Metabolomics Q and A Flashcards
Which molecules can be seen?
- Biochemicals (Metabolites)
-> organic or inorganic
What are typical applications of metabolomics?
-> investigate several human diseases, improve their diagnosis and prevention -> design better therapeutic strategies/”personalized metabolic phenotyping”
- nutritional analysis
- clinical blood/urine analysis
- cholesterol testing
- drug safety
Why is metabolomics analytically more complex than proteomics or genomics?
- no simple building blocks
- huge number of chemicals possible
If you compare genomics, proteomics and metabolomics:
a) Which has the greatest variety of compounds?
b) For which do we have the best analytical coverage, for which the worst?
A) metabolomics 8x10^5
B) metabolomics -> proteomics -> genomics
Which factors influence the human metabolome?
-> Nährstoffangebot, Effekte von Wirksubstanzen, Umweltfaktoren
Why and how is metabolomics used to assess food quality? Which information can be gained?
Zusammensetzung, Reinheit, Herkunft
Describe a metabolomics workflow
1) samples
2) record chemical data
3) process dataset
4) analyse/model data/identity
5) interpret the result
Describe the difference between targeted and untargeted metabolomics
UNTARGETED ANALYSIS
- no Prior knowledge of metabolites of interest
- finger printing (binned spectra) or profiling (concentrations of all quantifiable metabolites)
- statistical approach
and then multivariate analysis or univariate analysis
TARGETED ANALYSIS
- Prior knowledge of metabolites of interest
- statistical approach
How is the resolving power in MS defined?
delta M/M
-> the ability of an instrument or measurement procedure to distinguish between two peaks at m/z values differing by a small amount and expressed at the peak width in mass units
Name two common ionization methods for MS
- Chemical ionization (CI)
- electron impact (EI)
- electron spray ionization (ESI)
- matrix-assisted laser desorption ionization (MALDI)
Why does GC-MS require derivartization?
The compounds have first to be in the gaseous phase in order to get analyzed
What are the detection limits for GC-MS, LC-MS and NMR
GC-MS: LOD = 100 nM - organic and inorganic classes
LC-MS: LOD = 5 nM - organic and inorganic classes
NMR: LOD = 5 µM -quantitativ-organic classes
Which substances are seen in NMR spectra of blood serum that are strictly not metabolites?
Glycoproteins and lipoproteins
What is the difference between multivariate and univariate statistics?
multivariate = mehrere statistische Variablen zugleich
univariate = einzelne Variablen ohne sich um eventuell vorhandene Einflussgrößen oder Zusammenhänge zu kümmern
How can one get a homonuclear decoupled 1D?
J-RES: Tilt, symmetrise, projection
Which extra information can be gained from J-RES spectra
- couplings in 2D mode, decoupling after tilt and projection
What is the effect of CPMG?
Get rid of broad lines from proteins, T2 filter
NMR spectroscopy -> advantages and disadvantages
- solution state (plasma, urine, extracts)
- MAS (tissue extracts, tissue)
- MRI in vivo chemical shift imaging
- relatively robust
ADVANTAGES
-simple sample preparation
- highly reproducible
- quantitative
- can detect any metabolite above 5 µM
- structure elucidation of unknown compounds
- High throughout (50 samples per day)
DISADVANTAGES
- limited sensitivity (5 µM), limited number of metabolites (50 - 200)
- complex data deconvolution
Mass spectrometry (MS)
- GC-MS
- LC-MS
- more analytically< sensitive (5 nM)
- potentially truly global
- problems with ionization
- standardisation is challenging
ADVANTAGES
- structural elucidation of unknown compounds (accurate mass fragments)
- large number of metabolites detected and quantified
- automation requires massive quality control
- high throughput - 100 samples per day
DISADVANTAGES
- many different variants, data from different sources not comparable
- not as robust as NMR
- high level of QC needed
Which 2D spectra can be used for metabolomics?
HSQC before all others; HSQC-TOCSY, HMBC
Why can lipoproteins be separated by HPLC?
Size exclusion
Which groups can be detected by NMR lipoprotein analysis
VLDL, LDL, HDL, with subfractions, IDL, cholesterol, triglycerides, phospholipids
Which other protein class shows up in NMR spectra of blood?
Glycoproteins
How are these complex signals of lipoproteins analysed?
- Complex line-shape fitting, different versions
P-values - understand qualitatively
The probability that the observed result was obtained by chance (i.e. the H0 is true) -> if the p-value is small, it suppress the observed result cannot be easily explained by chance
What information does a t-test give
used to compare the mean of two independent groups
What is STOCSY?
- statistical correlation spectroscopy (STOCSY)
- generates a pseudo 2D-NMR spectrum that displays the correlation among the intensities of the various peals across the whole sample
PLS vs PCA
PCA
- unsupervised method
- used for clustering
- shows similarities in variables
- maximizing the variance that is explained by the model
PLS
- supervised method
- used for classification
- shows discrimination between variables
- maximizing the covariance
1D Noesy
water suppression
CPMG
removal of large proteins
diffusion-edited spectra
removal of small molecules
J-resolved spectra (tilted and projection)
2D spectrum with couplings in 2nd dimension -> after processing: 1D without couplings
Diffusion-ordered spectroscopy (DOSY)
separation of diffusion coefficient
Derivatization
- goal: make the analyte more volatile (pre-column)
- in the case of amino acids derivatization replaces the OH, NH2 and SH functional groups with a non-polar moiety
-> Silylation is a very common derivatization
-> Trimethylsilylation is the most common approach which can be achieved using bromotrimethylsilane (TMBS) or chloretrimethylsilane (TMCS)
Massenspektrometrie: Rund um den Molekülpeak werden Satelliten beobachtet -> erklären Sie den Effekt
ISOTOPENVERTEILUNG
- 12C -> 13C
- 1H -> 2H
- 14N -> 15N
- 16O -> 17O
PCA
- finds the largest correlations between variables within a data set
- the PCA is equivalent to fitting an m-dimensional ellipsoid to the data, where the eigenvectors of the covariance matrix of the data set are the axes of the ellipsoid -> the eigenvalues represent the distribution of the variance among each of the eigenvectors
C*V = gamma * V
C = covariance matrix
V = corresponding eigenvector
Therapeutic Ratio
LD50/ED50
Therapeutic Index
LD25/ED75
LD10/ED80
Statine
- Cholesterinsenker
- Lipidsenker
Massenspektrometrie
Basispeak = höchster Peak -> zeigt die Häufigkeit des häufigsten Ions
-> wird auf 100 % gesetzt und alle anderen peaks werden darauf bezogen
Molekülpeak = Molekülion, das die höchste Masse des Spektrums hat -> zeigt die relative Molekülmasse der Verbindung
Isotopenpeak = zeigt die natürliche Häufigkeit der Isotope -> rechts vom Molekülpeak (die kleinen Peaks)
- Molekülion zerbricht bevorzugt an chemischen Bindungen mit funktionellen Gruppen
Non-competitive antagonists
- decrease Emax of the agonist
- the ED50 of the agonist is NOT changed
Competitive antagonist
- lead to a parallel shift in the dose-response curve of the agonist (ED50 will increase)
- the Emax of the agonist is NOT affected
