week 5 part 1 Flashcards
Define metabolomics
systemic identification and quantification of the small molecule metabolic products (the metabolome of a biological system(cell, tissue, organ, biological fluid or organism) at a specific point in time
What are metabolites?
small (<1000 atomic mass unit (amu x10-24g) chemical compounds (<1000 atomic mass unit
Define metabolism
Ensemble of chemical transformation carried out in the living tissue
What is netabolomics essentially extension of?
proteomics to the activities of expressed enzymes
Define metabonomics
subset of metabolomics involving the quantitative measurement of the multi-parametric metabolic responses of living systems to pathophysiological stimuli or genetic modifications
What are metabolites seen to be?
Most revealing markers of disease or chronic expsoure to toxins from the environment and of the effect of drugs
What are metabonomics a subset of ?
Investigation of the absorption, distribution, metabolism and excretion (ADME) OF DRUGS
What does metabonomics include?
not only intracellular molecules but also the components of extracellular biofluids
What does metabonomics deal with?
A diverse set of metabolites even more varied than proteins
Why is metabolomics not a new idea?
- Ancient physicians tasted patient’s urine for sweetness to test for diabetes
- Urine charts were for diagnosis in middle ages
- Blood lactate is a diagnostic and prognostic tool for newborns with perinatal asphyxia
What is used to measure the degree of lactic acidosis?
- lactate and not base deficit
What did the study examine?
The relation between blood gas measurements and outcome following perinatal asphyxia (pre-term infants)
What can urine lactate be?
Novel biomarker of lactate production capacity but reliability has been unsatisfactory so far
What can metabolomics provide?
- unbiased approach to assess a process without prior assumption
What are metabolites?
- end products of all processes occurring in cells, including gene, transcription and protein function
- provide a snapshot of biological status
what causes substantial changes in metabolites levels?
- subtle changes in gene, transcripts and protein
What is used to analyse blood metabolites?
NMR spectroscopy
What is example of endogenous metabolite?
normal cellular function
What is example of Exogenous metabolites?
- Diet
2. Gut flora
how many known metabolites are there?
- 200,000
How are compounds identified?
comparison with known standards within library (National institute of standards and Technology)
How long does it take a laboratory to identify one metabolite
A day by looking at 500 spectra per day
What can recent algorithms do?
Sift through 1000x times more
What is mass spectrometer?
Instrument which can measure the masses and relative concentrations of atoms and molecules
What is GC-MS?
- separate compounds using gas chromatography
- Detection using mass spectrometry
- Separation by GC based on volatility and polarity
What is LC-MS?
- separate compounds using liquid chromatography
- Detection using mass spectrometry
- can use higher molecular weight compounds than GC
What is LC-MS/MS?
- liquid chromatography tandem-mass spectrometry
- the first MS analyses the precursor ion
- The precursor is fragmented and is analysed with a 2nd MS
What is advantage of MS/MS?
- increases sensitivity
2. more structural information of the analytes
What does LC/MS provide?
- further selectivity
- unbiased detection
- information about the structures of the separated compounds
How are metabolites introduced into MS?
- electron spray ionisation (ESI)
2. enter as charged molecules that are transported in an electrical field between end of columns and entrance of MS
How can ionisation occur?
- Deprotonation (ESI-)
- Protonation (ESI+)
- Results in single or multiple ions
- structural information is obtained by collision induced decomposition
What is mass spectrometry?
- intrinsically a highly sensitive method
2. powerful tool for detection, quantification and structure elucidation of metabolites
What is the most popular approach for metabolomics application?
- LC-MS
What does GC-MS achieve?
a better metabolite seperation and generally avoids ion suppression
What are steps to mass spectrometer?
- Ionisation
- Acceleration
- Deflection
- Detection
What is Ionisation?
- the atom is ionised by knocking one or more electrons off to give a positive ion
- mass spectrometers always works with positive ions
What is acceleration?
- the ions are accelerated so that they all have the same kinetic energy
What is deflection?
- The ions are then deflected by a magnetic field according to their masses
- the lighter they are, the more they are deflected
What is Detection?
- The beam of ions passing though the machine is detected electrically
What is NMR?
- less sensitive than MS
- Highly quantitative
- quantify metabolites in intact tissue and tissue extracts
What are the characteristics of NMR?
- rapid
- non-destructive
- Requires little sample preparation
What is NMR good for?
Determining structure of unknown compounds
What can certain nuclei(e.g. Proton (1H)) give?
distinct signal if excited by specific radio frequencies