Metabolomics 2 Flashcards
Metabolomics Technologies
NMR
- mg/mL - µg/mL
- organic acids
- lipids
- amino acids
- nucleotides
Mass Spectrometry (LC/MS, GC/MS, CE/MS)
- µg/mL
- ng/mL
- nucleotides
- steroids
- eicosanoids
- neurotransmitters
CUSTOM
- ng/mL
- pg/mL
- neurotransmitters
- peptides
- trace elements
Mass Spectrometry
-> Advantages and Disadvantages
Advantages
* Highly sensitive (5 nM)
* Structural elucidation of unknown compounds (accurate mass, fragments)
* Large number of metabolites detected and quantified
* Automation requires massive quality control
* HT, 100 samples / day
Disadvantages
* Many different variants, data from different source not comparable
* Lack of standardisation
* Not as robust as NMR
* High level of QC needed for quantification
NMR
-> Advantages and Disadvantages
Advantages
* Simple sample preparation
* Robust automation, HT (50 samples a day)
* Highly reproducible
* Truly quantitative (reference method for NIST standards)
* Can detect any metabolites above 5 μM
* Structure elucidation of unknown compounds
* Lipids: Total trigycerides, cholesterol, phosphatidylcholines, sphingomyelins
* Inblood: HDL, LDL, VLDL subclasses
Disadvantages
* Limited sensitivity (5μM), limited number of metabolites (50-200)
* Complex data deconvolution
Technology and sensitivity
-> NMR
Metabolites
1. Small water soluble molecules
2. Lipoproteins
Sample type
Biofluids (urine, blood, CSF), tissue extracts, plant extracts
Sample volume
5 mm: 600 µL
3 mm: 180 µL
1.7 mm: 35 µL
Run-time
10-30 min
Detection limit
5 µM
No. Metabolites
Blood: 40, 100
lipoproteins, glycoproteins
Urine: ca 100
Technology and sensitivity
-> GC-MS
Metabolites
- mainly water-soluble molecules
Sample Type
- biofluids, tissue extracts, plant extracts, bacterial, food
Sample volume
- 30-50 µL
Run-time
- 30-60 min
Detection limit
- 100 nM
No. metabolites
- 150 - 200 from any matrix requires sample prep
Technology and sensitivity
-> LC-MS
Metabolites
- biofluids, cell extracts
Sample Type
- mainly biofluids
Sample volume
- 10 µL
Run-time
- 1-2 h for 96 samples
Detection limit
- 5 nM
No. metabolites
- 2000-3000
- 300-500 quantifiable
- Lipidomics: >3000
-> Software: XCMS Online
Literature-assisted identification
MS data -> feature annotation -> MS filtering -> AI-NLP search -> MS2 identification of short list -> cognitive analysis of metabolomics
Biological pathway disease activity
MS data -> metabolites -> pathways -> AI gene search -> role in disease -> cognitive analysis of metabolomics
Metabolite prioritization
MS data -> dysregulated features -> AI network search -> categorize knowns -> select unknowns -> test unknowns
Metabolome-level Disease comparison
AI search diseases and compounds -> metabolites -> compare diseases 1 and 2 -> not shared = biomarker -> shared = drug target -> MS validation
Metabolome-level disease comparisons for drug repurposing
Iterative filtering of literature-based chemical-disease similarity network for CKD and AMI. Red dot: Vitamine D.
Drugs predicted for the diseases, with vitamin D as the top-ranked drug compound for CKD and ranked 70th for AMI.
Categorization of shared metabolites by types of drug, endogenous metabolites, hormones and well-known actors.
MetaboAnalyst
A unified and flexible workflow that enables end-to-end analysis of NMR and LC-MS metabolomics data.
Raw LC/MS data (LC-MS spectra) -> data processing (peak picking), (peak annotation) -> data processing (data cleaning) -> data analysis (statistical analysis) (knowledge based analysis)
What does NMR observe?
- The NMR phenomenon is based of a property of the nucleus of some atoms termed the spin
- Spins behave somehow like positively charged particles rotating in a magnetic field
- The rotation in the magnetic field induces a magnetic moment, as a consequence particles align in the magnetic field
- The two possible orientations of rotation lead to two energy states related to alignment with or against the outside magnetic field
- Most NMR applications use spin-1/2 nuclei with exactly two energy states
1H -> Net spin: 1/2 -> gamma/MHzT-1 42.58 -> Abundance/% 99.98 %
15N -> Net spin: 1/2 -> gamma/MHzT-1 4.31 -> Abundance/% 0.37
13C -> Net spin: 1/2 -> gamma/MHzT-1 10.71 -> Abundance/% 1.108
The NMR phenomenon
Spinning charge -> a magnetic field.
-> magnetic moment (μ).
External magnetic field (B0)
-> two spin state, +1/2 and -1/2.
+1/2 state, lower energy, aligned with the external field
-1/2 state, higher energy, opposed to the external field.
Energy Difference between spin states is dependent on the external magnetic field strength.
The Resonance Condition
The energy of the spin depends on its state: E = μzB0 = γħmIB0
Two possible states for I = 1/2 nuclei: E1/2 = 1⁄2μħB0. E1/2 = -1⁄2γħB0
Energy difference: ΔE = γħB0
Resonance condition: ω0 =γB0
Larmor Frequency ω0 ⇔ precession frequency of the spins about the axis of the static magnetic Field B0
classical:
B0 causes a torque on the magnetic moment -> Larmor precession
The RF probe
-> contains several coils for different nuclei
-> probe must be tuned like a radio by adjusting the eigenfrequency in a capacitor parallel to the coil
Phase sensitive detection
The sign of the signal can only be determined if a phase sensitive FID is available!
NMR spectra: Relaxation
Longitudinal relaxation T1 = spin lattice relaxation = relaxation of z-magnetisation
Transverse relaxation T2 = spin spin relaxation = relaxation of xy-magnetisation proportional to line width
S = ∑ exp(iωit) exp(t/T2)
T2 = signal decay
The NMR Spectrum: Chemical Shifts
The magnetic field induces magnetisations in the electron cloud surrounding the nucleus
=> Shielding, i.e. the magnetic field at the site of the nucleus is influenced by the electron density surrounding the nucleus
=> a higher magnetic field is required to meet the resonance condition
Blocal=B0(1-s)
s = shielding constant
The ppm scale = 𝛿 = (𝜈 − 𝜈ref)/v0
Field independent
=> same values of δ for spectrometers of different field strengths
The NMR Spectrum: Chemical Shifts
Depending on their chemical environment, protons in a molecule are shielded by different amounts.
Sauerstoff an ein Kohlenstoff gebunden = more shielded, absorb at a higher field
Sauerstoff an ein Wasserstoff gebunden = less shielded, absorbs at a lower field
Chemical Shift reference
TMS = Tetramethylsilane
TMSP = Trimethylsilylpropanoic acid
- TMS or TMSP is added to the sample
- Since silicon is less electronegative than carbon, TMS protons are highly shielded. Signal defined as zero.
- Organic protons absorb downfield (to the left) of the TMS signal.
The NMR Spectrum: Chemical Shifts
- NMR can distinguish spin-1/2 atoms in molecules based on their resonance frequency
- NMR spectroscopists called this the chemical shift
- The chemical shift changes as a consequence of the surrounding electron density which is directly related
to the chemicals bonds - The chemical shift is therefore highly sensitive to neighboring atoms (shiedling and deshielding) and to the
type of chemical bonds (single, double, triple bond, aromatic structures).
The NMR Spectrum: Signal Intensity
- The signal intensity is proportional to the number of spins
- I.e.: Proton spectrum of CH3 has 3 x intensity of CH
The NMR Spectrum: Spin-spin coupling
Nucleus A causes a weak magnetic polarisation of the bonding electrons, transmitted through overlapping orbitals to nucleus X.
=> local magnetic field for X varies
Two equally probable states => lines have same intensity
Energy of coupling is field independent => coupling constant is field independent
