72 -49 -49 154 497 227 276 183 142 153 7663 7539 15 3027 2903 5384 5318 61 2440 (raw data) KT succinate

45

50

55 Compound Decanoyl-CoA D-Galacturonate 1-phosphate D-Galacturonate Methyl decanoate trans-Dec-2-Enoyl-CoA beta-Alanyl-L-lysine 2-Ethyltoluene 2-carboxy-D-arabinitol D-Arabonate 2’-Deoxy-5-hydroxymethylcytidine-5’- diphosphate N6-(L-1,3-Dicarboxypropyl)-L-lysine 7,8-DihydrofolateD-Galactono-1,5-lactonelumazineDeoxy-5-methylcytidylateCyclopenta[b]pyridineD-Aldonolactonephosphate Octadecanoylglycerone 282 1064 86 158 940 73 122 0 2 286 -8 0 0 3046 162 0 0 14 2980 -8 2956 32 88 -34 0 2874 0 0 22 0 -101 94 16 7952,25 2482 0 4064 12 7864,25 0 2394 3976 168,5 1046 322,75 80,5 958 234,75 2’-Deoxy-5-hydroxymethylcytidine-5’- triphosphate D-Arabinitol 1,3-Diaminopropane 7,8-Diaminononanoate 3,4-Dihydroxy-2-butanone4-phosphate 2413 2289 12

292 EP 2 230 312 A1 98 218 139 179,5 228,5 440,5 5 (- L’A background) 306 227 10 186 267,5 316,5 528,5 436,25 348,25 4703,5 4615,5 649,75 561,75 3375,5 3287,5 3492,75 3404,75 2357,75 2269,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 15 0 12 3520,75 3432,75 29 24 6254,25 6166,25 (- VUL background)

20 17 -174 (raw data) VUL

25 0 (- KOL background)

30 -54 932 866 2399 2333 -90 3837 3771 78 4319 4199 149 8146 8080 90 1669 1603 50 (continued) (raw data) KOL 0 0 30766 30700 1 0 0 0 0 0 35 0 20 7144 7078 -115 99 1625 1514 24 108 884 804 -62 146 5582 5455 -22 156 3880 3814 -71 2440 (- KT succinate background)

40 4 61 70 32 -20 -85 232 270 280 223 144 -144 4605 4481 2593 2527 41 2440 (raw data) KT succinate

45

50

55 Dihydrouracil Octadecanal 2’-Deoxy-5-hydroxymethylcytidine5’- phosphate 2-Deoxy-D-arabinose (S)-2-Hydroxyglutarate Decanoyldolichol Decane Decanal Compound Dodecanoyl-CoA 1-Tetradecanoyl-sn-glycerol3-phosphate Dodecanoyldolichol -339 Decanoyldihydroxyacetonephosphate Dodecanoate dCMPdCDP3-Hydroxy-4-trimethylammoniohexanoateDihydrothyminedCTP 57O-Butanoylcarnitine3’,4’,5,7-Tetrahydroxy-3-methoxyflavonetrans-Dodec-2-Enoyl-COA 0dodecanoate Methyl 99 5403 603 1769 60 0 626 183 5279 388 1645 739 2710 0 502 1481 59 -38 3115 2644 2214 615 1415 -122 2211 3049 0 -94 2148 2404 -5 0 2034 1 2129 2338 51 0 118 0 55 0 2041 -77 189,75 0 0 423 0 101,75 0 424 1569,75 6743,25 335 84 1481,75 4309,75 6655,25 336 4221,75 0

293 EP 2 230 312 A1 173 2199 3809 398,5 326,5 5 (- L’A background) 261 3897 10 2287 414,5 638,75 550,75 207,75 119,75 5487,5 5399,5 196,75 108,75 4954,75 4866,75 5860,75 5772,75 3583,75 3495,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 15 0 (- VUL background)

20 4 28 35 65 -13 8411 8273 486,5 (raw data) VUL 9 0 0 0 0 25 0 (- KOL background)

30 -4 -14 -29 147 -385 -100 4027 3961 -99 4458 4392 -81 5888 5822 19 2833 2767 56 3673 3607 -52 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 6 0 35 0 628 4765 4699 -21 225 3494 3428 181 115 7005,25 6917,25 2440 (- KT succinate background)

40 8 81 59 12 30 -19 -28 -34 121 100 130 752 349 2440 (raw data) KT succinate

45

50

55 Compound D-FucoseD-Glucono-1,5-lactonedGDPD-GlucaratedGMP4-Bromophenol-2,3-epoxidedGTP1-Hydroxy-2-naphthoate -210hydroxyacetonephosphate Dihydroxyacetone 77(1S,3R,4S)-3,4-Dihydroxycyclohexane-1- 170 0carboxylate 7,8-Dihydrofolate 1125 2 0 351 46 -77 997 1001 0 1111 227 65 42 0 206 2802 873 1045 3782 73 0 -167 2736 82 0 -79 3633 45 7 0 50 4665 70 2645 0 59 0 4599 2549 -28 2577,5 0 4 -1 0 -151 456 68 2489,5 0 3357,25 461,5 0 0 558 3269,25 368 0 373,5 906 318,75 155,5 470 4677,25 818 230,75 4589,25 67,5 trans-2,3-Dihydroxycinnamate 3-Dehydrosphinganine 1,4-Dihydroxy-2-naphthoate 2-Amino-4-hydroxy-6-(D-erythro-1,2,3- trihydroxypropyl)-7,8-dihydropteridine (S)-Dihydroorotate Dihydropteroate (S)-4,5-dihydroxypentan-2,3-dione Dihydroneopterin phosphate Dihydroneopterin 2,3-Dihydroxyphenylpropanoate 3’,5-Dihydroxy-3,4’,7-trimethoxyflavone

294 EP 2 230 312 A1 54 732 329 541 275 2267 7366 1207 121,5 348,5 860,5 665,5 5 (- L’A background) 820 417 629 142 363 2355 7454 10 1295 209,5 436,5 948,5 645,75 557,75 491,25 403,25 2216,75 2128,75 6136,75 6048,75 1318,75 1230,75 18901,5 18813,5 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 15 0 62 1235,75 1147,75 37 (- VUL background) 1 0

20 -1 67 46 -49 948 882 (raw data) VUL 0 0 0 0 0 25 0 (- KOL background) 3

30 -7 -16 -73 448 259 134 -122 2823 2737 44 8583 8517 -44 1280 1214 103 2959 2881 2228 2150 2119 2053 -74 2180 2111 4289 4220 753,5 9727 9555 44 (continued) (raw data) KOL 0 0 0 0 4 0 0 0 0 0 0 35 0 45 1175 1032 -57 63 134 -76 654 3744 3678 504 5165 5066 161 580 3425 3287 3632 3494 5528,5 5440,5 130 2806 2673 19 2440 (- KT succinate background)

40 9 38 43 24 -10 -16 -41 778 120 628 704 254 128 258 169 123 187 -101 1810 1686 478 412 737 671 2440 (raw data) KT succinate

45

50

55 Compound Dibenzofuran (5R)-3,4-Dihydroxy-5-(hydroxymethyl) furan-2(5H)-one 2,4-Dibromophenol 2,6-Dibromophenol Deoxyguanosine Dibenzo-p-dioxin dTMP D-Iditol 4-Chlorophenylacetate Digallate Dihydrocoumarin 9-Hydroxynonanoate 2,4-Dinitrotoluene 10-Hydroxydecanoate Deoxyinosine Dioxybenzone 2,3-Bis(3-Hydroxytetradecanoyl)-D- glucosaminyl-1,6-beta-D-2,3-bis(3- hydroxytetradecanoyl)-beta-D- glucosaminyl 1-phosphate 2,3-Diketo-5-methylthiopentyl-1-phosphate4-Carboxymethylenebut-2-en-4-olide cis-2-Methyl-5-isopropylhexa-2,5-dienoic 41 acid

295 EP 2 230 312 A1 337 465 172 519 346,5 5 (- L’A background) 425 553 260 10 607 434,5 388,75 300,75 899,75 811,75 202,75 114,75 8388,5 8300,5 413,25 325,25 3178,5 3090,5 291,25 203,25 5784,75 5696,75 1155,75 1067,75 1558,25 1470,25 6509,75 6421,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 15 0 62 40 38 (- VUL background)

20 57 24 -72 -72 264 106 -140 -135 (raw data) VUL 0 0 0 0 0 0 25 0 67 (- KOL background)

30 -9 89 24 60 -91 -28 992 926 -75 676 564 174 133 5009 4800 71 6852 6768 3917 3833 510,25 422,25 1295 1229 23 3373 3307 104 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 35 0 91 15131 15065 -46 37 48 197 2475 2068 -23 734 2440 (- KT succinate background)

40 9 -5 86 86 75 73 321 112 858 161 215 107 172 -145 -137 4242 4118 9907 9841 -40 8590 8466 -45 2440 (raw data) KT succinate

45

50 ribityl)lumazine

55 Compound cis-2-Methyl-5-isopropylhexa-2,5-dienoyl- CoA D-Mannuronate D-Lyxose Dihydrolipoamide D-Mannonate o-dianisidine Dimethoxybenzidine 5,6-Dimethylbenzimidazole Dimethylglycine Nalpha,Nalpha-Dimethyl-L-histidine Sau3A + DNA lambda substrate DNA DNA resolvase substrate 5- GACGCTGCCGAATTCTGGCTTGCTAGG ACATCTTTGCCCACGTTGACCC-3 DNA substrate lambda DNA + HindIII + DNA lambda substrate DNA 4-alpha-ethylzymosterol 6,7-Dimethyl-8-(1-D- Dimethylallyl diphosphate DNA substrate TAAGCTCCGGATTGTCCGGGAGGTAAA GCCCTGAT CACAGGAAGCTCTACAGGTACTCCG DNA substrate

296 EP 2 230 312 A1 51 388 173 258 3682 703,5 5 (- L’A background) 139 476 261 346

10 3770 791,5 463,75 375,75 4754,5 4666,5 3156,5 3068,5 7282,5 7194,5 7771,25 7683,25 4991,25 4903,25 1186,25 1098,25 2571,75 2483,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 15 0 29 30 (- VUL background)

20 95 96 42 -12 -66 -19 -106 7668 7202 5703,25 5615,25 2499 2433 1387,75 1299,75 6002 5889 634,75 546,75 (raw data) VUL 0 0 0 2 0 0 0 25 0 (- KOL background)

30 85 29 25 -11 340 274 402 334 2507 2302 -23 5062 4996 217 151 1929 1863 -90 3772 3706 -82 2131 2065 -19 2474 2408 -68 1776 1710 5957 5891 5886,25 5798,25 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 0 35 0 43 77 26019 25929 6822 6732 221 -97 401 126 7213 7147 1646 1580 7316,75 7228,75 111 -82 2440 (- KT succinate background)

40 -5 76 65 50 47 20 47 -50 -80 -16 345 525 167 250 235 201 -100 20063680 1882 3556 72 68 2440 (raw data) KT succinate

45

50

55 Compound DNA Topoisomerase substrate 40-base single-stranded oligodeoxyribonucleotides DNA Gyrase substrate relaxed pBR322 relaxed substrate Gyrase DNA DNA Helicase substrate dsDNA: 5- GCACTGGCCGTCGTTTTACC-3 O-Propanoylcarnitine DNA substrate Sspl X174 O-Decanoyl-L-carnitine Deamino-NAD+ n-Dodecyl -D-maltoside Dehydrodolichol diphosphate Dephospho-CoA Dethiobiotin 2-[3-Carboxy-3-(methylammonio)propyl]-L- histidine Docosan-1-ol Dodecane Dodecanoyldihydoxyacetonephosphate Dolichol phosphate Dolichyl DNA substrate TGGTCATCAGGGCTTTACCTCCCGGAC AATCCGGAGCTTACGGAGTACCTGTAG AGCTTCCTGTGCAAGC Dolichyl D-mannosyl phosphate D-mannosyl Dolichyl

297 EP 2 230 312 A1 642 487 328 100 227 782 1165 150,5 464,5 156,5 123,5 5 (- L’A background) 730 575 416 188 315 870

10 1253 552,5 244,5 211,5 1754,5 1666,5 284,25 196,25 340,75 252,75 2071,5 1983,5 305,75 217,75 240,25 152,25 5389,25 5301,25 4423,25 4335,25 L’A (raw data) 0 0 0 0 2 0 0 0 0 0 0 0 0 0 15 0 22 30 66 (- VUL background)

20 8 94 45 68 14 -48 154 -151 -141 2917 2828 2724,5 2636,5 (raw data) VUL 0 0 0 0 0 0 0 0 0 25 0 (- KOL background)

30 0 51 83 52 65 -10 -42 -14 549 483 -12 -136 2935 2819 -64 5314 5248 88 3087 2880 7388 7181 238,5 5034 4968 96 4758 4662 -8 3361 3168 24 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 0 0 0 35 0 42 1147 1081 -32 90 20 4462 4396 -16 22 99 3500 3434 1676 1610 8395,75 8307,75 19 2874 2790 11 62 2440 (- KT succinate background)

40 0 25 29 38 59 51 -36 -87 -55 -39 -34 166 105 119 214 144 146 223 143 186 1479 1355 3093 2899 3781 3587 2967,75 2879,75 3192 3068 -34 2440 (raw data) KT succinate

45

50

55 Compound D-Ribitol 5-phosphateD-Ribitol Diethyl (2R,3R)-2-methyl-3-Diethyl hydroxysuccinate dTDP Phenanthrene-4,5-dicarboxylate Diethyl (2S,3R)-2-methyl-3-Diethyl hydroxysuccinate D-Ribonate D-Ribulose dTDP-4-Amino-4,6-dideoxy-D-glucose dTDP-4-Dehydro-6-deoxy-D-glucose dTDP-4-Dehydro-6-deoxy-L-mannose dTDP-4-oxo-6-deoxy-alpha-D-glucose 6-deoxy-L-talitol dTDP-D-galactose Deoxyribose 2-Acetamido-2,6-dideoxy-D-glucose dTDP-6-deoxy-L-talose dTDP-alpha-D-desosamine dTDP-glucose dTDP-D-fucose dTDP-L-rhamnose 4,5-Dihydroxy-4,5-dihydropyrene Dibenzothiophene

298 EP 2 230 312 A1 752 911 6506 288,5 162,5 5 (- L’A background) 840 999

10 6594 376,5 250,5 1205,5 1117,5 388,75 300,75 3649,5 3561,5 2530,25 2442,25 L’A (raw data) 0 0 0 0 0 0 0 0 15 0 (- VUL background) 7

20 -1 62 26 33 10 -56 -75 -31 (raw data) VUL 0 0 0 0 0 25 0 53 37 51 (- KOL background)

30 -12 -19 -74 -64 119 117 (continued) (raw data) KOL 0 0 35 0 39 91 38 184 -69 358 107 139 274 2440 (- KT succinate background)

40 57 37 -50 163 215 308 482 162 263 2440 (raw data) KT succinate

45

50

55 Compound 1,2-Epoxypropane 2,3-Dihydroxy-4’-chlorobiphenyl D-XylulosedTDP-D-galacturonate1-Deoxy-D-xyluloseThiamin triphosphateTropinone1,2-Dioctanoyl-sn-glycerolQuinazoline1,2-Dihexadecanoyl-sn-glycerol -1361-Deoxy-D-xylulose5-phosphate D-Xylonate -51 874,5-Dihydroxypyrene 1 0 2263-oxohexanoateEthyl 1353,4-Dihydroxyphenanthrene 83 0 03,6-Dichloro-cis-1,2-dihydroxycyclohexa- 8015 -70 102 03,5-diene 11L-Erythro-4-Hydroxyglutamate 54 430 2448 7949 0 5352 2855 0 6457 -99 5 2364 364 -3 -50 5286 239 -1 0 2789 6391 1857 0 78 1727 -17 0 173 0 101 3156 0 156 1791 0 1661 -34 0 3090 0 33 35 4221,5 28 -145 8 90 260 149,25 3529,75 0 60 4133,5 5819,25 5110,5 0 0 0 194 4527 0 61,25 3441,75 5731,25 5022,5 -89 0 1523,5 2947 11519 194 4439 -127 11392 0 2881 153,75 1435,5 0 106 361,5 6552,75 65,75 2359,25 5398 6464,75 2271,25 273,5 5310 2-Indanone oxime 2-Indanone (+)-Epicatechin Docosapentaenoic acid methyl ester methyl acid Docosapentaenoic D-erythro-1-(Imidazol-4-yl)glycerol3- phosphate Docosapentaenoic acid Docosapentaenoic

299 EP 2 230 312 A1 57 110 197 310,5 266,5 908,5 148,5 410,5 307,5 410,5 5 (- L’A background) 198 145 10 285 398,5 996,5 236,5 498,5 498,5 421,75 333,75 402,25 314,25 394,25 306,25 269,25 181,25 340,75 252,75 5584,75 5496,75 2651,75 2563,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 10 (- VUL background)

20 74 30 -94 -41 -64 -72 -70 -152 -108 4508 4291 395,5 (raw data) VUL 0 0 0 0 0 0 0 10999 10843 354,5 0 0 0 25 0 (- KOL background)

30 34 46 68 -47 -71 -23 -62 -75 2363 2278 -20 8009 7756 -68 1126 1022 -61 2015 1945 18 3672 3606 -190 (continued) (raw data) KOL 0 0 0 0 3 0 0 0 0 0 0 0 35 0 49 9596 9530 76 12 -131 412 -90 2440 (- KT succinate background)

40 0 39 45 61 32 26 36 -59 -57 -64 -58 -46 536 127 173 136 2310 2186 -42 2440 (raw data) KT succinate

45

50

55 Compound Ethylhexyl palmitate Ethyl heptanoate Ethylphenyl sulfide Ethyl (S)-3-HydroxyhexanoateEthyl N-Isopropylammelide Erythritol benzoate Ethyl Ethylguaiacol trans-2-Chlorodienelactone 1-Formyl-2-indanone Erythrose D-Erythrulose (R)-2-Hydroxystearate Ethyl butyrate EpimelibioseEpichlorohydrin1,2-Didecanoyl-sn-glycerolEpisterol(R)-3-HydroxyhexanoateEthyl trans-2-Methyl-5-isopropylhexa-2,5-dienoic acid 178 -189 -115 40 54 0 0 0 -93 7617 -104 84 -115 7551 0cinnamate Ethyl 0 0 11 0Ethanolamine 2205 2953 -72 0 2139 87 2874 0 9 407,75 21 1834 319,75 521,75 0 288 1746 433,75 5041,25 200 4953,25

300 EP 2 230 312 A1 142 158 210 311 494 1125 9009 1548 1842 544,5 175,5 5 (- L’A background) 230 246 399 298 582 1213 9097 1636 10 1930 632,5 263,5 372,75 284,75 370,75 282,75 220,25 132,25 698,75 610,75 118,75 30,75 5713,5 5625,5 220,75 132,75 301,75 213,75 L’A (raw data) 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 15 0 (- VUL background)

20 2 68 83 -24 -66 4377 4304 (raw data) VUL 0 0 0 0 25 0 (- KOL background) 3

30 -2 -60 -65 446 189 -25 -101 3427 3351 68 7661 7548 -61 1160 1048 5187 5075 3666 3526 35 5118 5039 -126 4471 4244 -73 1155 1076 -28 4334 4141 4017 3951 22 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 0 0 23505 23310 96 0 0 0 0 35 0 32 7950 7763 -59 231 9479 9303 86 2440 (- KT succinate background)

40 8 43 94 59 27 -21 -12 -13 -63 -62 -76 -43 -17 -48 156 355 -175 -332 1816 1692 4359 4293 -9 2440 (raw data) KT succinate

45

50

55 Compound Methyl ferulate Methyl 5-Hydroxyferulate Diphenylenemethane Formaldehyde 2,6-bisphosphate D-Fructose butylferulate FAXX 1-phosphateL-Fuculose EthylbenzenelactateEthyl Ethynylbenzeneacido-Hydroxybenzoic paraben Ethyl salicylate Ethyl 1-phosphate D-Fructose 192FAD -65 -94 -71-Hexanoyl-sn-glycerol3-phosphate 68N-Formylanthranilate 0Farnesol 0 0 -8 -56 -6 4111 0 0 4035 0 5592 63 -50 41 5356 0 0 507,25 0 2412,25 2847,5 419,25 203,75 2324,25 2759,5 115,75 FADH2 2-Hexadecanol Feruloyl-CoA L-Fuculose

301 EP 2 230 312 A1 379 124 274 337 213 632 2536 4112 5 (- L’A background) 467 212 362 425 301 720 2624 10 4200 440,25 4352,25 4767,5 4679,5 242,25 154,25 474,25 386,25 340,25 252,25 6598,5 6510,5 4981,5 4893,5 4231,75 4143,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 15 0 81 26 63 (- VUL background)

20 98 20 63 58 -48 -97 5719 5557 5588,75 5500,75 (raw data) VUL 0 0 0 0 0 25 7 52 (- KOL background)

30 -7 34 40 73 -11 165 4568 4502 21 3870 3804 -168 5240 5174 -142 4030 3934 11618 11522 1147,75 1059,75 2402 2152 58 5913 5847 129 (continued) (raw data) KOL 3 0 0 0 0 0 2 0 0 2 0 35 0 612 -83 160 2555 2432 -50 183 2599 2533 147 278 5017 4951 39 108 3702 3636 10415 10349 123 13937 13871 285 219 2440 (- KT succinate background)

40 19 65 40 44 -45 -43 -22 -74 736 284 127 307 402 102 126 232 126 247 2440 (raw data) KT succinate

45 p-coumarate 50 (4’-chlorophenyl)-hexa-

55 Fumarylacetoacetate D-Fuconate Farnesal 1-phosphateL-Fucose D-Fructose Fructosamine D-Fructuronate Compound Formate 2-Hydroxy-6-oxo-6- 2,4-dienoate N-Formimidoyl-L-glutamate (R)-2,3-Dihydroxy-3-methylpentanoate diphosphate Farnesyl 2-Dehydro-3-deoxy-D-fuconate 5-Formamido-1-(5-phospho-D-ribosyl) imidazole-4-carboxamide 4’-O-beta-D-Glucosyl-cis- Flavonol FlavoneFluorobenzeneFormiminoglycineN2-Formyl-N1-(5-phospho-D-ribosyl) glycinamide FMN 1088 -29 19 964 0 0 -25 3532 1367 0 3466 1111 97 37 -72 14 0 0 5663,75 250,75 375 5575,75 162,75 287

302 EP 2 230 312 A1 0 20 78 271 1632 5869 158,5 262,5 5 (- L’A background) 52 108 359 166 1720 10 5957 246,5 350,5 425,75 337,75 4209,5 4121,5 230,75 142,75 4998,5 4910,5 248,25 160,25 3758,5 3670,5 240,25 152,25 4252,5 4164,5 6139,75 6051,75 5318,25 5230,25 3518,25 3430,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 61 (- VUL background) -8 20 -8 47 24 56 79 -23 -23 127 (raw data) VUL 0 0 0 0 0 0 25 0 11 (- KOL background)

30 60 37 -69 -24 955 828 3196 3130 -58 3829 3763 9146 9080 3982,25 3894,25 3034 2912 -100 3580 3514 -47 5789 5723 -145 3165 3078 -15 2841 2751 21 2078 2012 2764 2698 3113 2775 2536 2198 299,25 211,25 2904 2838 45 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 0 0 0 0 0 35 0 14 5909 5843 -49 12 -153 191 231 120 -60 2440 (- KT succinate background)

40 40 14 55 70 -83 -95 -38 -36 315 102 138 136 100 115 244 -124 -116 3228 3104 -130 2708 2584 2481 2263 -156 2440 (raw data) KT succinate

45

50

55 Compound Furazan-3,4-diol Glycerol-3-phosphate sn-Glycero-3-phosphocholine sn-Glycero-3-phospho-1-inositol 6-phosphatebeta-D-Glucose Glutathionyl-OH-1,2-dihydronaphthaleneGlutathionyl-1,2-dihydronaphthalene D-Glucono-1,5-lactate -172 FumarateL-Fucoseacid 4-Formylsalicylic 1-phosphateD-Glucose 3-phosphateGlyceraldehyde 1825 17Glycerophosphoglycerol sn-Glycero-3-phosphoethanolamine 1701 0 3494 3428 230 15 63 0 -121 0 5087 4999 3896 3808 alpha-D-Galactose 1-phosphatealpha-D-Galactose D-Galactosamine Galactomannan D-Galactosaminoglycan Hexadecanoate 3-Aminobutanol gamma-Amino-gamma-cyanobutanoate -181 D-Galactose

303 EP 2 230 312 A1

5 (- L’A background)

10 L’A (raw data)

15 (- VUL background)

20 (raw data) VUL

25 (- KOL background)

30 (continued) (raw data) KOL

35 2440 (- KT succinate background)

40 2440 (raw data) KT succinate

45

50

55 Compound di-D-galactosyl-myo-inositolD-GalactarateD-Galactonate3-O-MethylgallateGallategallaten-Propyl -70GalactitolGalactitol 1-phosphate6-phosphateD-Glucosamine 01-phosphateD-Glucosamine -64D-Galacturonate 47 248D-Glucosamine 1463Octanoyl-CoA 0gamma-Butyrolactone 1308 -2 124 115 1285 04-Guanidinobutanoate 3520 -23Butyro-betaine 1184 -64 -34-Guanidinobutanamide -18 18 3396 0 0 -81Glycolaldehyde 0Cyclohexa-3,5-diene-1,2-dicarboxylate 90 68 0Glycolyl-D-mannosamine 0 0 47 0 32 4998 2824 0 62Glycolyl-D-mannosaminolactone 5589 -11 7 30acidOctanoic 24 0 4932 2758 -69 3636,5GDP-6-deoxy-D-mannose 33 5758 0 21 5523 4449 0 0 247 57 31623 0 -174 0 -25 0 3548,5 -5 5634 4363 0 -96 0 55 34 -56 0 7 6283 31557 123 2911 0 5752 0 874 0 -79 7644 378 0 4424,75 2 0 3557 6217 0 0 381,25 0 260 2845 0 5686 6105,75 808 -55 7578 10 4336,75 0 356,5 7796 1891 293,25 290 3469 7716,5 -10 8738 2820 297 187 0 -17 6017,75 119 21 633 7730 1779 268,5 3841 0 0 7628,5 8672 2754 -45 0 -84 209 53 0 4364,75 3775 -77 0 545 3669,25 343,75 26 -29 -113 0 4276,75 0 152,25 3581,25 2980 94 0 255,75 3109 0 0 0 64,25 61,5 -60 2892 823,75 3021 6 1019,75 -141 819,75 0 735,75 0 931,75 0 731,75 355,75 407,5 267,75 319,5

304 EP 2 230 312 A1 65 189 100 351,5 5 (- L’A background) 277 153 10 188 439,5 4164,5 4076,5 2476,5 2388,5 494,75 406,75 217,25 129,25 558,75 470,75 257,75 169,75 3365,5 3277,5 4232,25 4144,25 5819,25 5731,25 3474,75 3386,75 3828,75 3740,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 1 0 15 0 (- VUL background)

20 44 67 29 -53 -86 -37 -41 -49 103 770 524 4919,25 4831,25 -113 (raw data) VUL 0 0 0 0 0 0 0 0 0 25 0 (- KOL background)

30 1 13 64 38 -84 -76 -90 -65 131 556 490 4179 4113 -90 4718 4652 -27 4541 4475 -105 2518 2318 39 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 0 0 35 0 52 17 240 -33 2440 (- KT succinate background)

40 2 -5 36 76 40 40 77 80 43 54 -17 -10 176 364 141 3597 3473 3399 3333 62 2440 (raw data) KT succinate

45

50

55 Compound D-Glucuronate D-Gluconate Glutarate Geranic acid Geranic Nitrobenzene Geranial Geranylgeranyl diphosphate beta-D-Glucose GDP-6-deoxy-D-taloseGDP-4-dehydro-6-deoxy-D-mannoseGDP-D-mannoseGDP-L-fucose6-Deoxy-D-glucose 132GentiobioseGentisate -83acetate Geranyl 8 0 849 -155 -185 72 725 11 0 -92 0 0 104 414 0 37 0 -22 3883 0 290 163 0 3817 -16 0 82 48 56 136,25 0 0 118 374,75 0 0 48,25 0 52 24 286,75 -65 3686 327,25 417 0 3598 0 239,25 1179,75 329 1327,75 1091,75 1239,75 gamma-Hexachlorocyclohexane 1-alpha-D-Galactosyl-myo-inositol Geraniol trans-Geranyl-CoA Geranylate 3-beta-D-Galactosyl-sn-glycerol cis-Geranyl-CoA

305 EP 2 230 312 A1 0 310 369 445 385 7443 7399 5579 4326 5 (- L’A background) 398 457 533 473 74,5 7487 7531 5667 10 4414 283,75 195,75 520,75 432,75 265,25 177,25 747,75 659,75 2629,5 2541,5 337,75 249,75 4741,5 4653,5 6994,25 6906,25 5983,25 5895,25 2927,25 2839,25 2540,75 2452,75 4394,75 4306,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 (- VUL background) -2 20 -6 24 96 78 -57 -66 -119 -147 -148 -112 2379 2313 (raw data) VUL 0 0 0 0 0 0 11378 11270 0 0 0 0 25 0 52 31 (- KOL background)

30 -4 14 19 15 97 37 -80 -63 109 3595 3478 -27 1436 1370 -155 2716 2650 36 4735 4669 211 145 2295 2229 5136 5070 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 0 0 35 0 39 90 504 3129 3047 10310 10228 161 -36 316 7353 7287 -35 2440 (- KT succinate background)

40 3 91 51 10 56 29 -73 -90 -13 -48 -24 163 628 285 440 124 214 1686 1562 217 1388 1264 2686 2582 -2 3019 2895 -49 3031 2907 93 2440 (raw data) KT succinate

45

50

55 Compound L-Glutamate 5-semialdehydeL-Glutamate beta-D-Glucan Glycine 1-Hydroxy-2-naphthaldehyde D-Glyceraldehyde D-GlutamineL-Glutamine1-semialdehydeL-Glutamate 5-phosphateL-Glutamate D-Glucurono-6,2-lactone gamma-L-Glutamyl-D-alanine 126 4484 2 4360 5644 2252 5578 2186 51 58 0 0 379,75 3484 291,75 3396 alpha-D-Glucose D-Glutamate 3-Hydroxy-5-methylhex-4-enoyl-CoA phosphate alpha-D-Glutamyl 2,3-biphosphoglycerate Glutaryl-CoA D-Glucurono-3,6-lactone Glucomannan 2-Hydroxycinnamate L-Glutamate methylglyoxylate gamma-L-Glutamyl-L-cysteine

306 EP 2 230 312 A1 457 248 217 142 77,5 1634 5495 4077 5393 143,5 223,5 5 (- L’A background) 545 336 305 230 5583 1722 4165 10 5481 165,5 231,5 311,5 579,25 491,25 201,25 113,25 2739,5 2651,5 1816,5 1728,5 534,25 446,25 6448,25 6360,25 537,333 449,333 739,667 651,667 3188,75 3100,75 1113,25 1025,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 32 (- VUL background) 7

20 -7 87 24 -58 -95 -45 -148 -111 2246 2180 (raw data) VUL 0 0 0 0 0 0 0 0 25 0 (- KOL background)

30 -4 99 -38 -73 112 210 126 9265 9181 431,75 343,75 4019 3953 -240 3388 3322 -73 4359 4256 4777 4674 1463 1397 23 3376 3310 -111 4753 4687 98 2889 2823 19 5742 5676 -137 2946 2880 -12 2189 2123 (continued) (raw data) KOL 0 0 22469 22360 -93 0 0 0 9 0 0 0 0 0 0 0 0 0 35 0 24 343 -25 711 4237 4171 30 2440 (- KT succinate background)

40 64 11 41 25 57 64 79 24 -57 -22 -20 -27 -27 467 835 133 148 2715 2591 -4 2167 2043 -54 3235 3111 -15 2440 (raw data) KT succinate

45

50

55 Compound D-Glycerate 3-phosphateD-Glycerate L-Glutamate methylester L-Glutamate D-Glycero-D-manno-heptose1-phosphate D-Glycero-D-manno-heptose7-phosphate 114 85 Glycerol Glycolate 1,3-Bisphospho-D-glycerate P1,P4-Bis(5’-guanosyl)tetraphosphate D-Glycero-D-manno-heptose1,7- bisphosphate Glycogen Glycerone D-Glucosaminate D-Glucosaminide sulphate Glycerone Glyoxalate D-Galactono-1,4-lactone (R)-Glycerate Glutathione reduced Glutathione Geranyl diphosphate Guanosine oxidized Glutathione 2,3,4,5-Tetrahydrodipicolinate

307 EP 2 230 312 A1 322 6533 5581 128,5 5 (- L’A background) 410 6621 10 5669 216,5 421,75 333,75 238,75 150,75 445,75 357,75 333,75 245,75 323,25 235,25 5215,75 5127,75 5281,25 5193,25 3496,75 3408,75 3059,25 2971,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 15 0 (- VUL background) 9 1 20 3 20 -88 116 -106 8169 8067 4663,5 4575,5 4763 4697 (raw data) VUL 0 0 0 0 0 0 25 0 (- KOL background) 5

30 -5 74 48 73 18 110 242 166 -60 4543 4477 6866 6743 -105 4175 4109 (continued) (raw data) KOL 0 0 0 0 1 0 0 0 0 35 0 46 4536 4470 1793 1727 4303,5 4215,5 79 32 4016 3950 -85 694 3302 3236 5861 5795 6511,75 6423,75 2440 (- KT succinate background)

40 63 82 64 -77 170 203 125 156 818 -168 -226 2010 1886 4788 4568 -15 1825 1701 3759 3693 -13 2440 (raw data) KT succinate

45

50

55 Compound GlutathionylspermidineTetrahydropteroyltri-L-glutamateGuanidoacetateGuaiacolL-Gulose4-O-methyl-D-glucuronateGlucuronoxylan 98 -65 -140 0 0 -97 -42 52 198 0 -107 0 74 0 3812 0 -150 3746 108 42 2517 0 50 42 2450 0 780 0 -34 -31 163,25 692 0 0 75,25 299 229,5 399,75 211 311,75 141,5 1-Hydroxy-2-methyl-2-(E)-4-diphosphate -161 (S)-3-Hydroxy-3-methylglutaryl-CoA Glucuronoxylan D-glucuronate Glucuronoxylan Hydroxyacetone phosphate cis-4-(2-hydroxynaph-3-yl)-2-oxobut-3- enoic acid 3-Hydroxy-2-naphthoate (E)-4-Hydroxy-3-methylbut-2-en-1-yl diphosphate methylgalactarate But-1-ene-1,2,4-tricarboxylate methyl (15S)-15-Hydroxy-5,8,11-cis-13-methyl trans-eicosatetraenoate 2,2’,3,4’,5,5’,6-Heptachloro-4-biphenylol -215 L-Homocysteine alpha-D-Glucose 6-phosphatealpha-D-Glucose Hexadecenoic acid trans-Hexadec-2-enoyl-CoA

308 EP 2 230 312 A1 68 500 396 131 389 9912 3529 4357 4495 387,5 784,5 222,5 5 (- L’A background) 588 484 156 219 477 3617 4445 10 4583 475,5 872,5 310,5 408,25 320,25 360,25 272,25 563,75 475,75 3354,25 3266,25 1281,25 1193,25 5289,25 5201,25 4309,75 4221,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 15 0 20 53 (- VUL background)

20 9 29 15 -34 -57 2931 2865 4918,5 4830,5 4905 4839 3210 3059 (raw data) VUL 0 0 0 0 0 0 12656 12527 0 0 25 0 (- KOL background)

30 51 13 59 60 -95 -11 -77 115 -120 1040 912 1216 1088 10000 4362 4296 119 3592 3526 5416 5350 4075 4009 -39 3837 3565 19 2186 1156 -70 2557 2491 21 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 0 0 0 0 0 35 0 29 10763 10679 4 20 2088 1965 143 153 722 2599 2533 6195 6129 497,25 409,25 139 4688 4528 -60 2440 (- KT succinate background)

40 69 96 14 96 47 -32 -19 -57 -11 -11 277 108 153 109 846 122 105 263 144 -109 3380 3256 3080 2999 -136 2440 (raw data) KT succinate

45

50 Hydroxy)-thionaphthenyl]-2-oxo- 55 Compound 2,2’,4,4’,5,5’-Hexachlorobiphenyl 2,2’,4,4’,6,6’-Hexachlorobiphenyl Hexyl cinnamaldehyde Hexyl Hexanoic acid Hexacosanoate Hexanal 2,2’,3,3’,5,5’-Hexachlorobiphenyl RNA Helicase substrate 3-butenoate cis-4-[2-(3- Heptane Heptadecanoic acid Heptadecanoyldolichol Heptanal Heptanoic acid Heptanoic trans-2-Methyl-5-isopropylhexa-2,5- dienoyl-CoA Hexacosane Hexadecane 2,6-Dimethyl-5-methylene-3-oxoheptanoyl-CoA Heptadecane Heptadecanoyl-CoA all-trans-Heptaprenyldiphosphate

309 EP 2 230 312 A1 431 814 678 406 374,5 5 (- L’A background) 519 902 766 10 494 462,5 237,25 149,25 4262,5 4174,5 5957,5 5869,5 2043,5 1955,5 6824,75 6736,75 1040,75 952,75 3224,75 3136,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 15 0 45 (- VUL background)

20 -7 24 61 -32 -22 5455 5389 508,75 420,75 4899 4724 5853,75 5765,75 3566 3500 (raw data) VUL 0 0 0 0 0 0 25 0 10 67 14430 14261 2192,5 2104,5 94 52326 52216 698,75 610,75 (- KOL background) 1

30 -5 15 41 236 204 135 2148 2082 -168 2713 2600 1227 1114 387,25 299,25 2153 2087 36 2786 2643 -47 4649 4583 111 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 35 0 31 3094 3028 -40 40 75 18 111 -50 133 5131 5065 29 2440 (- KT succinate background)

40 0 92 94 48 84 71 89 66 235 108 155 164 199 142 1178 1054 159 2143 2019 -26 2440 (raw data) KT succinate

45

50

55 2-Hydroxy-6-oxonona-2,4-diene-1,9-dioate2-Oxohept-3-enedioate 257 3-Hydroxy-L-kynurenine Histone Compound Histamine (S)-3-Hydroxyisobutyrate D-Hexose L-Histidinol Hydroxymethylbilane (S)-3-Hydroxyisobutyryl-CoA L-Histidine Histone L-lysine 3,3’,4’,5,7,8-Hexahydroxyflavone 2,2’,3,3’,6,6’-Hexachlorobiphenyl3-hydroxybenzoatepropyl Hexadecanoyl-CoA1,2-EpoxyhexadecanephosphateHexadecanoylglycerone 154Hexanoyldolicholphosphate L-Histidinol -61 30 36 104 44677 237 0 0 44487 113 0 310 90 4336 39 6489 -39 244 4103 0 0 6328 130 0 -1 -47 -19 5451 64 86 0 0 0 5363 6055 20 1311,5 11 1129,75 5967 445,75 1223,5 1041,75 0 357,75 732,75 644,75 Homoisocitrate Histone-arginine Histone N(omega)-methyl-L-arginineHistone

310 EP 2 230 312 A1 174 7548 4867 6728 3520 766,5 5 (- L’A background) 262 7636 4955 6816 10 3608 280,25 192,25 5299,5 5211,5 430,75 342,75 716,75 628,75 3198,75 3110,75 3698,25 3610,25 7300,75 7212,75 1350,75 1262,75 2887,75 2799,75 2287,25 2199,25 1459,25 1371,25 L’A (raw data) 0 0 0 0 8 0 0 0 0 0 0 0 0 15 0 (- VUL background) 7 8 20 7 74 48 -79 -52 -78 105 -113 2807 2724 5372,5 5284,5 2270 2204 3206,75 3118,75 2507 2232 854,5 1287 1199 5212 4889 (raw data) VUL 0 0 0 6 0 0 0 0 0 25 0 35 13 (- KOL background)

30 7 16 79 38 71 -86 -49 101 185 168 102 4873 4725 50 2325 2259 37 (continued) (raw data) KOL 0 0 1 0 0 0 0 15832 15543 123 0 35 0 84 52 40 2921 2855 28 6167 6101 -52 43 158 -16 632 -72 847 2440 (- KT succinate background)

40 76 85 83 52 93 -39 -73 107 125 282 208 176 756 971 164 152 167 4377 4253 4981 4879 2460 2358 5213,5 5125,5 9580 9456 172 1459 1335 1759 1693 2440 (raw data) KT succinate

45

50

55 Compound trans-4-Hydroxy-L-proline methyl hexanoatetrans-4-[2-(3-Hydroxy)-thionaphthenyl]-2- oxo-3-butenoate 280 156 43 0 69 0 7099,5 7011,5 L-Homoserine (S)-3-Hydroxybutanoate Homogentisate 3-hydroxyvalerate 3-Hydroxypropionyl-CoA all-trans-Hexaprenyldiphosphate Hydroxypyruvate cis-4-Hydroxy-L-proline 2-Hydroxy-2H-benzo[h]chromene-2- carboxylate N6-methyl-L-lysineHistone Hypoxanthine trans-Hexacos-2-enoyl-CoA Hexadecanal 4-Hydroxy-2-oxohexanoate Hydroxyatrazine Hydantoin-5-propionate 5-amino-2-oxopentanoate 2-(Indol-3-yl)acetaldehyde

311 EP 2 230 312 A1 4220 4692 5 (- L’A background) 4308 10 4780 473,75 385,75 469,25 381,25 L’A (raw data) 2 0 15 0 (- VUL background)

20 109 834 705 (raw data) VUL 0 25 0 (- KOL background)

30 4 179 113 -42 -121 (continued) (raw data) KOL 0 0 0 10133 10067 -71 35 0 2440 (- KT succinate background)

40 19 94 -35 100 1646 1522 5180 5014 1139 973 5451,25 5363,25 2440 (raw data) KT succinate

45

50

55 Compound Gentisate aldehyde Gentisate Indole-3-acetyl-beta-1-D-glucoseIndole-3-acetyl-L-glutamineIndole-3-acetyl-myo-inositol2-PhenylacetamideIndole-3-acetamide -43(Indol-3-yl)pyruvatePentadecanal 29Indole-3-ethanol 0 94(Indol-3-yl)acetateIminoaspartate 02-Methylpropanoyl-CoA 4170 116 02-ethylpropanoyl-CoA 96256-Aminohexanoate 131 5313 4046Isochorismate 0 9501 90L-Idonate -1 5247Indole-2-acetaldehyde 2717 69 7 -10Isocitrate 103 742 305 245’-diphosphate Inosine 2651 161 93 -28 3099 0 0 646 0 0Indoleglycerol sulphate 181 132 20 4317cis-3,4-Dihydroxyphenanthrene-4- 95 3033carboxylate 0 1470 0 6956 -119 -110 2669 2154 8734,75 14 4251 66 7271,5 0 188 1404 6890 4992 142 8646,75 2603 2088 -84 0 7568,5 0 3214,25 0 7183,5 122 3675 5269 74 4926 -108 812 -2 18 7480,5 3527,75 3126,25 0 -20 3467 3182,75 -77 5145 130 0 8 3439,75 746 37 0 72 3094,75 -70 0 9 0 64 5553,75 5596,25 6949,5 3983,25 0 6 0 818 5465,75 5508,25 1419,5 -27 0 6861,5 3895,25 -79 6658,75 743 4464,25 1331,5 0 13 6570,75 4376,25 1038,75 0 1903,25 0 950,75 4192,75 1815,25 6062,25 4104,75 5974,25 L-Isoleucine

312 EP 2 230 312 A1 213 90,5 3095 2405 1180 5240 438,5 370,5 5 (- L’A background) 301 3183 2493 5328 10 1268 178,5 526,5 458,5 527,75 439,75 657,75 569,75 3867,5 3779,5 349,25 261,25 3975,5 3887,5 380,25 292,25 3967,5 3879,5 656,75 568,75 1437,75 1349,75 5140,75 5052,75 1027,75 939,75 15832,5 15744,5 8091,75 8003,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 34 1171,25 1083,25 19 (- VUL background) 6 20 6 16 -29 -71 100 -102 -138 (raw data) VUL 0 0 0 0 0 25 0 18 (- KOL background)

30 -5 84 52 -12 -32 -10 -46 225 159 -63 689 467 2397 2331 -217 3416 3350 6361 6295 3534 3465 88 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 0 23418 23352 -238 0 35 0 26 1916 1850 -15 28 91 3330 3264 2338 2272 58 2628 2562 33 25 7096 6753 1854 1511 212 1775 1709 -16 856 1550 1484 -62 352 4389 4323 178 112 4493,5 4405,5 2440 (- KT succinate background)

40 22 72 71 51 39 79 61 78 64 -26 -87 -59 336 150 152 215 182 980 149 476 3215 3091 2877 2811 -40 1869 1745 1667 1601 -61 2688 2564 3127 3061 -79 2440 (raw data) KT succinate

45

50

55 Compound L-Histidinal Indole-3-succinate Inosine Indoline 2,3-Benzopyrrole 5’-tetraphosphate Inosine 3-(Imidazol-4-yl)-2-oxopropylphosphate 4-Imidazolone-5-propanoate Indoxazene Indene 3-hydroxybenzoate Methyl Imidazo[1,5a]pyrimidine myo-Inositol 5’-triphosphate Inosine 2-isobutylmalate Indane Isopentenyl diphosphate Isopentenyl Isothiazoline Isomaltose Isochromen-3-one Isocoumarin Isobenzofuran 2-Methylpropanoate

313 EP 2 230 312 A1 5640 1717 6284 3416 262,5 5 (- L’A background) 5728 1805 6372 10 3504 350,5 6652,5 6564,5 L’A (raw data) 0 0 15 0 64 (- VUL background)

20 42 18 1719 1653 (raw data) VUL 0 25 0 71 2940 2776 7615,25 7527,25 29 8699 8622 4716,5 4628,5 (- KOL background)

30 9 28 285 219 1663 1597 -23 3919 3853 130 (continued) (raw data) KOL 0 0 0 35 0 25 28 4901 4675 1240 1014 138 235 176 106 2440 (- KT succinate background)

40 67 69 91 37 149 262 300 152 2440 (raw data) KT succinate

45

50

55 alpha-Ketoglutaric acid alpha-Ketoglutaric Compound (S)-2-Methylmalate 3-Deoxy-D-manno-octulosonate8- phosphate Ketoprofen methyl ester Kanosamine 6-Phosphate 3-Deoxy-D-manno-2-octulosonate Hexose-1,5-lactone IsoorientinIsopentanoyl-CoAIsopreneL-Carnitinamide2-BenzazineIsoscoparinImidazoloneEthyl isovalerateIsovaleryl-CoA 79Isoxazolidine 77Isoxazole7-Hydroxycoumarin 123 -66Carnitine 0 0 130(2S)-2-Isopropyl-3-oxosuccinate5-Carboxymethyl-2-hydroxymuconate 0 70semialdehyde 175 253 0 4 -2 6 16 -91 612 0 129 51 62 8 282 0 4005 0 55 0 0 488 4663 98 -37 3939 0 158 -123 0 113 -29 4597 6407 -132 -6 0 3895 0 25 0 0 -85 205 6263 0 0 50 0 5009 3825 0 0 0 0 16223 0 139 31 3239 3222 4943 0 36 194 317 16135 140,75 37 396,25 3137 0 0 0 42 3134 4324,75 0 128 229 52,75 308,25 45 0 365,75 603,25 4236,75 5925,25 0 1913,75 329,25 277,75 5837,25 515,25 0 467 1825,75 6438,5 241,25 1202 6350,5 379 1114

314 EP 2 230 312 A1 148 77,5 2575 2099 439,5 462,5 100,5 5 (- L’A background) 236 2663 10 2187 527,5 550,5 188,5 493,25 405,25 261,25 173,25 4967,5 4879,5 552,75 464,75 4652,5 4564,5 2228,25 2140,25 4960,25 4872,25 5203,75 5115,75 L’A (raw data) 0 0 0 0 0 9 0 0 0 0 15 0 13 27 (- VUL background)

20 3 93 27 75 11 -26 225 159 717,25 629,25 (raw data) VUL 0 0 25 0 24 10 7666 7600 42 68 1108 1042 101,25 13,25 31 (- KOL background) 0 30 8 90 35 76 97 108 134 9042 8912 -47 3086 3020 6544 6447 110 5417 5351 -43 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 35 0 40 5270 5182 -99 72 936 1989 1923 -60 353 140 2440 (- KT succinate background)

40 0 71 25 36 -73 -30 164 196 477 110 114 264 -137 1162 1038 10435 10309 -21 1060 7058 6934 2709 2591 23 1239 1115 5479 5413 8825 8759 165,5 2440 (raw data) KT succinate

45

50

55 Compound L-Lactaldehyde D-Lactate L-Arabinitol 2-carboxyl-L-arabinitolphosphate 1 KetosamineKetose5-Carboxymethyl-2-hydroxymuconateJasmonateLaminarinL-Arabinonate 60dihydrate Lactitol 0 127 159 52 3 -89 109 3 93 0 -27 0 0 48 0 48 0 2080 49 0 3425 2014 0 -5 3352 4937,25 0 -41 97 0 4849,25 6 -129 0 0 768 0 0 3290,25 1731,25 680 3202,25 729,5 4301 1643,25 641,5 4213 L-Leucine Itaconyl-CoA Itaconate p-nitroaniline Leucine L-Formylkynurenine L-Gulono-1,4-lactone L-Arabonate L-Arogenate 5-Aminoimidazole L-Lactate L-Ascorbic acid L-Ascorbic Lactose

315 EP 2 230 312 A1 3863 1568 5 (- L’A background) 3951 10 1656 455,75 367,75 L’A (raw data) 0 0 15 0 (- VUL background)

20 21 7262 7146 917,75 829,75 (raw data) VUL 0 25 0 (- KOL background)

30 6 -54 8244 8151 5764 5671 6148,75 6060,75 3783 3717 1794 1728 2050,5 1962,5 (continued) (raw data) KOL 0 0 35 0 45 10748 10651 12 32 136 18233 18167 -16 2440 (- KT succinate background)

40 42 31 -51 169 156 260 2440 (raw data) KT succinate

45

50

55 Compound 3-Methylsalicylaldehyde L-Leucyl-glycyl-glycineL-HomocitrullineL-Gulonate(R)-S-LactoylglutathioneLicodioneL-IditolLimonoate 108Limonene-1,2-diol(-)-(S)-LimoneneLimonene-1,2-epoxide 184 726 0LindaneLinolenate 3Linoleate 602 60 7204Linoleyl-CoA 1432,3-Bis(3-Hydroxytetradecanoyl)-D- 134 277glucosaminyl-1,6-beta-D-2,3-bis(3- 7138 54 0 101hydroxytetradecanoyl)-beta-D- -29 69 271glucosaminyl 1-phosphate 192,3-Bis(3-hydroxytetradecanoyl)-beta-D- -29 0 153 3944glucosaminyl 1-phosphate 0 0 0 147 0 22L-Kynurenine 4742 3878 0 99 80 -29 5379ester benzyl DL-Leucine, 4354 -39 -1096-Lactoyl-5,6,7,8-tetrahydropterin 18 145 -93 4676 -85 0 5192 288,5 0 0 4269 0 0 0 122 0 0 0 2630 762,25 0 200,5 -40 5575,5 15 11 -85 198 56 2502 107 674,25 -115 37 466,25 5487,5 0 0 0 2897,25 27 2367,5 0 0 378,25 0 0 2173,5 2809,25 6723 2279,5 1039,25 83 382,75 8346,75 -44 2085,5 71 6657 951,25 294,75 8258,75 17 0 5820 5 4453 793 5732 4185,25 4365 4097,25 705

316 EP 2 230 312 A1 2309 2228 293,5 5 (- L’A background) 2397 10 2316 381,5 399,25 311,25 2610,5 2522,5 6949,75 6861,75 L’A (raw data) 0 0 0 0 15 0 (- VUL background)

20 -35 -23 -15 197 131 2931,75 2843,75 3388 3322 392,75 304,75 3217 3060 (raw data) VUL 0 0 0 0 0 25 0 (- KOL background)

30 8 -13 -127 5159 5038 -14 4108 3900 7245 7037 5963,75 5875,75 3439 3127 1412 1100 10390,8 10302,8 1483 1389 -90 (continued) (raw data) KOL 0 0 0 0 0 35 0 256 582 3132 3011 2727 2606 8358,75 8270,75 2440 (- KT succinate background)

40 65 72 10 90 -48 -15 380 706 5298 5174 -17 1131 1007 37 3922 3798 48 2440 (raw data) KT succinate

45

50 S)-1-benzyl-1,2,3,4-

55 L-Lysine 3-Methylsalicylate Compound (alpha-D-Mannosyl)4-beta-D-mannosyl- diacetylchitobiosyldiphosphodolichol 5’-monophosphate Inosine Methyl-2-hydroxy-2-methylpropionate Methyl-3-bromopropionate Melibiitol Methyl 2,2-dimethyl-1,3-dioxane-4-Methyl carboxylate N-Methyl-(R, tetrahydroisoquinoline alpha-D-Mannosyl-beta-D-mannosyl- diacetylchitobiosyldiphosphodolichol 2-Methyl-3-oxopropanoate2-Methylhomogentisate(+)-(R)-LimoneneL-PalmitoylcarnitneL-Rhamno-1,4-lactoneL-Xylono-1,4-lactone 2492L-Xylo-Hex-3-ulono-1,4-lactoneL-Xylonate 86Methyl-2-bromopropionate 23682-hydroxybenzoate propyl 41 -82 47 -15 0 3 25 0 0 0 -41 49 0 0 0 56 174 1920 0 96 7 0 21 -21 1774 0 50 2932 30 0 87 0 0 -31 2861 3750,25 70 94 62 0 37 4005 3662,25 461,5 0 0 0 0 -50 3939 0 373,5 1012 -137 1189,25 2764,25 14382,8 0 1510,5 1101,25 924 2676,25 0 14294,8 497,75 0 2784 409,75 2696

317 EP 2 230 312 A1 417 418 2510 2461 7715 3502 7629 9479 8566 4620 702,5 661,5 234,5 5 (- L’A background) 505 506 2598 2549 3590 7803 7717 9567 8654 10 4708 790,5 749,5 322,5 935,75 847,75 459,75 371,75 2937,75 2849,75 2672,25 2584,25 6108,25 6020,25 7074,25 6986,25 L’A (raw data) 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 15 0 93 62 (- VUL background)

20 8 53 51 67 35 -69 -172 (raw data) VUL 0 0 0 0 0 25 0 (- KOL background)

30 8 16 93 48 21 -65 4250 4184 59 3254 3188 2827 2761 25477 25411 6421 6286 69 4967 4743 100 (continued) (raw data) KOL 0 0 10670 10501 -48 0 0 5 0 0 0 0 35 0 25 2843 2706 -51 98 3534 3366 -91 12 3519 3453 -176 85 1923 1709 152 46 3687 3621 159 107 3843 3571 -36 128 115 127 3402 3336 128 2440 (- KT succinate background)

40 0 -9 78 86 72 44 -97 -13 149 222 136 231 106 252 209 239 129 170 251 3783 3659 4833 4767 2065 1999 2817,25 2729,25 2440 (raw data) KT succinate

45

50

55 Compound Malonyl-CoA Maleate Melilotate Maltitol 6’-phosphate Maltose beta-Maltose Isomaltotriose Isomaltotetraose 3-Oxohexanoatecis-1,2-Dihydroxy-4-methylcyclohexa-3,5- diene-1-carboxylate 4-Maleylacetoacetate 4-hydroxybenzoate Methyl 2945 2821 -13 0 -162 0 464 376 beta-D- Mannosyldiacetylchitobiosyldiphosphodolic hol 2-Methylacetoacetyl-CoA Malathion Malonate L-Mannuronate ethylmalonate alpha-Maltose 2-Methylprop-2-enoyl-CoA 3-Methylmuconolactone

318 EP 2 230 312 A1 403,5 5 (- L’A background)

10 491,5 411,75 323,75 938,25 850,25 2804,25 2716,25 L’A (raw data) 0 0 15 0 36 (- VUL background)

20 (raw data) VUL

25 (- KOL background)

30 (continued) (raw data) KOL

35 143 2664 2598 -150 2440 (- KT succinate background)

40 267 2714 2590 7240 7101 -69 3743 3619 3288 3099 -78 1907 1783 3563 3497 102 2440 (raw data) KT succinate

45

50

55 Compound IsomaltopentaoseIsomaltohexaoseMethyl-3-bromo-2-methylpropionatepropylmalonate2-Ethyl-2-methyl-4-butyrolactoneMannobiose -65(R)-Mandelate(-)-Menthol 41D-Mannosamine -62 0D-Mannose 8(S)-MandelateMannotriose 0 -108 5974 0Mannotetraose 02-Methylbutyryl-CoA 5809 3606Malonate semialdehyde 66 5325 0 892-Methylbut-2-enoyl-CoA 93-hydroxy-4-oxoquinoline 4106 3540 -3 -58 5259Melitriose 205 0 01,2-Dihydroxy-3-methylcyclohexa-3,5- 125 3941 -36 87dienecarboxylate 9063 0 0 0 84 154 50 2426 -37 9 6567,5 -92 4098 8997 1 0 2612 295 0 5514 2241 28 2302 30 6479,5 0 3974 0 0 595 2546 2560 0 433,25 5448 4940 2120 171 71 0 1444 45 7215 2494 -75 -29 341 13 345,25 -117 507 4874 83 629,75 4220 1378 5 7149 -16 -62 0 0 -40 4120 541,75 0 0 253 17 0 64 3248,75 -75 0 3353 0 -149 -35 0 2094 3160,75 806,5 2072 18 0 3277 0 4137,25 1011,75 0 2028 0 718,5 518,5 1984 0 4049,25 1494,25 15714,8 923,75 7977,5 2777,25 4639,25 259 430,5 1406,25 15626,8 317 2689,25 7889,5 4551,25 171 251 695,5 607,5 meso-2,6-Diaminopimelate 1-phosphate D-Mannose D-Mannose 6-phosphate D-Mannose

319 EP 2 230 312 A1 812 1179 5 (- L’A background) 900

10 1267 1517,5 1429,5 L’A (raw data) 0 15 0 (- VUL background)

20 1243 1163 1551,75 1463,75 2036 1970 (raw data) VUL 0 25 0 (- KOL background)

30 -75 -46 4529 4269 128 (continued) (raw data) KOL 0 0 35 0 2440 (- KT succinate background)

40 98 96 110 6243 6119 2046 1980 -89 2440 (raw data) KT succinate

45

50

55 Compound (-)-Menthylacetate Mannan(-)-MenthoneMethyl3-hydroxybutyrateo-Methylbenzoate2-Keto-3-deoxy-6-phosphogluconateMethyl cinnamateD-Methionine 20022-Amino-2-methylpropanoate 19375,10-Methenyltetrahydrofolate 92 1878Methylmalonate 1813 621 328L-Methionine 2343-Methyl-cis,cis-muconate 2027 0 4905Benzylparaben 218 497 202 204 821Methylparathion 1813 110m-Methylbenzoate 4839 5 195Xanthosine 94 3781 1949 697 78 -113(S)-mevalonate 66 903 35Methyl jasmonate 644 3684 1883 4048 71 01-phosphate1D-myo-Inositol 8994 -54 0 18453-Methylglutaconyl-CoA 0 779 3543 1896 0 3982 5204-Methylmuconolactone 7191 352 8849 503N-Methyl-L-histidine 491 1721 0 256 3586 3446 1830 162 -25 -66 7040 4850,75 22 46 228 418 197 367 4325 1302,25 164 30 132 3498 4762,75 5002 49 55 0 38 0 0 1529,25 125 0 1214,25 77 100 4201 8025 40 0 1236 4914 1441,25 7239,75 2455,75 0 0 0 84 608,75 7959 71 -1 0 1123 47151,75 2312 2367,75 691,5 4699 3414,5 6686 -103 520,75 0 4640,75 2246 0 0 8 603,5 4633 3326,5 6494 4552,75 0 936 563,75 -80 5793 0 84 13859 475,75 1887,5 870 5705 0 14385,3 18 13771 1799,5 368,25 14297,3 7497,75 4200,25 280,25 7409,75 4112,25

320 EP 2 230 312 A1 311 579,5 5 (- L’A background)

10 399 667,5 815,75 727,75 351,75 263,75 349,75 261,75 311,75 223,75 3744,75 3656,75 6103,25 6015,25 15873,5 15785,5 3171,25 3083,25 7107,25 7019,25 3419,25 3331,25 6662,25 6574,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 15 0 58 22 7455,75 7367,75 (- VUL background)

20 -8 55 85 88 -89 -74 148 -183 2593 2527 1345,75 1257,75 4300 3894 966,25 878,25 (raw data) VUL 0 0 0 2 0 0 0 0 0 25 0 (- KOL background) 6 30 5 89 23 84 -36 -31 4775 4435 5222 4882 832,25 744,25 6096 6030 4941 4875 1361,25 1273,25 5811 5701 -72 (continued) (raw data) KOL 0 0 0 0 0 35 0 74 2708 2584 -41 60 1702 1588 55 42 2700 2576 98 36 44 67 6930 6864 4646 4580 6104,75 6016,75 401 -91 866 185 224 901 -13 138 2440 (- KT succinate background)

40 73 77 81 -30 -10 198 990 184 525 348 166 262 160 168 191 1994 1870 5814 5748 -60 1025 2529 2405 4143 4073 42 2440 (raw data) KT succinate

45

50

55 Compound L-Malate 5,10-Methylenetetrahydrofolate (R)-Methylmalonyl-CoA (S)-Methylmalonyl-CoA Mevaldate7-MethylxanthineMethylisocitrateD-ring-lactone Limonoate acid 7-Methyluric acid 1,7-Dimethyluric 41 204(S)-Methylmalonatesemialdehyde 3160L-Met-L-Met-L-Met 0 80 3036 4129 -56 188 4063 42 0 1903 2 785 1837 719 0 3062,5 1306,75 2974,5 9457,75 1218,75 9369,75 D-Mannitol 1-phosphateD-Mannitol N-Acetylmethionine 4-Hydroxyphenylacetate 2D-5-O-methyl-2,3,5/4,6- pentahydroxycyclohexanone Tetrahydro-1,4-oxazine Methylglyoxal D-Mannitol Menaquinol Menaquinone 5-Methyltetrahydropteroyltri-L-glutamate 69

321 EP 2 230 312 A1 0 1417 7527 357,5 224,5 5 (- L’A background) 1505 7615

10 -65,5 445,5 6640,5 6552,5 L’A (raw data) 0 9 15 0 25 (- VUL background)

20 178 112 575,25 487,25 -171 9663 9461 423,75 335,75 (raw data) VUL 0 0 25 0 (- KOL background)

30 7 673 563 119 6183 6117 91 (continued) (raw data) KOL 0 35 0 72 -135 783 1200 1134 459 393 174 2682 2557 -3 432 123 7987 7921 4470 4404 2536,75 2448,75 2440 (- KT succinate background)

40 19 907 298 556 247 196 -104 1290 1166 3752 3603 12022 11873 312,5 1132 1008 -28 2440 (raw data) KT succinate

45

50

55 Compound (S)-5-Oxo-2,5-dihydrofuran-2-acetateMucic acid(R)-MevalonatePhenanthrene-4-carboxylate 2752N1-Acetylspermidinecis,cis-Muconate 26284alpha-Methylzymosterol3-Hydroxyisovaleryl-CoA 5240N4-Acetylaminobutanal 622-OxohexanoateN1-Acetylspermine 5174 22 1382-Nonaprenyl-4-hydroxybenzoate 219 0N-Acetyl-4-O-acetylneuraminate 71 -133 1925 1998N-Acetyl-5-methoxytryptamine 14 0 95D-ribonucleoside Nicotinate 1560 64N-Acetylarylamine 181 0 1801 0 1874 3759N-Benzoyl-D-arginine-4-nitroanilide 200 3020 1436 15 -138 29 4464 0 177 996 4604,25 57 1233 3622 2954 2218 76 0 4398 0 4516,25 1167 857 18 53 32 0 35 12 1964 623 -39 1462 5465 -48 -14 -52 4816 0 0 0 87 0 1205 0 5399 0 4671 0 0 0 4349,75 5575 18 0 21 282 3647 497 2017,25 36 4261,75 6785 1182 9540 5487 0 1929,25 471 3559 0 194 0 0 9371 6697 1094 3578,25 383 3919,25 230,5 5288,25 282,75 3490,25 3831,25 5200,25 142,5 194,75 N-Acetyl-L-aspartate Nicotinate N-Acetylgalactosamine 1,2-Anthracenediol 2-Naphthaldehyde Nicotinamide adenine dinucleotide

322 EP 2 230 312 A1 0 0 0 988 4520 2042 5 (- L’A background) -46 81,5 38,5 1076 4608 10 2130 906,75 818,75 4623,5 4535,5 130,75 42,75 722,25 634,25 455,25 367,25 6158,5 6070,5 207,75 119,75 174,25 86,25 1455,75 1367,75 2802,75 2714,75 3772,25 3684,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 68 72 89 1116,25 1028,25 (- VUL background)

20 -9 38 20 -84 -83 -86 -60 -48 -19 155 (raw data) VUL 0 0 0 0 0 0 25 0 49 54 27 59 -161 (- KOL background)

30 6 79 17 58 42 115 161 1914 1690 -9 1784 1718 -100 1859 1767 -37 3171 3100 41 (continued) (raw data) KOL 0 0 0 0 0 34369 34188 -134 35 0 43 68 45 1601 1485 789 673 1105,25 1617,25 358 368 124 2837 2771 469 403 4283,5 4195,5 375 3764 3677 159 702 2440 (- KT succinate background)

40 98 52 26 61 54 167 482 492 248 192 499 108 826 169 1675 1551 -80 1914 1790 1875 1751 93 3115 2991 120 7722 7598 -24 1548 1424 6535 6449 154 2440 (raw data) KT succinate

45

50

55 N-Benzoyl-4-hydroxyanthranilate N-Benzoyl-4-methoxyanthranilate benzoate Ethyl N-Acetylserotonin N-Acetyl-L-tyrosineester ethyl Compound N-Acetyl-L-histidine Nonadecanoic acid methyl ester Nonadecanoyldolichol 2-Naphthalenesulfonate n-Butanol 2-Naphthalenol N6-Acetyl-L-lysine N-Acetyl-L-phenylalanine N-Acetylglycinamide N-Acetyl-D-galactosaminoglycanNADHNicotinamide adenine dinucleotide phosphate -75NADPH 0N-Acetylimidazole 5181 263 5115 139 41 -6 0 0 3070 213 2982 147 6577,5 6489,5 N-Acetyl-D-tryptophan N-Acetyl-N-hydroxy-L-lysine N-Ribosylnicotinamide

323 EP 2 230 312 A1 0 311 4132 3013 2361 8305 1949 150,5 511,5 5 (- L’A background) 54 399 4220 3101 2449 8393 10 2037 238,5 599,5 6505,5 6417,5 4933,5 4845,5 2331,5 2243,5 375,75 287,75 605,25 517,25 426,25 338,25 3933,5 3845,5 11082,8 10994,8 4726,25 4638,25 L’A (raw data) 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 15 0 17 4581,25 4493,25 (- VUL background)

20 49 79 32 61 43 -18 -69 -37 -69 -80 449 383 -137 3472 3406 (raw data) VUL 0 0 0 0 0 0 0 0 25 0 15 17 (- KOL background)

30 81 83 65 35 63 -44 -25 903 837 -104 1122 1056 -48 5848 5780 85 (continued) (raw data) KOL 0 0 0 0 0 0 0 35 0 52 4395 4309 43 38 2236 2120 -87 92 69 102 212 146 -32 882 -170 320 2440 (- KT succinate background)

40 3 78 -18 -47 -48 -39 226 176 162 216 193 444 -199 2851 2727 -19 2217 2093 621 555 3309 3185 24 4055 3931 2866 2607 54 1006 2440 (raw data) KT succinate

45

50

55 Compound N-Decanoyl-glycine Nerol Neral 7-Methylxanthosine N-Carbamoylputrescine 2,5-Dichloro-cis,cis-muconate N-Dodecanoyl-glycine Nicotinic acid amide acid Nicotinic N-Carbamoyl-beta-alanine 1,6-Dihydroxy-cis-2,4-cyclohexadiene-1- carboxylic acid gamma-L-Glutamylputrescine Neotetradecane 3,6-Dichlorocatechol (+)-Neomenthol Neooctane N-Carbamoylbeta-glycine N-Benzoylglycinecis-2-ChlorodienelactoneN-Butyryl-L-homoserinelactone lactoneN-(3-Oxododecanoyl)homoserine lactone N-(3-Oxooctanoyl)homoserine Naphthalene 119 122 892 114 0 768 0 3432 3794 2735 3366 3728 2669 17707 117 8053 17641 51 7987 153,25 237,75 468,5 65,25 149,75 380,5

324 EP 2 230 312 A1

5 (- L’A background)

10 L’A (raw data)

15 (- VUL background)

20 (raw data) VUL

25 (- KOL background)

30 (continued) (raw data) KOL

35 2440 (- KT succinate background)

40 2440 (raw data) KT succinate

45

50

55 Compound N-Heptadecanoyl-glycineL-4-HydroxyphenylglycineN-Hexadecanoyl-glycine(3R)-3-Isoprenyl-6-oxoheptanoyl-CoAN-Hexanoyl-glycineN-Hydroxy-L-lysine 135N-Heptanoyl-glycine 43 25(3R)-3-Isopropenyl-6-oxoheptanoateD-ribonucleotideNicotinate 212 11Nicotine 0Nitroglycerin 0 695N-Methylanthranilate 88 -92 6364D-Glucose 5668 25 3950N-Malonylanthranilate 571 -3 693 2300 6298N-Methylaniline 5448 0N-Methylindole 3884 6862 0N-Methylhistamine 2166 -18 569 0 133N-Methyl-L-glutamate 1105 -246 6753Pentadecan-1-ol -9 -64 4492 -8 169 257N-Methylphenylethanolamine 0 1039 1257 31 0N-Methyltyramine 110 0N,N-Dimethylaniline 4338 947 0 0 133 -114 45 0 7048,75N-Octadecanoyl-glycine 1133 375 0 575,5 1 457,25 325 -152 884 1566 6960,75 1866,25 823 -6 3522 -99 74 185 0 -77 251 2129 487,5 369,25 6349,25 179 1442 201 0 1778,25 760 3402 -91 119 0 3854,25 0 8 -100 2016 0 -2 6261,25 103 981 1956 18 200 6070,75 55 -123 3766,25 0 101 3664,75 6258 11 47 0 5400,5 1890 0 5982,75 857 0 3860 92 0 0 3576,75 35 6192 3 5312,5 0 99 -19 0 -136 -16 3794 2943 330 3947,25 -148 -4 8103,5 0 116 2625 5777,75 33 0 0 3859,25 0 646 222 0 8015,5 0 5689,75 -11 10595,5 10514,8 50 8234 1366,5 5669 558 7 5441,25 10507,5 10426,8 2758,5 8168 8706,5 1278,5 0 5353,25 5581 0 2670,5 376,75 8618,5 2640,5 2590 288,75 2552,5 2502

325 EP 2 230 312 A1 997 5 (- L’A background)

10 1085 609,75 521,75 776,25 688,25 2779,5 2691,5 841,75 753,75 2828,25 2740,25 5635,25 5547,25 4886,25 4798,25 2624,25 2536,25 L’A (raw data) 0 5 0 0 0 0 0 0 15 0 (- VUL background)

20 -8 74 71 -83 -65 -11 672 606 1944,75 1856,75 -117 5367 5301 1570,25 1482,25 (raw data) VUL 0 0 0 0 4 25 0 14 49 18 (- KOL background)

30 80 59 27 37 61 469 217 -16 118 102 (continued) (raw data) KOL 0 0 0 0 35 0 70 821 218 8187 8042 -50 405 2440 (- KT succinate background)

40 21 -22 -11 945 116 115 342 194 529 1149 1025 -17 2154 2030 4111 4045 1758 1692 1151,25 1063,25 2440 (raw data) KT succinate

45

50

55 Compound Nonylphenol aminopropylcadaverine N-Octanoyl-glycinelactoneN-(3-Oxooctanoyl)homoserine 2,4-Diamino-6-nitrotolueneNonadecaneNonadecanoic acid -8Nonanoyldolichol4-Acetamido-2-amino-6-nitrotoluenenonylateMethyl 159 0 3488NonanalNonane -302-Hydroxy-8-methylchromene-2- 3364 3029 35carboxylate 434 81 2786 -73 0 101 73 310phosphate Oleoylglycerone -101 -12 0 0 38ester methyl acid Oleic 0 0 1902all-trans-Nonaprenyldiphosphate 0 235 -63 -80 0 0 3171 1836 9 98 111 441 0 0 164 3047 54 -110 17 0 4255 353 4880,25 98 7 41 0 0 5553,75 48 4189 4792,25 1194,75 5465,75 4729,25 0 0 51 31 0 1106,75 4641,25 11798,3 30 0 0 6877,75 11710,3 6789,75 0 3585 911,5 1277,75 3497 823,5 1189,75 Nicotinamide mononucleotide Nicotinamide Phenanthrene-3,4-oxide N-Succinyl-Ala-Ala-Phe-7-amido-4-methyl coumarin N-Succinyl-Ala-Ala-Pro-Phep-nitroanilide 1188 1064 128 N-Succinyl-Ala-Ala-Alap-nitroanilide 3-Amino-2-oxopropylphosphate

326 EP 2 230 312 A1 263 1010 5307 8276 7020 9221 615,5 353,5 255,5 5 (- L’A background) 351 1098 5395 8364 7108 10 9309 703,5 441,5 343,5 11286 11198 1593,75 1505,75 5151,25 5063,25 7834,25 7746,25 3394,75 3306,75 3903,25 3815,25 5609,25 5521,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 8 0 0 15 0 (- VUL background)

20 -5 86 64 64 74 62 -81 -15 472 287 336,75 248,75 183 117 10351,3 10263,3 259 544 358 -126 4437 4290 5056,5 4968,5 1995 1825 4613,75 4525,75 1080 930 1971,25 1883,25 (raw data) VUL 0 5 0 0 0 0 0 0 0 0 0 0 25 0 37 13 (- KOL background)

30 3 71 46 33 79 37 -54 -148 7381 7251 237 107 (continued) (raw data) KOL 0 0 0 0 0 35 0 64 3114 2828 1098 812 11544,3 11456,3 14 113 4289 4206 -44 574 3085 3014 -97 135 700 119 210 3358 3270 26 295 2319 2253 2820 2754 1275,25 1187,25 314 217 127 -69 249 2440 (- KT succinate background)

40 62 14 70 77 38 61 237 698 259 188 824 334 419 438 138 373 1561 1437 400 320 4533 4409 -5 2402 2278 22 1414 1290 103 2779 2655 -44 1705 1581 32 4715 4591 32 2440 (raw data) KT succinate

45

50

55 o-Cresol Octane Octane-1,8-diol Compound Octadecane Octadecenoic acid acid 9-Octadecynoic Stearoyl-CoA Ethyl octanoate Octadecanoic acid N1-(5-Phospho-D-ribosyl)glycinamide 1-Octanal L-Octanoylcamitine Oxaloacetate O-Acetyl-L-homoserine acid 2-Butenoic Octadecanoyldolichol GDP dAMP Octanoyldolichol N-Heptanoylhomoserine lactone N-Heptanoylhomoserine Methyloxaloacetate 2-Oxohex-trans-4-enoate O-Acetylcholine

327 EP 2 230 312 A1 998 1759 1173 7991 5199 5 (- L’A background) 1847 1261 8079 1086 10 5287 3363,5 3275,5 6744,5 6656,5 5288,5 5200,5 107,75 19,75 5949,25 5861,25 4553,75 4465,75 1054,75 966,75 10025,8 9937,75 8619,75 8531,75 1431,75 1343,75 7681,75 7593,75 3852,25 3764,25 5506,25 5418,25 4147,75 4059,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 (- VUL background) 5

20 -2 -20 -80 -57 -34 -55 -92 166 100 -103 -103 6913 6805 18051,5 17963,5 (raw data) VUL 0 0 0 0 0 0 0 0 0 25 0 81 39 4629 4563 7515,5 7427,5 (- KOL background)

30 6 42 55 23 45 64 -19 105 -103 4669 4412 -82 1763 1697 -4 7583 7386 65 2690 2482 1541 1420 10 (continued) (raw data) KOL 0 5 0 0 0 0 0 0 0 0 35 0 23 5067 4969 45 53 1476 1410 53 208 147 220 6036 5860 68 188 138 2440 (- KT succinate background)

40 40 55 31 78 32 36 129 332 147 114 101 177 344 104 312 262 -118 3375 3251 2591 2525 -145 4745 4621 4225 4101 -133 2440 (raw data) KT succinate

45

50

55 Compound all-trans-Octaprenyldiphosphate cis-1,2-Dihydroxy-1,2-dihydro-7- hydroxymethylnaphthalene 2-Ethylphenol 1-Octene 4-Hydroxylamino-2,6-dinitrotoluene O-Demethylpuromycin O-Feruloylquinate Octyl salicylate Oleyl alcohol 2-Oxo-3-hydroxy-4-phosphobutanoate 10196 10072 -81 1,2-Dihydroxy-7- hydroxymethylnaphthalene Oleyldihydroxyacetonephosphate 3-(5-oxo-4,5-dihydro-3H-imidazol-4-yl) propanoate Oleic acid Oleic ethyl 3-oxobutanoate ethyl 2-Hydroxylamino-4,6-dinitrotoluene 2-Oxopent-4-enoate N-Propionylglycine alpha-N-Phenylacetyl-L-glutamine Pantetheine 4’-phosphate L-Omithine

328 EP 2 230 312 A1 712 909 339 8689 9349 7139 121,5 5 (- L’A background) 800 997 427 8777 9437 10 7227 209,5 1052,5 964,5 502,25 414,25 7151,5 7063,5 6413,5 6325,5 5234,25 5146,25 1426,25 1338,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 15 0 28 7567,75 7479,75 28 10530,3 10442,3 (- VUL background)

20 30 57 57 18 17 35 -77 -45 -43 -10 743 592 4370,75 4282,75 -135 1435 1369 2759 2693 4803,75 4715,75 (raw data) VUL 0 0 0 0 0 0 0 0 0 0 0 0 0 25 0 (- KOL background)

30 55 23 33 39 53 -98 -38 -133 7279 7213 4352 4286 16885 16797 4696 4630 94 (continued) (raw data) KOL 0 0 0 0 0 0 0 35 0 93 84 4128 4062 4483 4417 77 142 -72 905 -124 2440 (- KT succinate background)

40 -5 94 33 47 -37 217 113 124 266 208 201 103 7466 7342 -143 8822 8698 27 1435 1311 1906 1840 94 3804 3680 10 3853 3729 -145 1029 2440 (raw data) KT succinate

45

50

55 (R)-Pantolactone (R)-Pantothenate 3-Oxopropionyl-CoA Compound Panose 4-Hydroxyphenylacetyl-CoA Phosphatidate Orotate 2-Oxoadipate Oxomalonate Doxepin Oxirane Oxamate Oxazole Phenylacetaldehyde Oxalosuccinate Oxalic acid 4-Oxobutanoate (9Z,12Z)-Octadecadienoyl-CoAOrotidine 5’-phosphateO-SinapoylglucarolactoneOxalureateOxaloglutarate 95oxaloglycolate methyl 428 -25 0 304 -176 0 -3 -9 49 0 67 0 0 0 57 0 64 -37 146 0 33 0 0 0 4275,5 0 -183 -117 6637,5 4187,5 13822,8 6549,5 0 0 13734,8 5495,75 5050 5407,75 4962

329 EP 2 230 312 A1 0 31 832 628 896 6099 3468 10,25 5 (- L’A background) 716 920 119 984 6187 10 3556 98,25 15218 15130 10887 10799 -73,75 5401,5 5313,5 3370,5 3282,5 2471,5 2383,5 2860,25 2772,25 1277,75 1189,75 7568,75 7480,75 8809,75 8721,75 1746,75 1658,75 3660,75 3572,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 22 (- VUL background) 9 20 3 88 88 -11 -14 -83 -17 -16 -56 -72 -210 3227 3161 2543 2477 10960,3 10872,3 (raw data) VUL 0 0 0 0 1 0 0 0 0 1 0 0 0 25 0 12 -352 (- KOL background) 1 3 30 3 17 23 67 28 27 -62 663 484 -96 120 -135 3480 3414 3875 3809 2773,25 2685,25 2431 2365 -103 4203 4137 4569 4503 10043,3 9955,25 (continued) (raw data) KOL 0 0 0 0 0 0 35 0 84 85 19 3155 3089 14 19 834 -11 358 167 101 -52 545 307 133 101 4219 4153 6838 6772 101 235 127 663 2440 (- KT succinate background)

40 33 40 63 -64 -97 -71 958 482 208 669 209 143 431 225 225 124 359 251 787 143 2800 2676 146 3939 3815 35 2440 (raw data) KT succinate

45

50

55 Compound p-Cresol gamma-Phenyl-epsilon-caprolactam Pelargonic acid Palmitoyldihydroxyacetonephosphate Phosphatidyl-N-dimethylethanolamine Pectin p-Cymene Pentyl butyrate Phosphatidylethanolamine Pentachlorophenol Methyl pentadecanoate Benzaldehyde 3’-Phosphoadenylyl sulfate 3’-Phosphoadenylyl 5’-phosphatePyridoxine Phenethylamine Pentadecanoic acid Choline phosphate alcohol p-Coumaryl p-Cumate (R)-Pantoate2’,5’-bisphosphateAdenosine Parathion 474 350 220 154 4347 4281 3555,5 3467,5 3-sn-Phosphatidylcholine

330 EP 2 230 312 A1 926 573 974 742 903 6379 1019 6573 526,5 847,5 5 (- L’A background) 661 830 991 1014 6467 1107 1062 10 6661 614,5 882,75 794,75 10020,3 9932,25 2220,75 2132,75 1927,25 1839,25 8683,25 8595,25 1795,75 1707,75 1051,25 963,25 3389,75 3301,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 15 0 14 (- VUL background)

20 30 80 -11 -61 -53 -88 -10 -62 -89 109 128 -137 4376 4211 2477,5 2389,5 3523 3326 6612 6444 12375,3 12287,3 1166 962 3713 3647 5187 4917 2731,75 2643,75 (raw data) VUL 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 25 0 35 12 15903 15822 935,5 82 2479 2413 9776,5 9688,5 (- KOL background)

30 9 52 15 40 93 -83 -64 -18 -21 171 (continued) (raw data) KOL 0 0 0 35 0 20 26 38 565 -126 223 227 -25 742 151 213 966 145 148 2440 (- KT succinate background)

40 61 10 72 -89 689 347 144 351 866 150 275 162 269 2054 1930 -12 2164 2040 -2 2090 1966 187 121 1069 1003 1630 1506 34 1090 3830 3706 32 1860 1736 -77 1162 1038 84 10509 10385 7413 7297 -46 2440 (raw data) KT succinate

45

50

55 Compound 2,4,6/3,5-Pentahydroxycyclohexanone L-1-Pyrroline-3-hydroxy-5-carboxylate Phenylacetyl-CoA Phenylethanolamine 2-Aminobenzoyl-CoApentanoate Pentyl acid Pentanoic Pentadecane 5-Methylhexa-2,4-dienoyl-CoA 132 8 66 0dATP 8 0 9363 9275 n-Pentane Phosphatidylglycerophosphate Perdeuterobenzene Phosphoenolpyruvate Phenethyl alcohol cis-2-Oxohept-3-ene-1,7-dioate Phenol acid Phenylboronic Perillyl aldehyde Perillyl Pentacen alcohol Perillyl Ethanolamine phosphate alpha-tolualdehyde2-methyl-

331 EP 2 230 312 A1

5 (- L’A background)

10 6634,25 6546,25 12470,5 12382,5 7326,75 7238,75 L’A (raw data) 0 0 15 0 (- VUL background)

20 (raw data) VUL

25 (- KOL background)

30 500 396 -65 541 220 -23 1350 1284 2283 2217 7678,75 7590,75 1982 1837 477 332 1247,5 1159,5 (continued) (raw data) KOL 0 0 0 35 0 21 1195 1129 -59 2440 (- KT succinate background)

40 23 18 -58 145 2440 (raw data) KT succinate

45

50

55 Compound 1-PhenylethanolL-Phenylalanine2-PhenylglycineQuercetinPhenanthrenePhenazinePhenetolePhosphatidylglycerolL-Phe-Gly-Gly 110 5222PhenothiazinePhloroglucinol 87O-Phospho-L-homoseirne 0 50981-(3-sn-phosphatidyl)glycerol 136 91Phosphatidylinositol 0 29902-Hydroxyphenylacetate 124 -41 -633-Hydroxyphenylacetate 12 3Phenylpyruvate 0 2924 -20 13 52cis-3,4-Dihydroxy-3,4- 0 102 0 232 0dihydrophenanthrene 5729 -43 148 25 0 0 3708 0 0 66 138 0 117 108 5663 38 296 0 24 3925 0 3642 3226 -41 2336 65 -81 -85 14 0 0 0 172 4071 3700 402 415,25 3160 2338,5 79 2270 0 0 0 84 0 -26 41 4005 5577 156 327,25 -53 2250,5 738,25 278 -81 13 5439 10069 6969 313 -26 44 8 0 650,25 46 0 0 35 8929,75 0 2294 6903 9981 247 3130 0 0 11067 633,5 581 8190,25 8841,75 0 2156 4100,75 458 568,75 3054 8102,25 10979 6758,5 545,5 515 537,25 4012,75 11755,5 -111 8912,75 480,75 370 6670,5 449,25 3574 11667,5 0 8824,75 3486 1996,5 1908,5 phenyl hexanoate phenyl 1,2-Dihydroxy-8-methylnaphthale 2,4-Dihydroxy-hept-trans-2-ene-1,7-dioate1-Phosphatidyl-D-myo-inositol 37

332 EP 2 230 312 A1 6212 2523 5 (- L’A background) 6300 10 2611 946,75 858,75 9480,5 9392,5 6056,5 5968,5 1009,5 921,5 6868,5 6780,5 1377,5 1289,5 1709,5 1621,5 8267,75 8179,75 1391,75 1303,75 7403,75 7315,75 4578,75 4490,75 9530,75 9442,75 1842,25 1754,25 L’A (raw data) 0 6 0 0 0 0 0 0 0 0 0 0 0 15 0 12 7909,75 7821,75 (- VUL background) 4 0 20 5 72 28 78 -51 296 209 13672,5 13584,5 345 279 5694,75 5606,75 228 162 1076,75 988,75 1988 1803 (raw data) VUL 0 0 0 0 25 0 53 3015 2900 8574,5 8486,5 10 (- KOL background)

30 20 76 951 864 -26 543 477 3038 2972 6991,75 6903,75 135 169 101 5070 5004 -51 2971 2905 -132 2596 2530 3863 3797 (continued) (raw data) KOL 0 0 0 0 0 0 0 35 0 39 4561 4495 -102 22 35 3328 3194 -45 30 3632 3566 65 134 168 392 206 140 237 8183 7999 26 495 -82 310 3312 3147 319 402 4097 4031 31 2440 (- KT succinate background)

40 -1 55 87 51 22 58 48 -52 163 146 258 516 361 159 619 526 1749 1625 44 4347 4223 53 3475 3351 4424 4358 743 677 7538,75 7450,75 2440 (raw data) KT succinate

45

50

55 Compound Pimelic acid Pimelic O-Phospho-4-hydroxy-L-threonine 1-Phosphatidyl-1D-myo-inositol 3- phosphate 1-Phosphatidyl-1D-myo-inositol4- phosphate 2,6-Diamino-4-nitrotoluene Piperidine Piperideine Piperazine 4-Amino-2-hydroxylamino-6-nitrotoluenePimeloyl-CoA 434 (R)-5-Phosphomevalonate 2,4-Dihydroxylamino-6-nitrotoluene 2-Amino-4,6-dinitrotoluene alpha-D-arabinofuranosidep-Nitrophenyl 154 2-Phenyl-1,3-propanediolmonocarbamate galactoside p-Nitrophenyl 443 4-Fluorocatechol Propenoyl-CoA p-Nitrophenyl glucopyranoside p-Nitrophenyl p-Nitrophenyl cellobioside p-Nitrophenyl (3S)-3-Hydroxyadipyl-CoA beta-D-Glucosyl-2-coumarinate

333 EP 2 230 312 A1 589 695 9709 8589 5832 7680 2208 2904 137,5 5 (- L’A background) 783 677 9797 8677 5920 7768 2992 10 2296 225,5 611,25 523,25 685,75 597,75 635,25 547,25 1884,5 1796,5 4946,5 4858,5 3410,5 3322,5 736,25 648,25 1331,75 1243,75 11399,5 11311,5 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 9 87 17556,8 17468,8 75 5626,25 5538,25 45 (- VUL background)

20 7 26 47 65 83 -75 -48 153 872 806 2105,25 2017,25 -102 -105 3872 3765 4511 4390 (raw data) VUL 0 0 0 0 0 0 0 0 4 0 35578 35512 5882,75 5794,75 0 25 0 99 16 (- KOL background) 3 30 2 50 60 95 52 66 70 54 32 -71 701 635 -69 165 103 465 399 141 4197 4033 -25 1901 1694 58 (continued) (raw data) KOL 0 0 0 0 7 0 0 0 0 0 0 0 0 35 0 824 116 289 867 833 2923 2755 213 341 2440 (- KT succinate background)

40 -3 91 46 42 13 30 30 75 -64 131 948 413 991 957 465 -158 1983 1859 1893 1598 -46 1438 1314 3423 3346 16 3182 3058 1682 1569 6441 6328 1751,5 1663,5 2093 1969 3070 3004 14 2440 (raw data) KT succinate

45

50

55 Compound p-Nitrophenyl myristate p-Nitrophenyl 5-Hydroxyconiferyl alcohol 5-Hydroxyconiferyl p-Nitrophenyl phosphate p-Nitrophenyl p-Nitrophenyl alpha-L-rhamnosidep-Nitrophenyl p-Nitrophenyl alpha-L-rhamnopyranosidep-Nitrophenyl -20 4-Hydroxystyrene Propionate phosphate Propanoyl Phosphatidylinositol-4,5-bisphosphate (R)-Diphosphomevalonate Prephenate alanine methylester 3,5,7,3’,4’-Pentahydroxyflavone Phosphoramidate 3’-diphosphateGuanosine 5’-diphosphate 83 Pseudouridine 2-Hydroxy-4-hydroxymethylbenzalpyruvate -38 5-Phospho-beta-D-ribosylamine 1-(5-Phosphoribosyl)-AMP 1-(5-Phosphoribosyl)-ATP all-trans-pentaprenyldiphosphate N-(5-Phospho-D-ribosyl)anthranilate Propanoyl-CoA

334 EP 2 230 312 A1 5639 8666 1048 2844 7262 1081 5 (- L’A background) 5727 8754 1136 2932 7350 10 1169 11637 11549 1932,5 1844,5 8556,25 8468,25 3922,25 3834,25 2084,75 1996,75 2658,75 2570,75 1024,75 936,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 15 0 (- VUL background)

20 18 22 22 -10 -66 -63 -169 2883 2793 3230,75 3142,75 1667 1458 2969,75 2881,75 1385 1319 2207,5 2119,5 (raw data) VUL 0 5 0 0 0 0 0 9 25 0 98 3813 3747 (- KOL background)

30 4 45 95 75 -12 -94 -14 -49 164 242 176 -12 180 114 742 676 4870,75 4782,75 424 306 2770 2652 202 115 8372 8270 -99 4555 4277 1770 1492 6439,75 6351,75 2122 1918 -72 1109 987 -83 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 0 0 0 35 0 19 31 97 154 2440 (- KT succinate background)

40 6 54 51 92 27 76 24 64 91 80 -53 -10 -15 278 143 155 221 1589 1465 -88 2440 (raw data) KT succinate

45

50

55 Pentanoyl-CoA 3-butenoyl-CoA Protein substrate denatured 5 kDa Protein substrate denatured 10 kDa Protein substrate denatured 15 kDa 15 denatured substrate Protein Protein substrate denatured 20 kDa 20 denatured substrate Protein Protein substrate denatured 30 kDa Propanoic acid Propionyldolichol L-Prolyl-AMP Propyl paraben Compound L-Proline 1,4-Dipropoxybenzene Quinate 3-Phenyl-2-propen-1-ol 5-(5-Phospho-D-ribosylaminoformimino)- 1-(5-phosphoribosyl)-imidazole-4- carboxamide 1-(5-Phosphoribosyl)-5-[(5- phosphoribosylamino)methylideneamino] idazole-4-carboxamideim 5-amino-4-imidazolecarboxamide1-(5-Phosphoribosyl)-5-[(5- phosphoribosylamino)methylideneamino] idazole-4-carboxamideim 180 56 3226 2971 -38 0 920,5 832,5

335 EP 2 230 312 A1 731 615 8117 3720 2702 2629 229,5 5 (- L’A background) 819 703 8205 3808 2790 10 2717 317,5 9212,25 9124,25 1564,25 1476,25 1436,75 1348,75 2875,75 2787,75 8119,75 8031,75 1008,25 920,25 L’A (raw data) 2 0 0 0 0 0 0 1 0 0 0 15 0 (- VUL background)

20 -6 30 18 46 -13 -67 -184 -121 (raw data) VUL 0 0 0 0 0 0 0 25 0 27 -150 (- KOL background)

30 7 63 64 93 -54 -55 216 123 2391 2298 156 1039 940 100 1873 1403 -21 (continued) (raw data) KOL 0 0 0 0 0 0 35 0 18 30 111 2440 (- KT succinate background)

40 -9 96 22 -54 -11 122 142 235 113 154 2420 22962129 63 2005 584 439 147 1516 1392 108 2440 (raw data) KT succinate

45

50

55 Compound Purine Pyrano[3,4-b]pyrrole Pullulan Puromycin Quercitrin Pyridoxal 1-(5-Phospho-D-ribosyl)-5-amino-4- imidazolecarboxylate Pteridine Protein substrate denatured 40 kDaProtein substrate denatured 60 kDatrans-O-Hydroxybenzylidenepyruvate5-Phospho-alpha-D-ribose1-diphosphate 195Pseudocumene 1365’-phosphatePseudouridine 252 80Phosphatidylserine 71O-Phospho-L-serine 12Heptylparaben 128 01-Phosphatidyl-1D-myo-inositol3,4- 21bisphosphate 29 1529 25propyl-3-hydroxybutyrate -7 114 0 1202 0 0 6 215 0 99 0 34 -45 2670 178 91 0 -152 0 90 0 2604 0 54 108 62 0 1918,25 -28 24 9450,5 2555 413,5 0 1830,25 0 1950 9362,5 -32 0 2489 325,5 1331 4346 1862 164 0 4467 1109 4245 98 1466,75 2479,25 4379 4365,5 1378,75 2391,25 7385,5 4277,5 7297,5 Putrescine Phosphatidyl-N-methylethanolamine 3-Phosphatidyl-D-myo-inositol

336 EP 2 230 312 A1

5 (- L’A background)

10 1015,25 927,25 L’A (raw data)

15 0 (- VUL background)

20 9 (raw data) VUL

25 0 (- KOL background)

30 (continued) (raw data) KOL

35 2440 (- KT succinate background)

40 3036 2912 -14 2440 (raw data) KT succinate

45

50 1’-ribulosylformimino)-5-

55 Compound PyrimidinePyridoxine(R)-PhenyllactatePyruvatePyrazinePyrazolidinePyrazolePyrazolo[1,5-a]pyrimidinePyrenePyridazine -7 236Pyridine 271Pyridoxal5’-phosphatePyrogallol 112 2418 0Pyrrole 44 147 132Pyrrolidine -77Ubiquinone 2294 2 4106 564 315Ubiquinol 0 8Pyridine-2,3-dicarboxylate 0 1904 1916 4026Pyridoxamine 5’-phosphate 52 407 72 191 0dUTP 6611 1555 18385-phosphatealpha-D-Ribose 1792 318 17 1227N-(5’-Phospho-D- 394 0 6545 4603 344 0amino-1-(5"-phospho-D-ribosyl)-4- 0 1424 -71imidazolecarboxamide 364 1147 163 -56 193 1486 0 17 4537 328 29 93 220 324 0 119 7413,75 22 2576 297 2250 0 233 1362 2241,25 0 69 1239 14 0 1387 7325,75 0 193 8840,25 0 2095 3848 2452 0 0 2153,25 103 1079 109 1173 1979 28 1201 8752,25 2508 0 8824 5355,75 3781 5376 7996 5590 135 -47 -60 8867,75 1750 0 71 991 4133 5267,75 8630 2384 0 14441,5 5256 -10 7908 5502 8779,75 0 830 0 3947 0 136 14353,5 487 0 -65 108 6002 0 7477,25 934,25 601 4922,25 40 293 0 5936 -48 0 0 7389,25 846,25 5236 4834,25 990 3405,25 545,25 0 7041,5 2568 0 5148 5148 3317,25 457,25 902 6953,5 8584,75 2502 3893,5 5060 6570,75 1633,25 1879,5 1545,25

337 EP 2 230 312 A1 8955 1295 6092 1220 2542 2065 726,5 530,5 5 (- L’A background) 9043 6180 1383 1308 2630 10 2153 814,5 618,5 2512,5 2424,5 4154,5 4066,5 5636,5 5548,5 8426,5 8338,5 937,25 849,25 4619,5 4531,5 8021,25 7933,25 11212,3 11124,3 1194,25 1106,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 30 (- VUL background) 0

20 -3 19 32 -33 -32 -45 (raw data) VUL 0 0 0 0 25 0 (- KOL background)

30 -2 -25 353 163 -124 202 136 6162 6096 7088,25 7000,25 196 130 6800 6710 -8 5325 5259 -46 1476 1235 17 2336 2211 107 1824 1736 1629 1541 6110,75 6022,75 3531 3465 238 172 2653,5 2565,5 2796 2730 55 3994 3925 99 6810 6744 31255 31189 820,75 732,75 (continued) (raw data) KOL 0 0 0 4 0 0 0 0 0 0 0 35 0 21 23 1983 1917 818 106 -22 2440 (- KT succinate background)

40 4 6 89 48 26 26 56 72 88 34 942 230 128 145 147 123 8643 8519 -9 2253 2129 1893 1784 977 868 12279,5 12191,5 3982 3858 35 1923 1799 1629 1353 53 5936 5812 666 600 -71 2826 2702 1191 1099 1357 1265 2440 (raw data) KT succinate

45

50

55 Compound D-Ribose 1,5-bisphosphate D-Ribose alpha-D-Ribose 1-phosphatealpha-D-Ribose Quinone Quinoxaline Quinuclidine pyridine-2,3-dicarboxylatedimethyl 2,3-dihydro-2,3-dihydroxybenzoate Quinoline-3,4-diol D-saccharic acid 1,4-lactoneacid D-saccharic 2-Phenylbutanoic acid 2-Phenylbutanoic (R)-4-Hydroxymandelate Pyridoxamine ketoprofen methyl ester N1-(alpha-D-Ribosyl)-5,6- dimethylbenzimidazole acetate Resorufin N-benzyl ethylester N-benzyl Raffinose L-Ribulose Resorcinol Dihydroresveratrol Quinaldate Folate

338 EP 2 230 312 A1 496 1189 5817 1937 1239 6222 5087 1788 3252 2909 202,5 5 (- L’A background) 584 1277 5905 2025 1327 6310 1876 5175 3340 10 2997 290,5 6564,5 6476,5 2633,5 2545,5 539,25 451,25 2106,75 2018,75 2030,25 1942,25 6689,25 6601,25 3612,75 3524,75 15269,5 15181,5 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 36 (- VUL background) 3

20 -2 -31 -67 -75 -86 -49 -52 121 535 443 -130 -108 2512 2440 401,25 313,25 3336 3251 8885,75 8797,75 (raw data) VUL 0 0 0 0 0 0 0 0 0 0 0 25 0 17 (- KOL background) 4

30 -9 81 13 83 61 74 51 27 -34 -24 -41 127 394 298 132 2781 2715 -4 2418 2229 120 5889 5733 12792 12636 1714,5 1626,5 4760 4351 122 (continued) (raw data) KOL 0 0 0 0 0 0 3 5 0 0 35 0 40 86 10 26 8643 8469 58 98 97 105 4091 3974 -53 119 573 3212 3130 4597 4515 2440 (- KT succinate background)

40 31 14 11 66 65 49 83 89 -36 164 229 210 150 182 127 134 129 243 222 221 697 1504 1380 1707 1633 -42 2440 (raw data) KT succinate

45

50

55 Compound Retinoate acetate Retinol Retinol propionate Retinol Retinol butyrate Retinal Retinol (R)-3-Hydroxybutanoate L-Rhamnulose L-Rhamnulose 1-phosphateL-Rhamnulose Retinol octanoate Retinol Resorufin-beta-D-galactopyranoside Retinol hexanoate Retinol Riboflavin Ribitol L-Rhamnofuranose Resorufin-beta-D-glucopyranoside L-Rhamnonate D-Ribose (R)-Hydroxybutanoyl-CoA (S)-Ribosyl-L-homocysteine gamma-Glutamyl-gamma- aminobutyraldehyde (S)-2-Hydroxystearate

339 EP 2 230 312 A1 510 838 487 1377 523,5 645,5 5 (- L’A background) 598 926 575

10 1465 611,5 3230,5 3142,5 373,25 285,25 2720,5 2632,5 1975,25 1887,25 990,667 902,667 1015,25 927,25 1248,75 1160,75 2370,25 2282,25 4178,25 4090,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 15 0 11 (- VUL background) 0 6 20 8 60 50 -38 243 116 2125,75 2037,75 641 557 4522 4433 (raw data) VUL 0 0 0 0 0 0 0 25 0 (- KOL background)

30 9 30 62 11 68 -23 127 927 861 4714 4648 733,5 124 6131 5981 -124 4261 4195 -39 6103 6037 -18 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 35 0 61 1199 1129 -69 92 6497 6425 6799 6727 6797,25 6709,25 42 2947 2881 40 99 2025 1959 3132 3066 1191,25 1103,25 10 546 6098 6032 255 1878 1743 146 339 195 2440 (- KT succinate background)

40 44 73 40 87 60 35 -91 -18 118 670 216 379 166 463 319 223 134 1916 1792 167 101 4314 4248 705,75 617,75 2440 (raw data) KT succinate

45

50

55 -Sabinol cis Sedoheptulose 7-phosphate Sedoheptulose (+)-Sabinone 3-Chloro-2-hydroxymuconicsemialdehyde 185 (S)-4-Hydroxymandelate 6-phosphate Salicin N6-(L-1,3-Dicarboxypropyl)-L-proline Compound L-Ribulose 5-phosphateL-Ribulose D-Ribulose 5-phosphate D-Ribulose 2-Hydroxy-3-methylpyruvate benzal Sedoheptulose 1,7-bisphosphate Sedoheptulose cis-1,2-Dihydroxy-1,2-dihydro-7- methylnaphthalene (S)-2,3-Epoxysqualene L-RhamnoseResorufin butyrateR-S-Alanylglycine1,2-oxide (1R,2S)-Naphthalene 1296 57 191 1172 0 67 193 3904 2261 0 3838 2170 -144 -34 25 0 0 0 828 8903,25 52 8815,25 740 0 (S)-(+)-2-Phenyl-butyrate (S)-3-Hydroxyhexanoyl-CoA (S)-3-Hydroxyoctanoyl-CoA 1-(5’-Phosphoribosyl)-5-amino-4-(N- succinocarboxamide)-imidazole 2-Carboxybenzaldehyde (+)-

340 EP 2 230 312 A1 280 8209 5 (- L’A background) 368

10 8297 593,75 505,75 2155,25 2067,25 7899,75 7811,75 L’A (raw data) 0 0 0 15 0 (- VUL background)

20 15 -18 769 491 6856 6705 17318 17230 (raw data) VUL 0 0 0 25 0 (- KOL background)

30 0 -33 -27 -64 356 290 213 147 615,75 527,75 (continued) (raw data) KOL 0 0 0 0 35 0 89 62466 62400 -114 244 6602 6466 22 2440 (- KT succinate background)

40 72 -76 -57 -37 -27 213 368 2440 (raw data) KT succinate

45

50

55 Compound D-Serine L-Serine S-Formylglutathione (S)-Hydroxybutanoyl-CoA 1,4-Cyclohexanedione methylesterSalicylaldehydeSalicinS-Adenosyl-(5’)-3-methylthioethylamineN-Succinyl-2-amino-6-oxopimelateSarcosine 2076-phosphateD-Sorbitol D-Sorbitol 2586L-Sorbitol 83sec-Butylbenzene -31 92O-Succinylbenzoyl-CoA 2462Scytalone 2831N-Succinyl-L-2,6-diaminopimelate 45226 264 0 51 02-Hydroxy-4-hydroxymethylbenzalpyruvate 2716Sedoheptulose 45160 55 3862-Amino-5-oxocyclohex-1-enecarbonyl- 140 29 -77CoA 3691 0 72 128 2577 -169 727 2446 236 262L-Seryl-AMP 0 3567 0 0 -82 2453 0 0 4 603 2380 39 1765 112 3238 8096 4427 -53 6 0 6016,75 4017 191 149 -97 1486 2717 3172 1641 7969 0 3985 5928,75 3951 0 -25 0 0 2651 1398 5115 -124 0 25 -24 8848,5 2857 95 6127,5 5049 0 -85 17 4287 2244,75 0 60 8760,5 0 2770 6039,5 29 1453,25 2156,75 3988 6913,75 0 3358,25 0 18 4932,25 0 1365,25 2864,25 6825,75 3067,75 3270,25 4844,25 1368,75 6322,75 0 82 2776,25 2979,75 1280,75 6234,75 1769,75 16 1681,75 1517,75 1429,75

341 EP 2 230 312 A1 882 1575 7042 5 (- L’A background) 970 1663 10 7130 1540,5 1452,5 7669,5 7581,5 5375,5 5287,5 2969,5 2881,5 6624,5 6536,5 2263,25 2175,25 10777,8 10689,8 1000,25 912,25 3172,25 3084,25 4533,75 4445,75 L’A (raw data) 0 0 0 0 1 0 0 0 0 5 0 1 15 0 (- VUL background)

20 20 77 41 59 67 18 87 67 -60 -65 638 495 18562,8 18474,8 1078 874 8088,5 8000,5 (raw data) VUL 0 0 0 0 0 0 0 0 0 0 25 0 18049 17910 14653,8 14565,8 56 (- KOL background)

30 1 82 48 35 60 70 30 53 -64 -41 122 109 3561 3495 -5 (continued) (raw data) KOL 0 0 0 9 0 0 35 0 47 55 210 4441 3974 -3 471 164 427 159 2370 2222 153 104 2440 (- KT succinate background)

40 9 54 88 -17 -10 334 171 595 288 133 283 551 228 179 1423 1299 189 123 2440 (raw data) KT succinate

45

50

55 Compound (S)-Methylthioglycolate 2-Hydroxychromene-2-carboxylate N-Succinyl-2-L-amino-6-oxoheptanedioate 108 shikimate methyl (S)-beta-Methylindolepyruvate Sinapate Sinapoyl-CoA Shikimate N-Succinyl-LL-2,6-methyl diaminoheptanedioate (S)-2-Methyl-3-oxopropanoyl-CoA Sinapyl alcohol Sinapyl Sinapoyl-(S)-malate Sinapaldehyde Sinapoyltartronate Shikimate 3-phosphate S-Succinyldihydrolipoamide4-HydroxymethylsalicylaldehydeSphinganineSphingenineS-(Hydroxymethyl)glutathione(S)-2-Hydroxyoctadecanoate 1018 207cis-glycol Styrene 894 83 6530 178 112 73 2 6406 37 54 0 -26 7 0 0 3301 18 0 90 1406 3222 -30 0 -107 1340 24 45 0 2404,75 -15 0 1676,25 0 2316,75 -2 1588,25 0 244,25 752,25 0 156,25 10237,8 664,25 10149,8 2251,25 2163,25

342 EP 2 230 312 A1 523 7471 1840 5995 1914 5 (- L’A background) 611 7559 1928 6083 10 2002 8379,75 8291,75 8883,75 8795,75 1624,25 1536,25 2422,75 2334,75 5524,75 5436,75 2123,75 2035,75 7105,25 7017,25 1033,25 945,25 10011,8 9923,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 (- VUL background)

20 -2 94 13 22 -90 -22 -45 -89 247 181 8921,5 8833,5 -155 5010 4849 7322,75 7234,75 (raw data) VUL 0 0 0 0 18023 17953 2342,75 2254,75 0 0 0 7 0 25 0 49 36 35 3078 3012 790,25 702,25 (- KOL background)

30 4 60 36 56 74 16 79 53 -29 102 101 2016 1848 -35 6610 6544 -44 3736 3670 -24 4145 3981 5885 5721 3015,25 2927,25 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 0 35 0 18 2860 2794 -184 43 58 3565 3475 3941 3851 7546,75 5532,75 10 208 200 120 145 367 3762 3664 -62 2440 (- KT succinate background)

40 67 87 37 94 11 23 90 -16 142 332 244 167 269 105 182 491 102 134 -139 2426 2302 -17 2440 (raw data) KT succinate

45

50

55 Compound 2,5-Dichloroaniline (1R,4R)-Dihydrocarvone (1S,4S)-Dihydrocarvone semialdehyde 2-hydroxymuconic L-Sorbose1-phosphateSorbose Citronellate1-phosphateSphinganine Spermidine 3041 2338 2917 410 2214 -117 286 367 0 128 301 -15 0 46 0 147 0 5280,5 0 7641,25 5192,5 7553,25 2565,5 2477,5 N2-Succinyl-L-arginine Sucrose 6-phosphateSucrose Spermine Squalene 1,2-oxide (1S,2R)-Naphthalene 4-hydroxysphinganine Starch (S)-Squalene-1,2-oxide Stachyose heptanoate Stearyl (1S,4R)-1-Hydroxy-2-oxolimonene O-Succinylbenzoate-CoA 2-Succinylbenzoate m-Tolualdehyde

343 EP 2 230 312 A1 291 149 3802 3716 1660 2697 1376 4900 437,5 761,5 5 (- L’A background) 379 237 3890 1748 2785 1464 10 4988 849,5 525,5 3155,5 3067,5 290,25 202,25 3956,75 3868,75 3643,25 3555,25 6454,25 6366,25 1328,25 1240,25 2315,25 2227,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 (- VUL background)

20 -2 48 81 32 -93 -47 -17 -59 -57 970 904 (raw data) VUL 0 0 0 0 0 0 0 0 0 25 0 (- KOL background)

30 41 65 26 15 50 58 46 -83 -10 835 698 -129 -143 3693 3593 -57 5524 5458 22445 22379 3804 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 35 0 84 10 36 860 1965 1763 -66 132 204 138 -129 2440 (- KT succinate background)

40 20 75 30 76 95 -47 -46 121 119 984 208 256 134 160 7759 7635 3263 3197 -122 2440 (raw data) KT succinate

45 2,4-dienoate 36

50

55 Compound o-TolualdehydeHydroxycinnamoyl-CoA1,3,4,6-Tetrachloro-1,4-cyclohexadieneO-Succinyl-L-homoserineN2-Succinyl-L-ornithine 214Sucrosesemialdehyde 3-hydroxymuconic 76 90 194 104 2901 110 0 70 0 2776 0 -85 -185 3762 -4 7 68 0 0 3637 0 0 2057 1700,75 2 -58 3101 1612,75 1991 68 -114 0 3035 418,5 2 0 7036,75 2685 330,5 6948,75 -92 550,75 2597 462,75 0 1918,5 1830,5 Syringaldehyde trans-Cinnamate D-Tagatose 1,6-bisphosphate D-Tagatose trans-2,3-epoxysuccinate D-Tagatose 6-phosphateD-Tagatose D-Tagaturonate D-Talose cis,cis-2-Hydroxy-6-oxohept- D-Tagatose D-Tartrate acid L-Tartaric 2,3,4,5-Tetrachlorophenol GMP N-Succinyl-L-glutamate N-Succinyl-L-glutamate5-semialdehyde 1157 1033 5204 5093 -47 trans-1,2-Dihydrobenzene-1,2-diol

344 EP 2 230 312 A1 0 0 259 1829 1101 772,5 5 (- L’A background) 347 1917 10 3115 11970 11882 13667 11653 2192,5 2104,5 9685,5 9597,5 6867,5 6779,5 6782,5 4768,5 574,75 486,75 5730,75 5642,75 1667,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 15 0 (- VUL background)

20 15 -59 -55 -73 -81 2735 2669 7555,25 5541,25 4762 4696 2011,25 3545 3479 8367,25 6353,25 5138 4750 860,5 (raw data) VUL 0 0 0 0 0 0 0 25 0 84 (- KOL background) 4 30 8 62 42 -55 150 4484 4322 7070 6908 4865 4799 5338 5272 2000,5 1912,5 3336 3198 -277 5582 5516 -47 4739 4609 66 6126 6060 -31 (continued) (raw data) KOL 0 0 0 0 0 0 35 0 41 93 -126 35 71 889 2009 1937 -42 202 299 520 -63 2440 (- KT succinate background)

40 58 63 27 82 -13 165 159 108 326 217 423 109 644 195 1013 3914 3790 3737 3517 17 1398 1274 1740 1667 2139 2066 9188,25 7174,25 1918 1794 -88 2440 (raw data) KT succinate

45

50

55 Compound 2-Hydroxy-7-hydroxymethylchromene-2- carboxylate Tetracosane Tetrahydrothiophene Taurine2’,6,3’,4’-Tetrachlorobiphenyl2,4,4’,6-Tetrachlorobiphenyl2,2’,5,5’-Tetrachlorobiphenylketone methyl propyl 2,3,4,5-Tetrachlorobiphenyl 2,3,4,4’-Tetrachlorobiphenyl 122Succinate 144 72D-Threose 0 29823-hydroxybenzoate propyl 20Toluene-cis-dihydrodiol 0 29 2858 40N-Tetradecanoyl-glycine 3712 0 0 0 3575 0 3660 -99 3907 3594 -142 3770 0 1993 0 2476,75 4678,5 1905 2388,75 6930,5 4590,5 6842,5 Tetradecanoyldolichol Succinyl-CoA trans-Tetradec-2-enoyl-CoA Deoxycytidine Tetradecanoyl-CoA Tetrahydrofuran 3’,4’,5,6-Tetrahydroxy-3,7- dimethoxyflavone

345 EP 2 230 312 A1

5 (- L’A background)

10 L’A (raw data)

15 (- VUL background)

20 (raw data) VUL

25 (- KOL background)

30 (continued) (raw data) KOL

35 2440 (- KT succinate background)

40 2440 (raw data) KT succinate

45

50

55 Compound GTP5,6,7,8-TetrahydrofolateTHF-L-glutamateThymidine1,3-ThiazoleThiomorpholineThiopheneThiamin monophosphate 1009ThiaminThiamine diphosphate 8853-Ethylphenol 78 13440dUDP1,3,6,8-Tetrahydroxynaphthalene 1033 70 13316 11634 277Guanine 197 0L-Threonine 7268 909L-Threonate 11510 348 4 942 153alpha-Tocopherol -118 73 225,7,3’,4’-Tetrahydroxyflavone 7192 431 2253Thiepin 372 224 120 818 -7 4669 71 03-Hydroxy-3-isohexeneylglutaryl-CoA 0 -27 2124 1422-Chlorohexane 5154 248N,N,N-Trimethyl-L-histidine 4483 54 83 2486 0 -62,4,6-Trinitrotoluene 5067 299 0 107 3272 4553 0 11271 239 5088 -83 23 11773,8 157 1543 2362 17 4938 3206 128 4264 175 11147 0 99 0 15898,5 106 115 11685,8 3656 1419 0 309 0 3332 33 10270 114 3584 10937 62 4118 15810,5 9101,25 33 40 5366 -52 -54 38 3590 34 3266 4850 -207 185 3518 8256 8133,5 3956 54 48 8923 7087,25 9711,5 5197 10324,8 46 -32 0 2365,75 0 4726 0 -45 6119,5 0 0 -33 2 7811,25 7697,5 0 8310,75 -2 351,75 0 0 13 94 7723,25 -1 139 9949,25 -178 0 0 0 -56 0 2317,25 7935,25 2296 0 0 73 0 0 3489,5 6179 -39 0 303,25 6082,25 282 898,75 -28 -5 1475,5 13805,3 2 4165 4068,25 0 3288,75 11791,3 0 0 0 1274,75 0 5573,25 10662,5 766,5 3559,25 862 8648,5 0 0

346 EP 2 230 312 A1 0 0 138 479 493 7440 3184 5 (- L’A background) 226 567 581 7528 10 5198 193,75 105,75 1692,5 857,75 889,75 801,75 194,75 106,75 788,75 700,75 977,75 889,75 5266,25 5178,25 7194,75 5180,75 5780,75 3766,75 7070,25 5056,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 15 0 41 17 (- VUL background)

20 65 51 87 47 -71 107 -105 -141 -133 6383 6317 4800,25 2786,25 2364 2189 4351,75 2337,75 (raw data) VUL 0 0 0 0 0 0 0 0 0 25 0 89 10536 10470 65 (- KOL background)

30 -5 37 -85 -36 -47 -75 155 131 892 826 -79 5256 5190 47 3963 3805 175 (continued) (raw data) KOL 0 0 0 0 0 0 0 35 0 12 4898 4832 -21 86 -148 12 98 168 965 -18 2440 (- KT succinate background)

40 76 27 77 48 -53 -23 136 292 136 120 222 210 106 2443 2319 -110 1089 4724 4600 73 3375 3251 6525 6459 -23 2148 2024 2431 2365 -82 2440 (raw data) KT succinate

45

50

55 Compound Reduced thioredoxin Trehalose Trehalose 6-phosphate Trehalose Benzoylformate Triacetin Glyceryl tributyrate Isohexenyl-glutaconyl-CoA Oxidized thioredoxin Oxidized 7-Methyl-3-oxo-6-octenoyl-CoA 1,2-Dibromohexane Thyminegamma-TocopherolToluene-4-sulfonateToluene4-Ethylphenol1,4,5-trisphosphate D-myo-Inositol -112 63 1795 0 1671 0 95 86 3779 2213 -22 0 3713 0 2147 0 24 -164 5653 209 -70 5587 0 135 0 9916 0 74 70 5772 7902 6959 0 0 3758 4945 2686 9742 7728 672 3-Fluoro-cis,cis-muconate 3-Fluorocatechol Glyceryl tridodecanoate 1-Bromohexane 1-Chlorohexane Citronellyl-CoA tridecanoate Glyceryl

347 EP 2 230 312 A1 0 45 508 96,5 5744 6511 2472 5 (- L’A background) 596 7758 8525 4486 10 2059 2493,5 2405,5 135,25 47,25 6881,5 4867,5 1014,5 2935,5 2847,5 4224,5 4136,5 2741,5 2653,5 6046,5 4032,5 347,75 259,75 1450,25 1362,25 1012,75 924,75 2345,25 331,25 10252,3 8238,25 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 25 (- VUL background)

20 24 46 22 65 -57 -64 -34 -99 101 -120 -126 2095 1887 2110,5 1749 1683 2360,25 346,25 (raw data) VUL 0 0 0 0 0 0 0 0 0 0 0 25 0 26 -122 99 (- KOL background)

30 5 23 73 17 92 59 41 -79 -39 166 218 152 -119 -110 -140 1408 1195 -28 3359 3143 3482 3266 1680 1560 332 212 3850 3682 94 (continued) (raw data) KOL 0 0 0 0 0 0 0 0 0 0 0 6 0 0 35 0 53 3900 3727 -66 15 239 -27 811 -90 258 819 2428 2024 1568 1164 7896,25 5882,25 2440 (- KT succinate background)

40 1 24 71 56 24 54 79 49 90 -25 -61 177 363 139 935 382 943 102 130 -107 -112 2440 (raw data) KT succinate

45

50

55 Tetracosanoyl-CoA Tylactone Tetradecane Methyl tetradecenoate (9Z)-Tetradecenoicacid Tetradecanoate L-Tryptophan dUMP D-Tryptophan 1-Methyl-1-phenylethylene acid Tetracosanoic Compound Tropine Tryptamine Glycerol trioleate Glycerol 1,4-Lactone Glyceryl tripropionate Tropate tritetradecanoate Glyceryl Glyceryl trioctanoate Glyceryl Glyceryl trihexadecanoate 4,5,6-Trinitrotriazinetrihexanoate Glyceryl -118 0 75 0 -85 0 5996,25 5908,25

348 EP 2 230 312 A1 457 9067 1290 1551 8294 5 (- L’A background) 3304 2471 10 3565 11081 10308 12112 10098 3433,5 1419,5 3588,5 1574,5 9157,75 7143,75 2588,25 574,25 10219,5 8205,5 10796,5 8782,5 4764,25 2750,25 10435,5 8421,5 L’A (raw data) 4 0 0 0 0 0 0 0 0 0 0 0 15 0 28 (- VUL background)

20 -7 87 70 94 -64 -74 -81 -48 -18 -123 (raw data) VUL 0 0 0 0 0 0 25 0 31 85 84 (- KOL background)

30 -1 70 19 55 135 828 718 -68 167 -125 7208 7142 -70 2196 2130 -8 (continued) (raw data) KOL 0 0 0 0 0 35 0 76 32 4586 4492 -60 79 546 252 -84 941 4730 4625 4696 4591 12896,3 10882,3 218 659 284 2440 (- KT succinate background)

40 5 1 12 59 69 -10 376 342 200 670 783 156 203 1065 8210 8086 13 2440 (raw data) KT succinate

45

50

55 Compound UDP-N-Acetyl-3-O-(1-carboxyvinyl)-D- glucosamine Undecaprenyl-diphospho-N- acetylmuramoyl-(N-acetylglucosamine)-L- alanyl-D-glutamyl-meso-2,6- diaminopimeloyl-D-alanyl-D-alanine UDP-3-O-(3-hydroxytetradecanoyl)-N- acetylglucosamine UDP-2,3-bis(3-hydroxytetradecanoyl) glucosamine L-Tyrosine Tyramine UDP-3-O-(3-hydroxytetradecanoyl)-D- glucosamine UDP-N-acetyl-2-amino-2-deoxy-D- glucuronate UDP-N-Acetylmuramate UDP-N-Acetyl-D-glucosamine UDP-N-Acetyl-D-mannosamine UDP-N-Acetylmuramoyl-L-alanyl-D- glutamate UDP-N-Acetyl-D-mannosaminouronate Undecaprenyl-diphospho-N- acetylmuramoyl-L-alanyl-D-glutamyl-meso- 2,6-diaminopimeloyl-D-alanyl-D-alanine UDP-N-Acetylmuramoyl-L-alanine

349 EP 2 230 312 A1 1582 8299 5 (- L’A background) 3596 10 8387 7594,5 5580,5 2922,5 908,5 10113,8 8099,75 2976,75 2888,75 14824,8 12810,8 6316,75 4302,75 L’A (raw data) 0 0 0 0 0 0 0 15 0 (- VUL background)

20 34 29 49 -18 -33 (raw data) VUL 5 0 0 25 0 (- KOL background)

30 306 240 (continued) (raw data) KOL

35 0 384 -115 117 618 552 -34 408 2602 2510 -37 106 5402 5336 62 2440 (- KT succinate background)

40 -19 508 241 532 230 2580 2456 -9 1240 1116 83 1672 1548 41 2440 (raw data) KT succinate

45

50

55 Compound 4-ethyl-2-oxopentanoate Uridine 5’-diphosphateUndecaprenyl phosphateUndecaprenyl diphosphateUDP-D-glucoseMethylitaconateUDP-D-galactoseUDP-D-Galacto-1,4-furanose 1417UDP-D-xylose 199 178UDP-D-glucuronate 1293UDP-3-Ketoglucose 75UDP-L-Rhamnose 54UDP-N-Acetyl-D-galactosamine 172 3001 266UDP-N-acetylmuramoyl-L-alanyl-D- 272gamma-glutamyl-meso-2,6- 285 331diaminopimelate 97 2877 200 48UDP-N-acetylmuramoyl-L-alanyl-D- 148glutamyl-meso-2,6-diaminopimeloyl-D- 1262 197 522 207 63alanyl-D-alanine -43 199 7489 0 2540UMP 1131 1138 302,3-Didehydro-pimeloyl-CoA 114 398 1 74233-Oxopimeloyl-CoA 2474 0 0 75 1 1007 114 26 03-Hydroxy-5-oxohexanoyl-CoA 259 2458,25 1497 0 -23 288 -8 25 48 0 444,25 11165 193 1431 -33 178 -52 0 0 10462,3 0 -55 13106,8 9151 91 0 215 0 8448,25 11092,8 91 6410,5 0 13 25 105 3105 10687,5 4396,5 25 0 8698 4100,5 10501 8673,5 1091 10842,5 2086,5 6684 2284 8487 10754,5 270 Undecanoic acid Undecanoic

350 EP 2 230 312 A1 0 0 0 0 0 0 0 0 0 108 621 6034 1853 5 (- L’A background) 8048 2002 3867 2122 10 2635 401,75 985,75 1419,5 608,25 1339,75 2688,75 674,75 1986,75 1377,75 8853,25 6839,25 7944,75 5930,75 2422,75 408,75 7769,25 5755,25 4135,75 2121,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 (- VUL background) 8 20 3 42 51 19 27 -20 -17 -55 7318 7240 1143,75 3603 3522 5514 5346 2161,33 147,333 (raw data) VUL 0 0 0 0 0 0 0 0 25 0 72 22 78 29 35009 34943 6300,25 4286,25 (- KOL background) 2

30 -5 92 27 33 95 82 -51 138 232 166 -10 144 209 612 546 -50 3929 3863 50 4043 3938 -28 (continued) (raw data) KOL 9 0 3 0 0 0 0 35 0 76 85 4925 4859 -87 36 25 62 -105 606 141 120 6715 6649 -179 270 190 332 187 121 -91 2440 (- KT succinate background)

40 8 -4 94 83 -96 -46 200 133 209 265 730 160 149 394 244 127 314 186 456 1275 1151 -10 3946 3822 3008 2942 -75 2440 (raw data) KT succinate

45

50

55 Compound Cyclopentanol octanoate p-Nitrophenol N-propylurea CyclohexaamyloseUracil5-Ureido-4-imidazolecarboxylate 3’,5’-CyclicIMP Butanoyl phosphate Urocanate 61Vanillin 0 2337 129 2213 63 43 -53 0 0 162 11312,5 40 9298,5 1784,25 0 2-Oxovalerate Neuraminic acid Neuraminic delta-Valerolactone L-Valine Vanillate gamma-Undecalactone 5-Methyl-3-oxo-4-hexenoyl-CoA Vermelone 1,3,7-Trimethylxanthine salicylate Methyl 5’-phosphate Xanthosine Xanthene o-Xylene

351 EP 2 230 312 A1 0 0 0 0 792 295 3824 3311 5 (- L’A background) 1122 5838 2806 2309 10 5325 6601,5 4587,5 3341,5 1327,5 964,75 1423,25 1393,75 2973,75 959,75 L’A (raw data) 0 0 0 0 0 0 0 0 0 15 0 28 (- VUL background)

20 -2 33 94 -85 -40 -63 145 -119 -104 1626 1417 2152,25 138,25 (raw data) VUL 0 0 0 0 0 0 0 0 0 25 0 (- KOL background) Experiments Compound in row 1746 (Methyl-3-bromo-2-methylpropionate) is

30 25 96 -98 -75 -25 -54 -32 (continued) (raw data) KOL Pseudomonas putida 0 0 0 0 0 35 0 92 -123 333 296 282 216 -55 755 -79 2440 (- KT succinate background)

40 42 73 88 36 78 58 216 457 420 879 2367 2243 1302 1078 -33 1733 1609 -70 2440 (raw data) KT succinate

45

50

55 Compound Xyloglucan Xylitol Compound in row 243 (2,2-Dichloropropanoicthe acid) control for is Metagenomic the Experiments control YES*not found in KEGG and PubChem Databases. *B= block, C=columna, R=row represent for molecules which do not match exactly to KEGG but they are named generally in KEGG NO* represent molecules quinoline1-phosphateL-Xylulose 1,3-beta-D-Xylan5-phosphateL-Xylulose piperazine-2-carboxilicacid -113 174 43 0 50L-Xylulose 205 0 207 139 253 2457 141 187 2391 -131 -93 1125,25 0 0 0 8101,5 2447,5 6087,5 433,5 L-Xylose delta-aminovaleramide Zymosterol D-Xylulose 5-phosphateD-Xylulose 5-phosphate Xylitol 2-butanone D-Xylose

352 EP 2 230 312 A1

5

10

15

20

25

30 communities.

35

40 : Fw1: ACIACIGARGARGGIACIGGIGC; Rev1 45 Kulguev-1 F, ATGTGGATACCTCGCAACAAGGCTAAGTCG; Kulguev-1ATGTGGATACCTCGCAACAAGGCTAAGTCG; F, Kulguev-1 TTAAAACGCATTACTAATCCAATCGAAGATAACTGG R,

50 15452 TTEHGTGA & PPCRFDVM KOL1 alkane hydroxylase : KOL2

55 Table 4. Detailed information of hypothetical proteins identified in the present study through analysis KT2440 single cells and VUL, KOL L’A Enzyme name: DNA sequence: Protein sequence: Peptides Q-TOF: Primers for amplification from library CATNACNTCNAANCKRCANGGNGG PCR amplification primers: Enzyme class: pl: 9,77 Enzyme name Protein length: 134 Molecular weight (Da): DNA sequence: Protein sequence:

353 EP 2 230 312 A1

5

10

15

20

25

30 (continued)

35

40 Fw1: GGNGTNCCNAANTANGTNGARGTNCARGTNATG; Rev1: RAANACNGCNTTNSWNGC 45 Kulguev-2 F, ATGCATCAGTTGGCAGTCACCGGC; Kulguev-2ATGCATCAGTTGGCAGTCACCGGC; F, Kulguev-2 TTAATTGACCAGAAAGCGCCTGCTGCC R,

50 19311 GVPKYVEVQVM & ASKAVF : PYAGLAYAG: WVGDLQAGIA & KOL7 haloalkane dehalogenase

55 PCR amplification primers: Primers for amplification from library: Enzyme class: pl: 5,34 Enzyme name: DNA sequence: Protein sequence: Peptides Q-TOF Peptides Q-TOF: Protein length: 172 Molecular weight (Da):

354 EP 2 230 312 A1

5

10

15

20

25

30 (continued)

35

40 : Fwd1: CCNTANGCNGGNYTNGCNTAYGCNGG; Fwd2: AANGANGANGANMGNCCNTAYGCNGG and Rev1: : Fw1: GTNGANGANNTNCANCARWSNGG; Fw2: GAIGCIGCIMGIWGIATG; Rev1: CNCKNTTNACRTCNGG; Rev2: 45 Volcano-9 F, ATGTTGCGCTTGGTGCAGGGG; Volcano-9ATGTTGCGCTTGGTGCAGGGG; F, Volcano-9 CTAGATAGTTTTCCCGAGACC R, : Kulguev-7 F, ATGTGGCCAGCTATCGCTTCGGCC; Kulguev-7ATGTGGCCAGCTATCGCTTCGGCC; F, Kulguev-7 : TCAAAACGCCCGAGCCACCACCAG R, 32750 50 16892 VEELHQSG &EAARSM PDVNRA & &QFAIGF 157 VUL9 L’A62 S-ribosylhomocysteine lyase phosphatidylinositol-bisphosphatase : 295

55 Primers for amplification from library GCNATNCCNGCYTGNARYTCNCCNACCC PCR amplification primers Enzyme class: Protein length Molecular weight (Da): pl: 5,55 Enzyme name: DNA sequence: Protein sequence: Peptides Q-TOF: Primers for amplification from library RAANCCNATNGCRAAYTG PCR amplification primers: Enzyme class: Molecular weight (Da): pl: 5,71 Enzyme name: DNA sequence: Protein length:

355 EP 2 230 312 A1

5

10

15

20

25

30 (continued)

35

40 pET41 L’A62Fwd: 45 pET41 L’A62Fwd, GACGACGACAAGATGATGTTATTGATGGCAGAGATGACAG; L’A62Rev,

50 18006 LLMAEMTEEKL & KEIALGLNLSLSQV & PEKVPEIMAKYK 162 Hydrogenase

55 GACGACGACAAGATGATGTTATTGATGGCAGAGATGACAG; pET41 L’A62Rev: GAGGAGAAGCCCGGTTATTTATACTTTGCCATAATTTCAGGAAC PCR amplification primers: Primers or amplification from library: GAGGAGAAGCCCGGTTATTTATACTTTGCCATAATTTCAGGAAC Enzyme class: Molecular weight (Da): pl: 4,55 Peptides Q-TOF: Protein length:

356 EP 2 230 312 A1

Claims

1. A probe compound comprising a transition metal complex, and a reactive component comprising a test component and an indicator component, wherein the test component and the indicator component are linked to form the reactive 5 component, and wherein the reactive component is linked to the transition metal complex.

2. The probe compound according to claim 1, wherein the transition metal complex comprises a cobalt or copper atom.

3. The probe compound according to claim 1 or 2, wherein the transition metal complex comprises a multidentate 10 ligand, preferably comprising nitrotriacetic acid.

4. The probe compound according to one of the preceding claims, wherein the transition metal complex comprises a nitrotriacetic acid cobalt(II) complex.

15 5. The probe compound according to one of the preceding claims, wherein the transition metal complex additionally comprises an anchoring component.

6. The probe compound according to one of the preceding claims, wherein the transition metal complex is a cobalt(II)

complex of Nα,Nα-bis-(carboxymethyl)-L-lysine. 20 7. The probe compound according to one of the preceding claims, wherein the indicator compound comprises a fluorescence dye.

8. The probe compound according to one of the preceding claims, wherein the test component comprises a compound 25 selected from the group listed in Table 1.

9. A method of preparing a probe compound according to any one of claims 1 to 8, which comprises the steps of:

Preparing a transition metal complex by reacting a salt of a transition metal with a ligand molecule, 30 Preparing a reactive component by linking a test component to an indicator component, and Linking the reactive component to the transition metal complex.

10. An array for detecting enzymes comprising a plurality of different probe compounds according to any one of claims 1 to 8. 35 11. A method for producing an array according to claim 10, comprising the production of a plurality of different probe compounds according to the method of claim 9, wherein an anchoring component is linked to the transition metal complex of the probe compound, and the arrangement of the different probe compounds in an array.

40 12. An isolation means comprising a nanoparticle and a probe compound according to any one of claims 1 to 8.

13. A method for producing an isolation means according to claim 12, comprising the production of a probe compound according to the method of claim 9, wherein an anchoring component is linked to the probe compound, and the probe compound is attached to a nanoparticle. 45 14. A method for detecting enzymes, using the probe compound according to any one of claims 1 to 8, or the array according to claim 10.

15. A method for isolating enzymes, using the isolation means according to claim 12. 50

55

357 EP 2 230 312 A1

358 EP 2 230 312 A1

359 EP 2 230 312 A1

360 EP 2 230 312 A1

361 EP 2 230 312 A1

362 EP 2 230 312 A1

363 EP 2 230 312 A1

364 EP 2 230 312 A1

365 EP 2 230 312 A1

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Non-patent literature cited in the description

• Abad et al. J. Am. Chem. Soc., 2005, vol. 127, 5689 • J. M. Abad et al. J Am Chem Soc, 2005, vol. 127, [0067] [0101] 5689 [0103] • U.K. Laemmli. Nature, 1970, vol. 227, 680 [0083] • M. Ferrer et al. Nature, 2007, vol. 445, 91 [0115] • F. Stephen et al. Nucleic Acids Res, 1997, vol. 25, • T. Fawcett. Pattern Recogn Lett, 2006, vol. 27, 861 3389 [0083] [0118] • Ferrer et al. Tetrahedron, 2000, vol. 56, 4053-4061 • De Lacey et al. J Biol Anorg Chem, 2004, vol. 9, 636 [0097] [0123] • Plou et al. Journal of Biotechnology, 2002, vol. 96, • J.C. Fontecilla-Camps et al. Chem Rev, 2007, vol. 55-66 [0097] 107, 4273 [0132] • Callahan et al. Bioorganic and Medical Chemistry • A. De Lacey et al. Biochem Biophys Acta, 1985, vol. Letters, 2006, vol. 16, 3802 [0097] 832, 69 [0132] • Bull. Chem. Soc. Jpn., 1981, vol. 54, 1879 [0099] • V. M. Fernandez et al. Biochim Biophys Acta, 1985, [0108] vol. 832, 69 [0132] • Tetrahedron Letters, 1996, vol. 37, 5119-5122 • P.M. Vignais ; B. Billoud. Chem Rev, 2007, vol. [0099] 107, 4206 [0132]

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