Presentation is loading. Please wait.

Presentation is loading. Please wait.

The Impact of Metabolomics on Flavor Chemistry Josephine Charve and Gary Reineccius Department of Food Science and Nutrition University of Minnesota.

Similar presentations


Presentation on theme: "The Impact of Metabolomics on Flavor Chemistry Josephine Charve and Gary Reineccius Department of Food Science and Nutrition University of Minnesota."— Presentation transcript:

1 The Impact of Metabolomics on Flavor Chemistry Josephine Charve and Gary Reineccius Department of Food Science and Nutrition University of Minnesota

2 Flavoromics My definition - The application of chemometrics to the study of a broad array of chemical stimuli involved in forming human flavor perception. My definition - The application of chemometrics to the study of a broad array of chemical stimuli involved in forming human flavor perception.

3 Historically – volatiles were the primary interest of the flavor chemist

4 Chewing gum - menthone, sucrose and perceived intensity (Davidson et. al, 2000) Aroma Sensory and Sucrose

5 Perception is multimodal

6 Perception Olfaction Taste Mouthfeel Appearance Sound Experience Texture

7 Why take this approach – value? Improved prediction of sensory properties Improved prediction of sensory properties Better product characterization Better product characterization Discovery - statistically linking stimuli to perception Discovery - statistically linking stimuli to perception New contributors to perception New contributors to perception Understanding of pathways leading to stimuli Understanding of pathways leading to stimuli

8 Why now? Developments in “omics” are driving the development (and availability) of instrumentation, approaches, and data handling and analysis. Developments in “omics” are driving the development (and availability) of instrumentation, approaches, and data handling and analysis. Our University Our University Two new metabolomics faculty Two new metabolomics faculty Over $2,000,000 in advanced MS and some nmr instrumentation Over $2,000,000 in advanced MS and some nmr instrumentation Staffing with data handling/analysis experts from Super Computing Center Staffing with data handling/analysis experts from Super Computing Center

9 Presentation What is metabolomics bringing us? What is metabolomics bringing us? Sample preparation/isolation for analysis Sample preparation/isolation for analysis Data collection (instrumentation) Data collection (instrumentation) Data handling Data handling Data analysis Data analysis Many similar challenges we face Many similar challenges we face

10 Preparation/Isolation Volatiles – not much help. Volatiles – not much help. We recognize the limitations of any extraction/isolation method We recognize the limitations of any extraction/isolation method Help with instrumentation for volatiles Help with instrumentation for volatiles

11 Non-volatiles – helping us Searching for “best” method for us. Searching for “best” method for us. Going through a host of methods evaluating each for sensitivity and breath Going through a host of methods evaluating each for sensitivity and breath

12 Non-volatiles – common approach Solvent extraction of solid tissues Solvent extraction of solid tissues Mechanical disruption of the tissue (grinding, vibration or other methods on a frozen sample.) Mechanical disruption of the tissue (grinding, vibration or other methods on a frozen sample.) Solvent selection varies widely with compounds of interest; Solvent selection varies widely with compounds of interest; polar compounds being best extracted with isopropanol, ethanol, methanol, acidic methanol, acetonitrile, water, and methanol:water. polar compounds being best extracted with isopropanol, ethanol, methanol, acidic methanol, acetonitrile, water, and methanol:water. Non-polar compounds are most often extracted with chloroform or ethyl acetate Non-polar compounds are most often extracted with chloroform or ethyl acetate (Dettmer et al., 2007). (Dettmer et al., 2007).

13 Polar substances in potatoes Polar substances – (potatoes) best extraction method Polar substances – (potatoes) best extraction method methanol and heating (for enzyme deactivation) methanol and heating (for enzyme deactivation) Methoxylation of sugars and silylation Methoxylation of sugars and silylation GC-MS profiling resulted in 150 polar compounds, 77 of which were identified GC-MS profiling resulted in 150 polar compounds, 77 of which were identified (Roessner et al. 2000) (Roessner et al. 2000)

14 Secondary plant metabolites Extraction with acidified aqueous methanol. (75% methanol, 0.1% formic acid, water from the sample or added as necessary). Extraction with acidified aqueous methanol. (75% methanol, 0.1% formic acid, water from the sample or added as necessary). Key factor is simplicity Key factor is simplicity De Vos et al. (2007) De Vos et al. (2007)

15 Analysis MS –GC/GC, LC (UPLC), direct analysis MS –GC/GC, LC (UPLC), direct analysis Long history of application of basic techniques to foods Long history of application of basic techniques to foods Impressive evolution in methods Impressive evolution in methods nmr nmr Applied to foods in related work for more than two decades. Applied to foods in related work for more than two decades. Historically, emphasis was to detect adulteration and fraud Historically, emphasis was to detect adulteration and fraud

16 GCxGC Better resolution Better resolution Better sensitivity – no back ground Better sensitivity – no back ground Better quantitative data Better quantitative data Going from unique use to standard method Going from unique use to standard method

17 GC-MS Broad use of TOF instruments Broad use of TOF instruments Improved sensitivity (4X vs. quad) Improved sensitivity (4X vs. quad) Fast scans – permits peak deconvolution (peaks with 1 sec difference in peak apex) Fast scans – permits peak deconvolution (peaks with 1 sec difference in peak apex) Also minor peaks not well separated from major peaks Also minor peaks not well separated from major peaks Accurate mass at fast scan Accurate mass at fast scan Leco – GC/MS and GCxGC/MS - low resolution MS but peak deconvolution due to high sampling rate Leco – GC/MS and GCxGC/MS - low resolution MS but peak deconvolution due to high sampling rate Thermo – triple quad also high resolution magnetic sector Thermo – triple quad also high resolution magnetic sector Waters – GCxGC with accurate mass TOF Waters – GCxGC with accurate mass TOF

18 UPLC or Capillary LC UPLC – sub -2μm stationary phase and high linear velocity of the mobile phase UPLC – sub -2μm stationary phase and high linear velocity of the mobile phase Faster analysis (high throughput) Faster analysis (high throughput) Better peak resolution Better peak resolution Higher peak capacity Higher peak capacity Must be interfaced with fast MS – often TOF Must be interfaced with fast MS – often TOF (Plumb et al., 2005). (Plumb et al., 2005).

19 Ambient ionization – gas analysis PTR-MS or API-MS PTR-MS or API-MS Ideally suited to providing an ion profile of a gaseous sample Ideally suited to providing an ion profile of a gaseous sample Many current applications in flavor chemistry Many current applications in flavor chemistry

20 Ambient Ionization (AI) Methods Ionization Method Ionizing Agent Sample State Desorption ElectroSpray Ionization (DESI) Charged droplets/ions Condensed phase, gas phase Desorption Atmospheric Pressure Chemical Ionization (DAPCI) Primary ions via corona discharge or charged droplets Condensed phase, gas phase Direct Ionization in Real Time (DART) Metastable atoms Condensed phase, gas phase Atmospheric Pressure Matrix Assisted Laser Desortion Ionization (AP-MALDI) Laser irradiation/Matrix Condensed phase Atmospheric Pressure Solids Ionization Probe (ASAP) Primary ions via corona discharge Condensed phase Electrospray Laser Desorption Ionization (ELDI) Laser irradiation/Charged droplets Condensed phase Ionization methods allow for analysis of samples under ambient conditions Ionization methods allow for analysis of samples under ambient conditions Many AI methods require no sample preparation Many AI methods require no sample preparation Currently, AI methods are receiving considerable attention Currently, AI methods are receiving considerable attention

21 DESI Instrumentation Implementation of DESI Implementation of DESI DESI spray head – generation of charged droplets/primary ions DESI spray head – generation of charged droplets/primary ions Atmospheric pressure ionization compatible mass spectrometer – mass analysis of generated secondary ions Atmospheric pressure ionization compatible mass spectrometer – mass analysis of generated secondary ions Some ancillary equipment/supplies – either necessary for experiment or convenience Some ancillary equipment/supplies – either necessary for experiment or convenience To date, majority of DESI data has been generated using linear quadrupole ion traps To date, majority of DESI data has been generated using linear quadrupole ion traps DESI has been demonstrated on nearly all mass analyzers DESI has been demonstrated on nearly all mass analyzers Several different API interfaces Several different API interfaces   surface d t-s gas jet spray capillary spray nebulizer capillary inlet of mass spectrometer sample desorbed ions solvent N2N2 HV

22 Example - Detection of lysozyme DESI spectrum of lysozyme present on PTFE surface; average surface concentration 50 ng/cm 2 Microbial contamination on food processing surfaces?

23 nmr MS offers sensitivity and capacity to detect compounds in mixtures but are limited to ionizable species, have difficulties resolving isomers, and usually require standard compounds for quantification. NMR has the capacity to characterize chemical structure and quantity but is limited to the most abundant compounds in a given sample without isotope labeling. (several other limitations) Quantitative and reproducible – statistical analysis Sensitivity not limited by same factors as MS High throughput – 500 samples per day (Pan and Raftery, 2007; (Lindon et al., 2004 Hegeman et al. Anal. Chem. 2007, 79, , Hegeman for isotope labeling studies; (Pan and Raftery, 2007; (Lindon et al., 2004

24 Simple sample preparation Freeze sample Freeze sample Freeze dry Freeze dry Reconstitute in 80:20 D 2 O:CD 3 OD containing 0.05% w/v TSP-d4 (sodium salt of trimethylsilylpropionic acid) Reconstitute in 80:20 D 2 O:CD 3 OD containing 0.05% w/v TSP-d4 (sodium salt of trimethylsilylpropionic acid) Sample heated (50C – 10 min) Sample heated (50C – 10 min) Micro centrifugation Micro centrifugation Ward J, Harris C, Lewis J, Beale MH, Phytochemistry 62 (2003) 949–957 Ward J, Harris C, Lewis J, Beale MH, Phytochemistry 62 (2003) 949–957

25 Data handling/Analysis Multivariate statistical projection methods (partial least squares, principle component analysis) are commonly used (as starting point) Lend themselves well to biological data because of their ability to correlate multiple variables in a robust and easily interpretable fashion (Jonsson et al., 2005).

26 Statistical heterospectroscopy (SHY) New technique used to correlate nmr data with UPLC-MS results by cross-assigning the signals. New technique used to correlate nmr data with UPLC-MS results by cross-assigning the signals. (Crockford et al., 2006). (Pan and Raftery, 2007). (Crockford et al., 2006). (Pan and Raftery, 2007). Technique used to combine nmr with DESI-MS to correlate known biomarkers with specific metabolites Technique used to combine nmr with DESI-MS to correlate known biomarkers with specific metabolites (Pan et al., 2007 ). (Pan et al., 2007 ). CARA CARA

27 MetAlign TM Used in numerous publications for data extraction from GC-MS and LC-MS data sets. Used in numerous publications for data extraction from GC-MS and LC-MS data sets. Pulls out all of the masses and sorts which are the same in all of the data sets and which could differentiate the data. Pulls out all of the masses and sorts which are the same in all of the data sets and which could differentiate the data. Allows the user to look for unique peaks in the sample set above a chosen noise threshold Allows the user to look for unique peaks in the sample set above a chosen noise threshold (Lomen et al., 2006). (Lomen et al., 2006).

28 MetAlign “MetAlign is a software program for full scan LC-MS and GC-MS comparisons and was designed and written by Arjen Lommen of RIKILT-Institute of Food Safety. It has been extensively tested in collaboration with Plant Research International.” “MetAlign is a software program for full scan LC-MS and GC-MS comparisons and was designed and written by Arjen Lommen of RIKILT-Institute of Food Safety. It has been extensively tested in collaboration with Plant Research International.”

29 Commercial packages For example – Marker Lynx (Waters), Xaminer™ Thermo and ? For example – Marker Lynx (Waters), Xaminer™ Thermo and ?

30 Umetrics SIMCA-P name/software_simcapplus/c/4 name/software_simcapplus/c/4 name/software_simcapplus/c/4 name/software_simcapplus/c/4 Select compounds to focus efforts on – not try to identify everything Select compounds to focus efforts on – not try to identify everything

31 Identifications Most existing metabolite libraries are either proprietary, insufficiently comprehensive, collected under non-standardized conditions or unsearchable by computers. Exceptions: Human Metabolome Database (HMDB; (>20,000 metabolites) and

32 Madison Metabolomics Consortium (MMC) Database (MMCD; nmrfam.wisc.edu/), a web-based tool that contains data pertaining to biologically relevant small molecules from a variety of species.

33 Conclusions Not much help in isolation methods for volatiles – information on semi or non— volatiles Not much help in isolation methods for volatiles – information on semi or non— volatiles New instrumentation – GC/GC, MS and sample interfaces, nmr, LC-MS New instrumentation – GC/GC, MS and sample interfaces, nmr, LC-MS New tools for data handling and analysis New tools for data handling and analysis Also combining instrument data e.g. nmr w/ MS Also combining instrument data e.g. nmr w/ MS Establishing public databases Establishing public databases

34 Greatest contribution? Availability Availability Methodologies Methodologies

35 Will demand collaboration

36

37 Descriptive Sensory Analysis Volatiles/Semi-volatiles Extraction SAFE ? SPME Non-volatiles/Semi- volatiles Extraction MeOH/H20 Instrumental Analysis LC-MS-TOF MS/MS NMR ? Instrumental Analysis GC-TOF-MS Accurate mass Collect LC fractions for descriptive sensory analysis Data Analysis MetAlign PLS with DA PCA SHY

38

39 Figure 1. GC-MS-based metabolomics. A, Analytical approach used B, Conventional approach. C, Alternative, unbiased approach to GCMS data analysis. Tikunov Y, Lommen A, Ric de Vos CH, Verhoeven HA, Bino RJ, Hall RD, Bovy AJ Plant Physiology, November 2005, Vol. 139, pp. 1125–1137

40

41

42

43

44

45 Formatted to export data For example - to SIMCA-P For example - to SIMCA-P

46 Case study

47 Strawberry metabolites Fourier Transform Ion Cyclotron Mass Spectrometry (FTMS) Fourier Transform Ion Cyclotron Mass Spectrometry (FTMS) FTMS - only MS system capable of routinely achieving ultra high resolution at high acquisition rate (100–1,000 amu scan/sec) allowing multiple scans in (1–2 min). FTMS - only MS system capable of routinely achieving ultra high resolution at high acquisition rate (100–1,000 amu scan/sec) allowing multiple scans in (1–2 min). Separation of the metabolites achieved solely by ultra-high mass resolution, eliminating the need for time consuming chromatography and derivatization. Separation of the metabolites achieved solely by ultra-high mass resolution, eliminating the need for time consuming chromatography and derivatization. Aarón A, Ric De Vos, Verhoeven HA, Maliepaard CA, Kruppa G, Bino R, Goodenowe DB. Omics 6(3), 2002 ( )

48 Method 4 stages of ripeness for berries 4 stages of ripeness for berries Extraction –50/50 MeOH/0.1% formic acid or 100% acetonitrile (AN) Extraction –50/50 MeOH/0.1% formic acid or 100% acetonitrile (AN) Direct injection into FTMS Direct injection into FTMS MS ionization - electrospray (ESI + or -) or atmospheric pressure chemical ionization (APCI + or -) MS ionization - electrospray (ESI + or -) or atmospheric pressure chemical ionization (APCI + or -)

49 Strawberry metabolite 1ppm mass accuracy 10 ppm mass accuracy (2 possible only 1 is logical)

50 Result total of 5,250 unique 12C masses were obtained from extracts of the four different developmental fruit stages, ionized in the four ionization modes. total of 5,250 unique 12C masses were obtained from extracts of the four different developmental fruit stages, ionized in the four ionization modes. In the red stage extract, 55% of the masses were assigned a single empirical formula, 10% two formulae, and 35% three or more formulae. In the red stage extract, 55% of the masses were assigned a single empirical formula, 10% two formulae, and 35% three or more formulae. Went to data databases for help - 159,000 natural products (Chapman and Hall, Dictionary of Natural Products) Went to data databases for help - 159,000 natural products (Chapman and Hall, Dictionary of Natural Products)

51

52 Next steps Look for changes or relationships of data to some attribute via multivariate statistics Look for changes or relationships of data to some attribute via multivariate statistics Identify the compounds of interest. Identify the compounds of interest. HPLC, mass spectrometry (MS/MS) and/or nuclear magnetic resonance (NMR) must be employed. HPLC, mass spectrometry (MS/MS) and/or nuclear magnetic resonance (NMR) must be employed. Evaluate result for value Evaluate result for value


Download ppt "The Impact of Metabolomics on Flavor Chemistry Josephine Charve and Gary Reineccius Department of Food Science and Nutrition University of Minnesota."

Similar presentations


Ads by Google