Metabolomics Flashcards
What is metabolomics?
- Metabolomics is the study of small molecules, commonly known as metabolites in a biological system (within biofluids, tissues, cell or organisms).
- Just as genomics is the study of DNA and genetic information within a cell, and transcriptomics is the study of RNA and differences in mRNA expression; metabolomics is the study of substrates and products of metabolism, which are influenced by both genetic and environmental factors.
- Metabolites and their concentrations directly reflect the underlying biochemical activity and state of cells/tissues. Thus metabolomics best represents the molecular phenotype
What is the metabolome?
Metabolome is consist of endogenous metabolites that are produced by an organism (such as amino acids, organic acids, nucleic acids, lipids amines, sugars) and exogenous metabolites that are not produced endogenously (such as drugs, xenobiotics, dietary metabolites, etc)
- The metabolome is the complete set of metabolites within a cell, tissue or biological sample at any given time point.
- Many reactions take place continuously within cells, so concentrations of metabolites are considered to be very dynamic, and may change rapidly from one time point to the next.
- The metabolome is inherently very dynamic. Metabolites are continuously absorbed, synthesised, degraded and interact with other molecules (both within biological systems, and with the environment) .
- Capturing the entire metabolome is impossible
What are lipoproteins?
What are the 4 main groups?
Lipoproteins are complex particles of lipids and proteins (apoproteins)
There are 4 main groups based on their size and density:
- chylomicrons (CM)
- very low density lipoproteins (VLDL),
- low density lipoproteins (LDL)
- high density lipoproteins (HDL)
What are Metabolic Pathways?
Metabolic pathway is essentially a series of chemical reactions, catalysed by enzymes, whereby the product of one reaction becomes the substrate for the next reaction These reactions can be divided into anabolic and catabolic
What are the four main applications of metabolomics?
Medicine:
- Biomarkers
- Disease mechanisms
- Drug target identification
Biochemistry and bioengineering:
- Pathway mappin
- Enzyme regulation
- Yield enhancement
Food and agriculture:
- Nutrient composition
- Crop fitness
- Dietary choices
Environment:
- Microbial communities
- Nutrient cycles
- Bioremediation
What are the main challanges in metabolomics?
Pre analytics:
- Study Design
- Sampling
- Storage
Analytics:
- Diversity
- Dynamic range
- Stability
Data analysis & Interpretation
How do NMR and MS compare in metabolomics?
How does the MS-based metabolomics work flow look like?
What are the most common statistical analysis approaches in metabolomics?
Grouped into univariate and multivariate methods
Univariate analysis takes only one variable into account, resulting in differently weighted results.
Multivariate analysis works on a matrix of variables and highlights characteristics based on the relationships between all variables.
What are possible analytical setups for metabolic profiling and what does it depend on?
- Preanalytics: choice of tests/samples, sample collection-transport-processing-storage
- Sample preparation: Matrix decomplexification Separation
- Inject
-
Column chromatography:
- GC
- HPLC-RP
- HPLC-HILIC
- IC
Goal of chromatography:
–> Improve separation, gain in specificity
–> Improve peak shape, gain in sensitivity
- Detection: mass spectrometry
- Ion Source
- EI
- ESI
- Mass analyser:
- Q, QQQ, QIT
- Q-TOF. Orbitrap
- Detection:
- Retention time
- m/z
- Intensity
Targeted –> small DATA
Non targeted –> Big DATA
- Other detection techniques:
- UV/VIS
- ECD
- Conductivity, …
- Ion Source
- Data interpretation:
- Quantification
- Identification
- Peak-annotation
Choice of setup –> choice of metabolite class:
–> Not all metabolites can be detected simultaneously:
- diversity in physico-chemical properties (pKa, polarity…)
- wide range of concentrations
- isobaric metabolites (separation before detection)
How can sample preparation be done in metabolomics?
What is the goal?
Possiblities:
- Protein precipitation
- Extraction
- Derivatisation
Goal:
- Quench metabolism
- Remove proteins (& lipids)
- Ev. derivatisation (improve stability, volatility, ionisability, detection…)
–> Must be compatible with analytical setup:
chromatography and MS (e.g. volatile solvent - no phosphate buffer)
Why are humans interested in specialized metabolites?
To heal themseves:
Specialized metabolites are privileged structures:
«Because of their three-dimensional structural complexity and inbuilt affinity for biological surfaces, natural products are in fact privileged structures for drug discovery from both a chemical and a biological standpoint and qualify nature not only as the ultimate synthetic chemist but also as the ultimate pharmacist.»
To understand the world they live in:
Studying metabolites allows to understand the interactions of organisms in ecological niches and between species. In fact, chemistry in the common language of Nature.
What is Systems Biology?
What questions does it pose?
The relationships and elements of biological information with respect to one another must be determined and all of this information need to be integrated to obtain a view (model) of the system as a whole.
–> So we need to study metabolism as a whole and account for as many levels of information as possible
Where can we find all of this information?
–> Databases
However, even if we focus on just the metabolic reaction network of an organism it can be extremely complex
Questions:
- What are the tools that can help us navigate it?
- How can we use it to help us analyze metabolomics data?
What two main tools are there for analysing metabolomic datasets?
Over-representation analysis (ORA)
Pathway analysis (PA)
What is Over-representation analysis (ORA)?
Tool to analyse metabolomic datasets
- Traditionally designed for transcriptomics data
- Purely statistical method – does not account for pathway topology
