protocol for nmr based metabolomics. tips and...
TRANSCRIPT
![Page 1: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/1.jpg)
Seminar 5 Protocol For NMR Based Metabolomics. Tips And Tricks
Dr. Dimitra Benaki School of Pharmacy, Dept. Pharmaceutical Chemistry
![Page 2: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/2.jpg)
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
Systems Biology
![Page 3: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/3.jpg)
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
What will or may happen
happened
is happening
Systems Biology
![Page 4: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/4.jpg)
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
Microbial biotechnology Microbial improvement Fermentation Biotechnological compounds
Plant biotechnology Crop improvement Transgenic breeding Plant breeding Improve stress tolerance
Systems biology Dynamics in biological systems Explore metabolic networks
Enzyme discovery Discovery of biochemical pathways Link changes in metabolite levels to catalytic activity Improve catalytic efficiency of enzymes
Applications of Metabolomics
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 5: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/5.jpg)
GC-MS
Metabolomics’ Methods
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
• NMR • GC, • HPLC, • UPLC, • HPTLC • CE (capillary electrophoresis) Hyphenated • LC-MS, • GC-MS • GC-MS/MS • UHPLC-MS
![Page 6: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/6.jpg)
NMR in Metabolomics
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
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
![Page 7: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/7.jpg)
Collection & Storage
Sample Preparation
Data Acquisition
Multivariate Statistical Analysis
Biomarkers identification
Experimental design
Validation
Metabolomics Workflow
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 8: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/8.jpg)
Collection & Storage
Sample Preparation
Data Acquisition
Multivariate Statistical Analysis
Biomarkers identification
Experimental design
Validation
Metabolomics Workflow
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 9: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/9.jpg)
Experimental Design
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
KEY ELEMENTS OF THE SCIENTIFIC METHOD
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 10: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/10.jpg)
Experimental Design
Avoid the fridge temptation
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 11: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/11.jpg)
Experimental Design
Avoid the fridge temptation
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 12: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/12.jpg)
Collection & Storage
Sample Preparation
Data Acquisition
Multivariate Statistical Analysis
Biomarkers identification
Experimental design
Validation
Metabolomics Workflow
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 13: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/13.jpg)
Collection & Storage
A single person should collect/harvest the initial material
stop enzymatic processes
store at -80 °C
transfer in dry ice
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 14: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/14.jpg)
Collection & Storage
Sample Preparation
Data Acquisition
Multivariate Statistical Analysis
Biomarkers identification
Experimental design
Validation
Metabolomics Workflow
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 15: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/15.jpg)
NMR in Metabolomics
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 16: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/16.jpg)
Metabolomics sample preparation
metabolites found within:
• Cytosolic metabolites • Released metabolites
• Urine, • Blood (serum, plasma), • Saliva, • Breath, • CSF, • Amniotic, etc
• Mammals: liver, kidney, heart, tumour, muscle, brain, fat tissue, etc.
• Insects (Flies, etc) • Marine organisms • Worms, etc.
• Whole organism
• Tissue, Organ
• Biofluid
• Cell
• Plants • Leaves, roots, fruits, etc.
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 17: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/17.jpg)
Metabolomics sample preparation
metabolites found within:
• Cytosolic metabolites • Released metabolites
• Urine, • Blood (serum, plasma), • Saliva, • Breath, • CSF, • Amniotic, etc
• Mammals: liver, kidney, heart, tumour, muscle, brain, fat tissue, etc.
• Insects (Flies, etc) • Marine organisms • Worms, etc.
• Whole organism
• Tissue, Organ
• Biofluid
• Cell
• Plants • Leaves, roots, fruits, etc.
Non- invasive
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 18: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/18.jpg)
pH adjustment: phosphate buffer pH 7.4; NaN3 to eliminate bacterial growth
axis calibration: Internal Standard (TSP, DSS; 0.01%)
& “QUALITY CONTROL” # Blood samples
field lock: deuterated solvent (10% D2O 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
Metabolomics sample preparation
Standardized Protocols
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 19: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/19.jpg)
Metabolomics sample preparation
metabolites found within:
• Cytosolic metabolites
• Released metabolites
• Urine, • Blood (serum, plasma), • Saliva, • Breath, • CSF, • Amniotic, etc
• Mammals: liver, kidney, heart, tumour, muscle, brain
• Insects (Flies, etc) • Marine organisms • Worms, etc.
• Whole organism
• Tissue, Organ
• Biofluid
• Cell
• Plants • Leaves, roots, fruits, etc. “tedious”
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 20: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/20.jpg)
Metabolomics sample preparation
Frozen sample
Homogenization in liquid N2 (manually), high throughput tissue homogenizer
with beads weight (<100 mg) and store at -80 °C till extraction
homogenizer probes (in extraction solvent; 1st step extract.)
Extraction 3 solvent system MeOH – CHCl3 – dH2O (-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% D2O buffered; pH 7.4; NaN3)
centrifuge and transfer 550 μL in NMR tube
Standardized Protocols
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 21: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/21.jpg)
Metabolomics sample preparation
Frozen sample
Homogenization in liquid N2 (manually), high throughput tissue homogenizer
with beads weight (<100 mg) and store at -80 °C till extraction
homogenizer probes (in extraction solvent; 1st step extract.)
Extraction 3 solvent system MeOH – CHCl3 – dH2O (-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% D2O buffered; pH 7.4; NaN3)
centrifuge and transfer 550 μL into NMR tube
Standardized Protocols
INCLUDE BLANK SAMPLES (reconstitution buffer)
CHECK SOLVENTS BEFORE RECONSTITUTION
CHECK SOLVENTS BEFORE EXTRACTION
INCLUDE BLANK SAMPLES
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 22: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/22.jpg)
4rth Workshop on Holistic Analytical Methods , 17-19 April 20116 D. Benaki
Metabolomics sample preparation
Solvent check before NMR sample preparation
D2O + 10% Urine Buffer
D2O
![Page 23: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/23.jpg)
Solvent check before extraction
Metabolomics sample preparation
MeOH lyophilized , reconstituted in 100% CD3OD (new bottle)
![Page 24: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/24.jpg)
NMR Reproducibility PC3 cell line
![Page 25: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/25.jpg)
Glutathione (GSH)
Glu
DMA
Ace
NMR Reproducibility PC3 cell line
![Page 26: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/26.jpg)
Glutathione (GSH)
Glu
DMA
Ace
NMR Reproducibility
Control 1
Control 2
PC3 cell line from 2 different persons
![Page 27: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/27.jpg)
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 1EA – 5EA Sample 1-5 person E buffer B 1EB – 5EB Sample 1-5 person S buffer A 1SA – 5SA Sample 1-5 person S buffer B 1SB – 5SB
NMR Reproducibility
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 28: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/28.jpg)
NMR Reproducibility
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 29: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/29.jpg)
NMR Reproducibility PCA
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 30: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/30.jpg)
NMR Reproducibility
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 31: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/31.jpg)
NMR Reproducibility
accepted sample preparation
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 32: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/32.jpg)
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 !
✓ ✗
Basic analytical issues - NMR
![Page 33: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/33.jpg)
Collection & Storage
Sample Preparation
Data Acquisition
Multivariate Statistical Analysis
Biomarkers Identification
Experimental Design
Validation
Metabolomics Workflow
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 34: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/34.jpg)
Nuclear Magnetic Resonance
o Reproducible results on concentration critical / crucial parameters:
![Page 35: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/35.jpg)
Nuclear Magnetic Resonance
o Reproducible results on concentration critical / crucial parameters:
Acquisition
relaxation delay,
![Page 36: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/36.jpg)
Nuclear Magnetic Resonance
o Reproducible results on concentration critical / crucial parameters:
Acquisition relaxation delay,
pulse width,
![Page 37: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/37.jpg)
Nuclear Magnetic Resonance
o Reproducible results on concentration critical / crucial parameters:
Acquisition relaxation delay,
pulse width,
acquisition time,
![Page 38: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/38.jpg)
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…
![Page 39: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/39.jpg)
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…
![Page 40: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/40.jpg)
Acquisition Parameters
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 41: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/41.jpg)
Acquisition Parameters
Processing Parameters
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 42: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/42.jpg)
NMR Standard Operation Procedure
makes science easier
Field drift (CDCL3)
Sensitivity test: 0.1% Ethylbenzene in CDCl3
1H resolution test: 1% CH Cl3 in Acetone-d6
Water suppression test: 2m M Sucrose with 0.5mM DSS, 2 mM NaN3 in 10% D2O and 90% H2O
Temperature calibration: CD3OD 99.8%
Temperature long term stability
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 43: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/43.jpg)
NMR Standard Operation Procedure
Sensitivity test: 0.1% Ethylbenzene in CDCl3
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
makes science easier
![Page 44: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/44.jpg)
NMR Standard Operation Procedure
1H resolution test: 1% CDCl3 in Acetone-d6
The AU hwcal program determines the resolution at the half height of the chloroform line.
makes science easier
![Page 45: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/45.jpg)
NMR Standard Operation Procedure
2 mM Sucrose with 0.5mM DSS, 2mM NaN3 in 10% D2O and 90% H2O
makes science easier
Water line width at 50% and 10% of DSS, S/N, and resolution calculations by typing suppcal
![Page 46: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/46.jpg)
NMR Standard Operation Procedure
2 mM Sucrose with 0.5mM DSS, 2mM NaN3 in 10% D2O and 90% H2O
makes science easier
Water line width at 50% and 10% of DSS, S/N, and resolution calculations by typing suppcal
![Page 47: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/47.jpg)
Temperature Calibration edte window
NMR Standard Operation Procedure
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 48: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/48.jpg)
![Page 49: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/49.jpg)
Temperature Calibration Temperature : 300 K 310 K Biological Samples Plasma CD3OD, 99.8% (Bruker std) 1H 1D; ns 1; ds 0; zg30
NMR Standard Operation Procedure
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 50: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/50.jpg)
Source: Bruker Instruments, Inc. VT-Calibration Manual
100% Methanol: T = -23.832D2 -29.46D +403.0
(D is the shift difference (ppm) between CH3 & OH peaks)
Δ
![Page 51: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/51.jpg)
Δ
![Page 52: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/52.jpg)
Δ
![Page 53: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/53.jpg)
![Page 54: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/54.jpg)
WEEKLY
![Page 55: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/55.jpg)
Instrument Performance
Long Term Stability Test CD3OD, 99.8% (Bruker std) 1H 1D; ns 1; ds 0; zg2D
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 56: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/56.jpg)
Profiling Optimization
1st sample transfer
Temperature equilibration; 5 min
Tuning and matching
Pulse calibration
Lock
Shimming (rsh; tsg)
TSP line width
O1 optimization
temperature stabilization unit robotic sample changer of 60 sample positions (B-ACS 60)
and supporting automation software
![Page 57: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/57.jpg)
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 58: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/58.jpg)
![Page 59: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/59.jpg)
manual tuning-matching optimization
4rth Workshop on Holistic Analytical Methods , 17-19 April 2016
![Page 60: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/60.jpg)
![Page 61: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/61.jpg)
![Page 62: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/62.jpg)
![Page 63: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/63.jpg)
![Page 64: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/64.jpg)
![Page 65: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/65.jpg)
![Page 66: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/66.jpg)
![Page 67: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/67.jpg)
![Page 68: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/68.jpg)
![Page 69: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/69.jpg)
![Page 70: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/70.jpg)
![Page 71: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/71.jpg)
noesygppr1d.comp
![Page 72: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/72.jpg)
noesygppr1d.comp
![Page 73: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/73.jpg)
jresgpprqf
![Page 74: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/74.jpg)
jresgpprqf
![Page 75: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/75.jpg)
jresgpprqf
![Page 76: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/76.jpg)
noesygppr1d.comp
![Page 77: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/77.jpg)
noesygppr1d.comp
![Page 78: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/78.jpg)
jresgpprqf
![Page 79: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/79.jpg)
jresgpprqf
![Page 80: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/80.jpg)
Metabolic Profiling Automation
![Page 81: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/81.jpg)
![Page 82: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/82.jpg)
![Page 83: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/83.jpg)
![Page 84: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/84.jpg)
![Page 85: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/85.jpg)
![Page 86: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/86.jpg)
![Page 87: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/87.jpg)
![Page 88: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/88.jpg)
Calibrated to TSP
signal at δ 0.0 ppm
Phase and Baseline
corrected
Resulting 1D NOESY spectra of urine
![Page 89: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/89.jpg)
600MHz 1H-13C DEPT135-HSQC
600MHz 2D TOCSY
Assignment process 2D spectra Literature and web databases
(HMDB, BMRB) Chenomx
![Page 90: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/90.jpg)
Collection & storage
Sample Preparation
Data Acquisition
Multivariate Statistical analysis
Biomarkers identification
Experimental design
Validation
Metabolomics Workflow
![Page 91: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/91.jpg)
Cladobotryum genus species Batch Effects
![Page 92: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/92.jpg)
Cladobotryum genus species Batch Effects
![Page 93: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/93.jpg)
Cladobotryum genus species Batch Effects
![Page 94: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/94.jpg)
Cladobotryum genus species Batch Effects
![Page 95: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/95.jpg)
Cladobotryum genus species Batch Effects
![Page 96: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/96.jpg)
-20
-10
0
10
20
-30 -20 -10 0 10 20 30
t[2]
t[1]
bt02TI_set1&2tries.M20 (PCA-X)
t[Comp. 1]/t[Comp. 2]
Colored according to Obs ID (species)
R2X[1] = 0.426144 R2X[2] = 0.198183 Ellipse: Hotelling T2 (0.95)
C. apic C. dend C. fungi C. myco C. rubr
C. rubr-t g C. tenu C. vari C. verti
C. apicC. apic
C. apicC. dendC. dend
C. dend
C. dendC. dendC. dend
C. dend
C. dend
C. dend
C. dendC. dendC. dend
C. dend
C. dendC. dend C. dend
C. dend
C. dend
C. fungi
C. fungi
C. fungi
C. myco
C. myco
C. mycoC. myco
C. mycoC. myco
C. myco
C. myco
C. myco
C. myco
C. myco
C. myco
C. rubr
C. rubr
C. rubr
C. rubr-t
C. rubr-t C. rubr-t
C. rubr-t C. rubr-t
C. rubr-t
C. rubr-t
C. rubr-t C. rubr-t
C. rubr-t
C. rubr-t
C. rubr-t
C. rubr-t
C. rubr-t
C. rubr-t
C. rubr-t C. rubr-t
C. rubr-t
C. tenuC. tenuC. tenu
C. variC. vari
C. vari
C. variC. vari
C. vari
C. variC. variC. vari
C. vari
C. variC. vari
C. variC. vari
C. vari
C. vari
C. vari
C. vari
C. vari
C. variC. variC. vari
C. vari
C. vari
C. verti
C. verti
C. verti
C. vertiC. verti
C. verti
C. vertiC. verti
C. verti
SIMCA-P+ 11.5 - 11/4/2016 7:51:36 ìì
Cladobotryum genus species Batch Effects
![Page 97: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/97.jpg)
Batch Effects
![Page 98: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/98.jpg)
Peak Alignment
![Page 99: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/99.jpg)
Peak Alignment CSF, MS patients
![Page 100: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/100.jpg)
Peak Alignment CSF, MS patients
![Page 101: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/101.jpg)
![Page 102: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/102.jpg)
-0.3
-0.2
-0.1
-0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
AA-buta005wo273.M8 (PLS-DA): Validate Model
$M8.DA1 Intercepts: R2=(0.0, 0.186), Q2=(0.0, -0.303)
100 permutations 3 components
R2 Q2
SIMCA-P+ 11.5 - 31/3/2016 11:15:09 ðì
![Page 103: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/103.jpg)
Heterocovariance based metabolomics
a powerful tool accelerating bioactive natural products identification
Morus alba case
![Page 104: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/104.jpg)
30 fractions from Morus alba by CPC
Heterocovariance based metabolomics
Tyrosinase Inhibition Activity 1H NMR profile of Morus alba fractions 1-30
10 mg/mL
2 mg/mL
![Page 105: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/105.jpg)
Heterocovariance based metabolomics
![Page 106: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/106.jpg)
1H NMR spectrum of purified 2,4,3´-trihydroxydihydrostilbene
HETCA covariance of biological activity with corresponding NMR data
STOCSY NMR peak correlation
SHY plot correlation of mass at m/z 229.086
at RT = 7.89 min and NMR
![Page 107: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/107.jpg)
1H NMR spectrum of purified oxyresveratrol
HETCA covariance of biological activity with corresponding NMR data
STOCSY NMR peak correlation
SHY plot correlation of mass at m/z 243. 066
at RT = 7.70 min and NMR
![Page 108: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/108.jpg)
total extract
oxyresveratrol
2,4,3´-trihydroxydihydrostilbene <1%
Heterocovariance based metabolomics
![Page 109: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/109.jpg)
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 Schafer, 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
![Page 110: Protocol For NMR Based Metabolomics. Tips And Tricksbioanalysis.web.auth.gr/metabolomics/files/sunday/Seminar_Dimitra... · Standardized Protocols 4rth Workshop on Holistic Analytical](https://reader034.vdocument.in/reader034/viewer/2022042805/5f5ccd02db4400651d4fd50c/html5/thumbnails/110.jpg)
THANK YOU
for your attention