Metabolomics. Innovation with Integrity. Powering Comprehensive Studies. NMR & Mass Spectrometry

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1 Metabolomics Powering Comprehensive Studies Innovation with Integrity NMR & Mass Spectrometry

2 Powering Comprehensive Metabolomics Studies The accurate determination of changes in small molecule profiles related to a disease, therapeutic intervention, genetic modification or environmental variation is central to all metabolomics studies. Bruker remains the leader in integrated solutions for metabolomics with its latest innovations for hyphenated NMR, LC-MS and GC-MS technologies. These systems can be used for a variety of metabolomics studies and are very well equipped to detect, identify, and measure a range of metabolites. Many different pieces of information have to be linked in metabolomics experiments. Bruker provides all you need in a comprehensive, integrated system adjustable to your growing requirements. Bruker s analytical systems for targeted and untargeted metabolomics are used in: Drug discovery & development Clinical research Public health studies Plant science Nutrition & food Microbial research Environmental & ecology research Biochemistry Systems biology

3 Analytical Approaches Bruker offers systems for the following metabolomics studies: Untargeted profiling Maximum coverage of the metabolome s chemical space can be achieved by utilizing Bruker s high performance LC-MS, GC-MS, CE-MS and NMR systems in conjunction with dedicated software for data evaluation. These systems combine all necessary tools for feature extraction, statistical evaluation and compound identification. The unique hyphenation of high resolution MS and NMR, the MetabolicProfiler TM, enables rapid biomarker detection and identification by combined statistical evaluation of MS and NMR data. Targeted analysis - biomarker validation Identified biomarkers can be validated in targeted metabolomics experiments, where a limited number of compounds are quantified. With its excellent sensitivity and powerful quantitation capabilities, Bruker s GC-MS triple quadrupole system is ideal for this task. Additionally, Bruker s ESI-UHR-TOF system provides highly accurate data with a wide dynamic range and excellent resolution for the simultaneous analysis of both high- and low-abundance metabolites. Bruker`s NMR based JuiceScreener TM is best suited for sample classification e.g. for food quality control and can be combined with Bruker s MS solutions for food safety studies. Biomarker visualization Bruker s renowned MALDI Imaging solution literally brings small molecule biomarkers into their biological context, opening the door to visualizing the spatial distribution of potential biomarkers within biological tissues.

4 Meet an Extensive Portfolio MetabolicProfiler: The only fully integrated NMR & LC-MS system for maximum metabolite coverage and de novo structure elucidation utilizing complementary techniques GC-APCI Source: Unique combination of high-resolution accurate mass MS with GC enables identification of unknowns in GC-MS based metabolomics Metabolomics 300 series Quadrupole GC-MS: Class leading detection performance for multi-compound quantitation in targeted metabolomics solarix FTMS: The ultimate in MS performance for definitive sum formula determination. Also equipped with MALDI capabilities for in situ metabolite imaging maxis and microtof series: High performance ESI-TOF LC-MS instruments featuring full sensitivity and resolution at fast scan speeds to detect, resolve and identify even low-level metabolites

5 Handling the Entire Workflow Untargeted Metabolomics Targeted Metabolomics Study design Hypothesis Study Design Study design Acquisition Experiment Acquisition Data Processing Quantification Statistical analyses Data Evaluation Statistical analyses Data preprocessing Interpretation EICs / Da + Propionate Identification Knowledge Propionate Validation Validation / quantification 9 10 Time [min] Visualization / interpretation

6 Transforming Data into Knowledge Untargeted metabolomics - detect possible biomarkers Unsupervised statistical analysis Find Molecular Features extracts all relevant information from complex data sets and combines ions belonging to one compound Retention time alignment Different scaling and normalization options Supervised and unsupervized statistics: PCA, t-test, ANOVA; etc. PCA scores and loadings plot De novo identification of target compounds Unique sum formula of precursor SmartFormula3D Sum formulae of corresponding fragments SmartFormula3D - unique sum formula generation capabilities by combining accurate mass and isotopic pattern information in MS and MS/MS spectra CompoundCrawler - query correct sum formula in public databases FragmentExplorer - confirm structure by correlation of precursor structure with fragment ions in measured MS/MS spectrum Direct link to MetFrag (IPB Halle, Germany) for in-silico fragmentation CompoundCrawler FragmentExplorer

7 Identification by integrated MS and NMR analysis MS and NMR provide complementary data MS delivers sum formula on precursor and fragment ions NMR enables de novo structure elucidation Correct sum formula accelerates identification by NMR Full hyphenation of LC-SPE-NMR-MS allows structure elucidation of lowabundance compounds NMR and MS covariance matrix Biomarker confirmation Rapid screening for known metabolites Easily customizable database Selectivity based on high-resolution EIC traces Confirmation based on accurate mass, isotopic pattern, retention time and qualifier ions Absolute quantitation of target compounds Targeted metabolomics Screening and quantitation LibrarySearch: ID of known compounds Identify by library search LibraryEditor Known compounds are readily identified by comparison to information stored in the LibraryEditor: MS/MS spectra MS n spectra Retention time Ionization mode Instrument type

8 Find, Identify and Validate Biomarkers A B Identifying a plant s response to insect attack Metabolic changes in Nicotiana attenuata during insect herbivory. C Study Design O untreated sim. herbivory Plants protect themselves from insects through complex and well-regulated defence mechanisms. LC-ESI-TOF (HPLC-micrOTOF and U-HPLC-maXis) based metabolic profiling comparing defence-elicited and control leaf extracts revealed significant changes in small molecule profiles Time (h) (A) Nicotiana attenuata leaf attacked by Manduca sexta. (B) Simluating insect herbivory by wounding leaves with fabric pattern wheel and applying insect s oral secretions. (C) Experimental setup - 5 biological replicates per time point and treatment were analysed by HPLC-micrOTOF and U-HPLC-maXis analyses. Read more: E. Gaquerel et al., J Agric Food Chem Sep 8;58(17): Identification of Caffeoylputrescine as a compound strongly induced upon simulated insect herbivory A O untreated Δ sim.herbivory 120h 86h 1h Loadings B Bucket Statistic Plot s 14h 1h 4h 86h 120h C MS MS/MS D Caffeoylputrescine [M+H] + = C 13 H 19 N 2 O 3 C 13 H 19 N 2 O 3 (A) PCA scores and loadings plot of extracted features revealed a clustering of samples according to treatment time (trend for late time points in treated samples highlighted by red arrow). Selected loading ( s) displayed in bucket statistics plot (B) depicts higher amounts for this compound in treated samples after 86 and 120 h simulated herbivory. SmartFormula3D generates one sum formula hit for this molecule (C). Compound Crawler database query provides caffeoylputrescine as likely structure for the target compound (D).

9 Plant, Ecological, Microbiological and Clinical Research Opening new areas to find biomarkers Metabolomics studies based on gas chromatography-mass spectrometry (GC-MS) are well-established and typically employ electron impact (EI) ionisation. Unfortunately, many possible biomarkers detected in these experiments cannot be identified due to the lack of EI reference spectra for a majority of biologically relevant compounds. Hyphenating GC with high resolution TOF-MS technology by soft atmospheric pressure ionisation (APCI) can preserve the molecular ion information and deliver accurate mass and isotopic pattern information. This data enables identification of possible biomarkers which remained unknowns up until now. GC-APCI-MS: Identification by accurate mass and isotopic pattern Intens. x hreics / Da + Propionate Intens. x Propionate 9 10 Time [min] C 9 H 24 NO 2 Si 2 1.9ppm Sigma Rank # m/z Derivatized metabolite extracts from Corynebacterium glutamicum ΔprpD2 grown with or without propionate were analyzed by GC-APCI-micrOTOF-QII analysis. High resolution EIC traces with 2 mda mass window reveal that alanine shows a higher abundance in bacterial cells grown on propionate (red trace). The atmospheric pressure ionization preserved the precursor ion and enabled the unambiguous sum formula generation by SmartFormula. Quantification in clinical studies Following biomarker identification, an absolute quantification of the target compounds is usually performed. To minimize matrix effects and obtain superior sensitivity levels, Bruker s GC-MS triple quadrupole instruments are the instruments of choice. Read more: Bruker Daltonics Application Note # ET-22 Read more: Bruker CA Application Note # CA

10 Metabolomics in Food & Nutrition Identification of m-coumaric acid by library search -MS -MS/MS Metabolite identification by accurate mass and library search An MS/MS spectral library of dietary phenolic compounds was created using data acquired with a microtof-q. The established LC-Auto/MS/MS method is fast and sensitive, enabling the characterization of even low-abundance compounds, and provides mass accuracy and true isotopic pattern for both precursor and product ions. Positive identifications of dietary phenolics were obtained in a variety of samples analyzed under the same conditions used to obtain the reference spectra. Read more: M. Gómez-Romero et al., Food Chemistry Jan; 124(1): Integrated use of NMR and MS - Identification of phloridzin as major compound differentiating two sets of apple juice samples A B C D Chinese juice Polish juice juice Phloridzin Phloridzin in acetonitrile-d3 from reference database (A) PCA scores and loadings plot of LC-MS data from a set of Polish (black) and Chinese (red) apple juices. (B) Sum Formula of the most differentiating peak (m/z = ) calculated as C 21 H 23 O 10 by SmartFormula. The upper part of (C) represents a cross-covariance of the NMR data with m/z = , i.e. the chemical shifts which correlate to mass These correlating signals are in accordance to the spectrum of the reference standard of phloridzin. (C lower part) Comparing two selected 1D-NMR spectra of Chinese (red) and Polish (black) apple juices shows a higher abundance of phloridzin in Polish apples juices. The identification of phloridzin (D) demonstrates that LC-MS and NMR provide complementary data and enable biomarker discovery and identification.

11 Selected Metabolomics Publications Plant Metabolomics The multifunctional enzyme CYP71B15 (PHYTOALEXIN DEFICIENT3) converts cysteine-indole-3-acetonitrile to camalexin in the indole-3-acetonitrile metabolic network of Arabidopsis thaliana. C. Böttcher et al.; The Plant Cell 2009 Jun 30; 21: LC-MSMS Profiling of Flavonoid Conjugates in Wild Mexican Lupine, Lupinus reflexus. M. Stobiecki et al.; J. Nat. Prod. 2010; 73, Bacterial Metabolomics Discovering the Hidden Secondary Metabolome of Myxococcus xanthus: a Study of Intraspecific Diversity. D. Krug et al.; Appl Environ Microbiol May; 74(10): Clinical Metabolomics Amino acid profiling in urine by capillary zone electrophoresis - mass spectrometry. O.A. Mayboroda et al.; Journal of chromatography A Aug; 1159 (1-2): Gas chromatography/atmospheric pressure chemical ionization-time of flight mass spectrometry: analytical validation and applicability to metabolic profiling. A. Carrasco-Pancorbo et al.; Anal Chem Dec 15; 81(24): Lipidomics Combined Reversed Phase HPLC, Mass Spectrometry, and NMR Spectroscopy for a Fast Separation and Efficient Identification of Phosphatidylcholines. J. Willmann et al.; J Biomed Biotechnol. 2011; 2011: In summary, GC/APCI-TOF MS is an analytical procedure, which combines the best of chromatography with one of the most robust MS interfaces, and as such, it has a potential to become one of the standard methods in metabolic profiling. T. Pacchiarotta, et al. J Biomol Tech December; 21(4): Based on the very accurate mass information and the isotopic pattern provided by the TOF MS analyzer, together with other available information, ten of these biomarkers and more than 50 metabolites, obtained through phase I and phase II biotransformation reactions, were tentatively identified. R. García-Villalba, et al. Analytical and bioanalytical chemistry Sep; 398(1): Food & Environmental Exploratory analysis of human urine by LC-ESI-TOF MS after high intake of olive oil: understanding the metabolism of polyphenols. R. García-Villalba et al.; Analytical and bioanalytical chemistry Sep; 398(1): Unraveling different chemical fingerprints between a champagne wine and its aerosols. G. Liger-Belair et al.; PNAS Sept. 29; 106(39): Single Cell Metabolomics Capillary electrophoresis with electrospray ionization mass spectrometric detection for single-cell metabolomics. T. Lapainis et al.; Anal Chem Jul 15; 81(14): Small Molecule MALDI Imaging Localization of secondary metabolites in marine invertebrates: contribution of MALDI MSI for the study of saponins in Cuvierian tubules of H. forskali. S. Van Dyck et al.; PLoS One Nov 10; 5(11):e Identification of Unknowns/ Hyphenated Metabolomics Solutions A strategy for the determination of the elemental composition by fourier transform ion cyclotron resonance mass spectrometry based on isotopic peak ratios. D. Miura et al.; Anal Chem Jul 1; 82(13): Combined NMR and LC-MS analysis reveals the metabonomic changes in Salvia miltiorrhiza Bunge induced by water depletion. H. Dai et al.; J Proteome Res Mar 5; 9(3):

12 Bruker Daltonics is continually improving its products and reserves the right to change specifications without notice. BDAL , # A Comprehensive Portfolio Powering Metabolomics Studies Mass spectrometry and NMR systems maxis series: UHR-Qq-TOF systems deliver unique full sensitivity at full resolution for complex sample analysis; high mass accuracy and isotopic fidelity in MS and MS/MS enable unknown identification. microtof and microtof-q II: Benchtop TOF and Qq-TOF for robust and easy-to-use accurate mass determination. amazon series: The first ion traps with dual ion funnel based sensitivity, zero-delay polarity switching and up to 20 Hz MS n acquisition speed. solarix FT-MS: Simply the most versatile mass spectrometer available, with unbeatable mass accuracy and resolution for studying ultracomplex samples and small molecule MALDI imaging capabilities. 300 series GC-MS: Enabling GC-MS metabolomics workflows. NMR: The gold-standard in NMR based metabolomics and structure elucidation. MetabolicProfiler: Unique and wellestablished integration of NMR and LC-MS systems providing the ultimate profiling platform. Integrated software solutions ProfileAnalysis: Data preprocessing and multivariate statistics based on MS data. DataAnalysis: A complete set of tools for sum formula generation, database query and structure elucidation. TargetAnalysis: Rapid screening for known metabolites. AMIX: Combined evaluation of NMR and MS data including combined covariance matrix. For research use only. Not for use in diagnostic procedures. Bruker Daltonik GmbH Bremen Germany Phone +49 (0) Fax +49 (0) sales@bdal.de Bruker Daltonics Inc. Billerica, MA USA Fremont, CA USA Phone +1 (978) Phone +1 (510) Fax +1 (978) Fax +1 (510) ms-sales@bdal.com ms-sales@bdal.com