Seminar 5 Protocol For NMR Based Metabolomics. Tips And Tricks. Dr. Dimitra Benaki School of Pharmacy, Dept. Pharmaceutical Chemistry

Transcription

1 Seminar 5 Protocol For NMR Based Metabolomics. Tips And Tricks Dr. Dimitra Benaki School of Pharmacy, Dept. Pharmaceutical Chemistry

2 Systems Biology 4rth Workshop on Holistic Analytical Methods, April 2016

3 will or may happen What 4rth Workshop on Holistic Analytical Methods, April 2016 Systems Biology is happening happened

4 Applications of Metabolomics Human diseases Diagnosis Determination of disease state Prevention Biomarker discovery Risk determination Pharmacology Drug discovery Treatment Doses evaluation Toxicology Toxicity assessment Toxic effects of drugs Food technology Food safety, quality Nutrigenomics Applications of Metabolomics Plant biotechnology Crop improvement Transgenic breeding Plant breeding Improve stress tolerance Microbial biotechnology Microbial improvement Fermentation Biotechnological compounds Enzyme discovery Discovery of biochemical pathways Link changes in metabolite levels to catalytic activity Improve catalytic efficiency of enzymes Systems biology Dynamics in biological systems Explore metabolic networks 4rth Workshop on Holistic Analytical Methods, April 2016

5 Metabolomics Methods NMR GC, HPLC, UPLC, HPTLC CE (capillary electrophoresis) Hyphenated LC-MS, GC-MS GC-MS/MS UHPLC-MS GC-MS 4rth Workshop on Holistic Analytical Methods, April 2016

6 NMR in Metabolomics Minimal sample preparation (biofluids) No extra steps, i.e. separation Measures multiple metabolites simultaneously Non-destructive Fast analysis High reproducibility Highly quantitative High throughput Steadily increasing sensitivity 4rth Workshop on Holistic Analytical Methods, April 2016

7 Metabolomics Workflow Data Acquisition Multivariate Statistical Analysis Biomarkers identification Validation Collection & Storage Sample Preparation Experimental design 4rth Workshop on Holistic Analytical Methods, April 2016

8 Metabolomics Workflow Data Acquisition Multivariate Statistical Analysis Biomarkers identification Validation Collection & Storage Sample Preparation Experimental design 4rth Workshop on Holistic Analytical Methods, April 2016

9 Experimental Design KEY ELEMENTS OF THE SCIENTIFIC METHOD In the absence of a proper design it is essentially impossible to distinguish biological variation from technical variation. When these two sources of variation are confounded, there is no way of knowing which source is driving the observed results 4rth Workshop on Holistic Analytical Methods, April 2016

10 Experimental Design Avoid the fridge temptation 4rth Workshop on Holistic Analytical Methods, April 2016

11 Experimental Design Avoid the fridge temptation 4rth Workshop on Holistic Analytical Methods, April 2016

12 Metabolomics Workflow Data Acquisition Multivariate Statistical Analysis Biomarkers identification Validation Collection & Storage Experimental design Sample Preparation 4rth Workshop on Holistic Analytical Methods, April 2016

13 Collection & Storage A single person should collect/harvest the initial material store at -80 C stop enzymatic processes transfer in dry ice 4rth Workshop on Holistic Analytical Methods, April 2016

14 Metabolomics Workflow Data Acquisition Multivariate Statistical Analysis Biomarkers identification Validation Sample Preparation Collection & Storage Experimental design 4rth Workshop on Holistic Analytical Methods, April 2016

15 NMR in Metabolomics 4rth Workshop on Holistic Analytical Methods, April 2016

16 Metabolomics sample preparation metabolites found within: Biofluid Urine, Blood (serum, plasma), Saliva, Breath, CSF, Amniotic, etc Cell Cytosolic metabolites Released metabolites Tissue, Organ Mammals: liver, kidney, heart, tumour, muscle, brain, fat tissue, etc. Plants Leaves, roots, fruits, etc. Whole organism Insects (Flies, etc) Marine organisms Worms, etc. 4rth Workshop on Holistic Analytical Methods, April 2016

17 Metabolomics sample preparation metabolites found within: Biofluid Cell Urine, Blood (serum, plasma), Saliva, Breath, CSF, Amniotic, etc Cytosolic metabolites Released metabolites Non- invasive Tissue, Organ Mammals: liver, kidney, heart, tumour, muscle, brain, fat tissue, etc. Plants Leaves, roots, fruits, etc. Whole organism Insects (Flies, etc) Marine organisms Worms, etc. 4rth Workshop on Holistic Analytical Methods, April 2016

18 Metabolomics sample preparation Standardized Protocols ph adjustment: phosphate buffer ph 7.4; NaN 3 to eliminate bacterial growth axis calibration: Internal Standard (TSP, DSS; 0.01%) & QUALITY CONTROL field lock: # Blood samples deuterated solvent (10% D 2 O in Urine; 50% in plasma) Centrifuge (+4 C) and transfer 550 μl in NMR tube Plasma samples: gentle handling, no vortex, no centrifuge, remove protein particles with a needle 4rth Workshop on Holistic Analytical Methods, April 2016

19 Metabolomics sample preparation metabolites found within: Biofluid Urine, Blood (serum, plasma), Saliva, Breath, CSF, Amniotic, etc Cell Cytosolic metabolites Released metabolites Tissue, Organ Mammals: liver, kidney, heart, tumour, muscle, brain Plants Leaves, roots, fruits, etc. tedious Whole organism Insects (Flies, etc) Marine organisms Worms, etc. 4rth Workshop on Holistic Analytical Methods, April 2016

20 Metabolomics sample preparation Frozen sample Standardized Protocols Homogenization in liquid N 2 (manually), high throughput tissue homogenizer with beads weight (<100 mg) and store at -80 C till extraction homogenizer probes (in extraction solvent; 1 st step extract.) Extraction 3 solvent system MeOH CHCl 3 dh 2 O (-20 C) WORK ON ICE 2 phase system; collection; repeat Lyophilisation; store at -80 C Reconstitution axis calibration (Internal Standard; TSP, DSS; 0.01%) field lock (100% D 2 O buffered; ph 7.4; NaN 3 ) centrifuge and transfer 550 μl in NMR tube 4rth Workshop on Holistic Analytical Methods, April 2016

21 Metabolomics sample preparation Frozen sample Standardized Protocols Homogenization in liquid N 2 (manually), high throughput tissue homogenizer with beads weight (<100 mg) and store at -80 C till extraction homogenizer probes (in extraction solvent; 1 st step extract.) Extraction CHECK SOLVENTS BEFORE EXTRACTION 3 solvent system MeOH CHCl 3 dh 2 O (-20 C) WORK INCLUDE ON ICE BLANK SAMPLES 2 phase system; collection; repeat Lyophilisation; store at -80 C Reconstitution axis calibration (Internal Standard; TSP, DSS; 0.01%) CHECK SOLVENTS BEFORE RECONSTITUTION field lock (100% D 2 O buffered; ph 7.4; NaN 3 ) centrifuge and transfer 550 μl into NMR tube INCLUDE BLANK SAMPLES (reconstitution buffer) 4rth Workshop on Holistic Analytical Methods, April 2016

22 Metabolomics sample preparation Solvent check before NMR sample preparation D 2 O + 10% Urine Buffer D 2 O 4rth Workshop on Holistic Analytical Methods, April D. Benaki

23 Metabolomics sample preparation Solvent check before extraction MeOH lyophilized, reconstituted in 100% CD 3 OD (new bottle)

24 NMR Reproducibility PC3 cell line

25 NMR Reproducibility PC3 cell line DMA Glutathione (GSH) Ace Glu

26 NMR Reproducibility PC3 cell line from 2 different persons Control 2 DMA Control 1 Glutathione (GSH) Ace Glu

27 NMR Reproducibility Urine samples recorded in 600 MHz Bruker AVANCE III, Athens Buffer A (PBS ph=7.4): original from Bruker Buffer B (PBS ph=7.4): local preparation Urine NMR samples Sample 1-5 person E buffer A Sample 1-5 person E buffer B Sample 1-5 person S buffer A Sample 1-5 person S buffer B 1EA 5EA 1EB 5EB 1SA 5SA 1SB 5SB 4rth Workshop on Holistic Analytical Methods, April 2016

28 NMR Reproducibility 4rth Workshop on Holistic Analytical Methods, April 2016

29 NMR Reproducibility PCA 4rth Workshop on Holistic Analytical Methods, April 2016

30 NMR Reproducibility 4rth Workshop on Holistic Analytical Methods, April 2016

31 NMR Reproducibility accepted sample preparation 4rth Workshop on Holistic Analytical Methods, April 2016

32 Basic analytical issues - NMR Run tube and buffer blanks Use the same tube type for a project Take care = consistent sample preparation Sample presentation give the spectrometer a chance!

33 Metabolomics Workflow Sample Preparation Data Acquisition Multivariate Statistical Analysis Biomarkers Identification Validation Collection & Storage Experimental Design 4rth Workshop on Holistic Analytical Methods, April 2016

34 Nuclear Magnetic Resonance o Reproducible results on concentration critical / crucial parameters:

35 Nuclear Magnetic Resonance o Reproducible results on concentration critical / crucial parameters: Acquisition relaxation delay,

36 Nuclear Magnetic Resonance o Reproducible results on concentration critical / crucial parameters: Acquisition relaxation delay, pulse width,

37 Nuclear Magnetic Resonance o Reproducible results on concentration critical / crucial parameters: Acquisition relaxation delay, pulse width, acquisition time,

38 Nuclear Magnetic Resonance o Reproducible results on concentration critical / crucial parameters: Acquisition relaxation delay, pulse width, acquisition time, spectrum analysis, number of scans, receiver gain

39 Nuclear Magnetic Resonance o Reproducible results on concentration critical / crucial parameters: Acquisition RELAXATION DELAY, pulse width, acquisition time, spectrum analysis, number of scans, receiver gain

40 Acquisition Parameters 4rth Workshop on Holistic Analytical Methods, April 2016

41 Acquisition Parameters Processing Parameters 4rth Workshop on Holistic Analytical Methods, April 2016

42 NMR Standard Operation Procedure makes science easier Water suppression test: 2m M Sucrose with 0.5mM DSS, 2 mm NaN 3 in 10% D 2 O and 90% H 2 O Temperature long term stability Temperature calibration: CD 3 OD 99.8% 1 H resolution test: 1% CH Cl 3 in Acetone-d6 Sensitivity test: 0.1% Ethylbenzene in CDCl 3 Field drift (CDCL 3 ) 4rth Workshop on Holistic Analytical Methods, April 2016

43 NMR Standard Operation Procedure makes science easier Sensitivity test: 0.1% Ethylbenzene in CDCl 3 Good sensitivity can be obtained with good resolution and good lineshape only. The splitting between the two central lines of the methylene quartet shoulg go lower than 15% (using a lb of 1 Hz) Automated S/N calculation is performed using sinocal

44 NMR Standard Operation Procedure 1 H resolution test: 1% CDCl 3 in Acetone-d6 makes science easier The AU hwcal program determines the resolution at the half height of the chloroform line.

45 NMR Standard Operation Procedure makes science easier 2 mm Sucrose with 0.5mM DSS, 2mM NaN 3 in 10% D 2 O and 90% H 2 O Water line width at 50% and 10% of DSS, S/N, and resolution calculations by typing suppcal

46 NMR Standard Operation Procedure makes science easier 2 mm Sucrose with 0.5mM DSS, 2mM NaN 3 in 10% D 2 O and 90% H 2 O Water line width at 50% and 10% of DSS, S/N, and resolution calculations by typing suppcal

47 NMR Standard Operation Procedure Temperature Calibration edte window 4rth Workshop on Holistic Analytical Methods, April 2016

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49 NMR Standard Operation Procedure Temperature Calibration Temperature : 300 K 310 K Biological Samples Plasma CD 3 OD, 99.8% (Bruker std) 1 H 1D; ns 1; ds 0; zg30 4rth Workshop on Holistic Analytical Methods, April 2016

50 Source: Bruker Instruments, Inc. VT-Calibration Manual 100% Methanol: T = D D (D is the shift difference (ppm) between CH 3 & OH peaks) Δ

51 Δ

52 Δ

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54 WEEKLY

55 Instrument Performance Long Term Stability Test CD 3 OD, 99.8% (Bruker std) 1 H 1D; ns 1; ds 0; zg2d 4rth Workshop on Holistic Analytical Methods, April 2016

56 Profiling Optimization temperature stabilization unit robotic sample changer of 60 sample positions (B-ACS 60) and supporting automation software O1 optimization TSP line width Pulse calibration Shimming (rsh; tsg) Lock Tuning and matching Temperature equilibration; 5 min 1 st sample transfer

57 4rth Workshop on Holistic Analytical Methods, April 2016

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59 manual tuning-matching optimization 4rth Workshop on Holistic Analytical Methods, April 2016

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71 noesygppr1d.comp

72 noesygppr1d.comp

73 jresgpprqf

74 jresgpprqf

75 jresgpprqf

76 noesygppr1d.comp

77 noesygppr1d.comp

78 jresgpprqf

79 jresgpprqf

80 Metabolic Profiling Automation

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88 Resulting 1D NOESY spectra of urine Calibrated to TSP signal at δ 0.0 ppm Phase and Baseline corrected

89 Assignment process 2D spectra Literature and web databases (HMDB, BMRB) Chenomx 600MHz 1 H- 13 C DEPT135-HSQC 600MHz 2D TOCSY

90 Metabolomics Workflow Sample Preparation Data Acquisition Multivariate Statistical analysis Biomarkers identification Validation Collection & storage Experimental design

91 Batch Effects Cladobotryum genus species

92 Batch Effects Cladobotryum genus species

93 Batch Effects Cladobotryum genus species

94 Batch Effects Cladobotryum genus species

95 Batch Effects Cladobotryum genus species

96 t[2] Batch Effects Cladobotryum genus species bt02ti_set1&2tries.m20 (PCA-X) t[comp. 1]/t[Comp. 2] Colored according to Obs ID (species) C. apic C. dend C. fungi C. myco C. rubr C. rubr-t g C. tenu C. vari C. verti C. myco C. myco C. verti C. myco C. C. verti verti C. verti C. myco C. C. verti C. myco verti C. verti C. C. C. verti dend dend C. myco C. dend C. C. dend C. apic C. myco dend C. dend C. dend C. fungi C. apic C. dend C. rubr-t C. myco C. dend C. myco C. rubr-t C. C. myco dend C. rubr C. rubr-t C. C. fungi fungi C. rubr-t C. rubr-t C. rubr C. vari C. rubr-t C. vari C. myco C. rubr-t C. vari C. tenu C. dend C. dend C. dend C. rubr C. C. rubr-t tenu C. tenu C. dend C. dend C. vari C. rubr-t C. rubr-t C. vari C. rubr-t C. rubr-t C. rubr-t C. C. C. C. vari dend vari C. C. varic. vari vari vari C. rubr-t C. rubr-t C. rubr-t C. vari C. vari C. rubr-t C. rubr-t C. vari C. C. vari vari C. vari C. C. vari vari C. vari C. vari C. vari t[1] R2X[1] = R2X[2] = Ellipse: Hotelling T2 (0.95) SIMCA-P /4/2016 7:51:36 ìì

97 Batch Effects

98 Peak Alignment

99 Peak Alignment CSF, MS patients

100 Peak Alignment CSF, MS patients

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102 -0.3 AA-buta005wo273.M8 (PLS-DA): Validate Model $M8.DA1 Intercepts: R2=(0.0, 0.186), Q2=(0.0, ) R2 Q permutations 3 components SIMCA-P /3/ :15:09 ðì

103 Heterocovariance based metabolomics a powerful tool accelerating bioactive natural products identification Morus alba case

104 Heterocovariance based metabolomics Tyrosinase Inhibition Activity 1 H NMR profile of Morus alba fractions mg/ml 2 mg/ml 30 fractions from Morus alba by CPC

105 Heterocovariance based metabolomics

106 1 H NMR spectrum of purified 2,4,3 trihydroxydihydrostilbene SHY plot correlation of mass at m/z at RT = 7.89 min and NMR STOCSY NMR peak correlation HETCA covariance of biological activity with corresponding NMR data

107 1 H NMR spectrum of purified oxyresveratrol SHY plot correlation of mass at m/z at RT = 7.70 min and NMR STOCSY NMR peak correlation HETCA covariance of biological activity with corresponding NMR data

108 Heterocovariance based metabolomics oxyresveratrol 2,4,3 -trihydroxydihydrostilbene <1% total extract

109 Heterocovariance based metabolomics as a powerful tool accelerating bioactive natural product identification Nektarios Aligiannis, Maria Halabalaki, Eliza Chaita, Eirini Kouloura, Aikaterini Argyropoulou, Dimitra Benaki, Eleftherios Kalpoutzakis, Apostolis Angelis, Konstantina Stathopoulou, Stavroula Antoniou, Maria Sani, Oliver Werz, Verena Krauth, Birk Schütz, Hartmut Scha fer, Manfred Spraul, Emmanuel Mikros * Leandros A. Skaltsounis. School of Pharmacy University of Athens, Greece Bruker BioSpin, Rheinstetten, Germany Dept. of Pharm. Med. Chemistry, Inst. of Pharmacy, Friedrich-Schiller-University Jena, Germany

110 THANK YOU for your attention