metabolics Flashcards
what is metabolics?
what is happening now, it allows the study and characterisation phenotype within living organism
what elements are metabolites made from?
H, C, N, O and S
what is MS2
if know elements and their weights and atomic mass it is a direct refection of make up
which AA are associated with detoxification reactions
cysteine and methionine- sulphur containing AA
what are metabolites
anything that can be metabolised
what is the citric acid cycle
where you make ATP, ADP and AMP implication in cancer and aging
what does it mean if see genomic expression but no metabolimics?
the event isn’t stable enough
what is primary metabolites
biomolecules that are synthesised by the cell because they are essential for growth and maianataince of a biological system
what are secondary metabolites
organic compounds produced by bacteria, fungi, or plants which aren’t directly involved in normal growth and mainatance of a biological system
what are examples of primary metabolism
amino acids, carbohydrate and alchohols and vitamins B2 and B12
what are examples of secondary metabolites
Phenolic, steroids, essential oils and alkaloid metabolites
what is 2HG
muatations in IDG result in the conversion of isocitrate to 2-HG instead of alpha ketoglutarate. This leads to altered methylation in cell and promotes tumour growth
how is tyoe 2 diabetes caused
icreased level of TAG which leads to saturation of acetyl-coA leading to desythestization
what is the Warburg effect?
cancer and tumourgenesis are due to tumour cells generating energy through the non oxidative breakdown of pyruvate.
why do plants have largest metabolome
because they can’t. move
why do plants have largest metabolome
because they can’t. move
what are all lipids made from
C, O, H double bond
what is metabolite target analysis s
quailitive and quantitive anaylsis of one, or several metabolites related to a specific matabolic reaction
what is metabolic foot printing
analysis of the metabolites screted or excreted by an organism; if the organism is growing in culture it would include its envioemnt
what is metabolic profiling
Identification and quantification of a selective number of predefined metabolites, which are related to a specific pathway
metabolic finger printing
global, high throughput, rapid analysis to provide sample classification. Also utilised as a screening tool to discriminate between samples from different biological status or orgin.
what are the main steps of untergetting metabolomics work flow
- experimental design
-sample prep
-sample anaylis by MS and NMR
-pre processing data analysis
-metabolite identification
-hypothesis
-ecperimental validation
what should you do in discovery work
identify a panel of biomarkers taht can refine disovery to validation
what is a spectroscopic technique
a non destructive technique taht uses radiowae laser so can profile samples at a decent enough reoliutio
what are the main spectroscopic techniques that are used?
NMR and FTIR
what is FTIR
it gives out a vibartional signature but is sensitive to water so needs to be processed properly
what is the gold standard in metabolic?
LC/MS
Which mass analyses are good for discovery work
QTOF and orbi trap
what is the advantages if quadrouple mass analyser
Robust, simple, low maintaince, relatively inexpensive
what are the disadvantages of the quadruple mass analysers
low mass resolution, low sensitivity, slow scan speed
what is the disadvantage of having a too long. TOF
the ions can diffuse
how does orbital work
the heavier the ion the longer it takes to orbit, ms2 analyses the results
what is the simplist mass spec analyser
quadropole
how does quadropole Give you the mass
the ratio of voltage is directly proptional to the mass
what is MS3 used for
when you know the fragments after further smashing up fragments after using low res mass spec
how do we know that the sample is fragmenting in MS2?
precursor intensity decreases with higher eV as more % is converted into fragments
what does MS1 do
quick ID based on hi res ion mass measurement
what does MS2 do
structural fragments for confirmation
what does electro spray ionisation do
converts liquid samples to gas phase ions
what is the mobile phase in GC
a coil
what is the mobile phase in LC
pac column
how does chromotogrpahy work
through plate theory and van deemter
what does the van deemter equation state
the plates height can be decreased or enlarged by temp flow
what is the most basic type of LC
TLC
When would you use GC
if you sample was a gas, anything with a smell
how does gas chormotgrpahy work
uses volatility go the gases. The most volatile are eluted quicker. It is then fragmented by election impact
what is the major cost of gas chromoatgrpahy
electricity and gas
what would not be detected in NMR
low abundance metabolites
which chromatography is the gold standard
LC
which types of chromatography would you use to separate complex liquid mixtures
PR, HILIC and lipidomics
what analysis is required after LC
DDA or DIA
What are the disadvantages of LC
- easily broken
-long downtime
-high operational skill needed
-sophisticated downstream analysis needed
what makes the high pressure in LC
solvent, column, flow rate and gradient composition
what would you use GC for
sweat analysis, breath analysis, pesticides
what disadvantage of GC
low coverage can only look at low molecular weigh metabolites
what are the 3 different phases of LC metabolimics
reverse pahse- non polar. helix phase- polar and ion chomrotgarphy- sugar
what are the advantages and disadvantages of polarity switching
reduced the time but reduces quality of data as one mode will be surpassed and other will be over expressed
why is plastic a contimanation an issue and how is it over come
use plastic tools, have to use low bais to stop plastic leaching from the solvent. Would show as a bleed across the results. At proeitn would see much less as would be absorbed
what are QCs
10% of samples go into a master pool. It is themes reproducible data point. Inject aliquots of QC at regular intervals to demonstrate stability and quality of aquisition
what is unsupervised anaylisis
clustering algortm which doesn’t now you labelling or class structures. Allows you to see outliers
what is the most commonly used data analysis in unsupervised
pca, which removes noise from data set. Anything after PC1,2, and 3 is noise
