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Personalized Medicine Week 4 Lecture Notes
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PROTEOMICS AND METABOLOMICSo Proteome the entire complement of proteins in a
given biological system such as cell type, biological
fluid or tissue.
o Dynamic: a living being has just one genome but many
proteomes (one per each cell type) and these change
during development and as a result of environmental
factor (e.g., fed versus starved conditions and in
disease).
o Proteomics: technology to study the proteome. Analysis
of proteins in a genomic scale.
Proteomics
* Aims to provide biological information not attainable with genomic technology.
* With the aim to identify protein alterations in disease; proteins can be drug targets and biomarkers.
* Involves identification and comparative analysis of proteins across sample groups.
* Emphasis is in comprehensiveness of analysis in an unbiased/untargeted manner.
Technological Challenges
* Chemistry of proteins is more complex than that of nucleic acids.
- DNA has only four bases with similar chemistry: acidic (-ve charged) sugars.
- 20 different a.a. with large differences in chemistry: polar, hydrophobic, basic, acidic.
* Two copies per gene; large dynamic range of protein expression in cells and bio-fluids.
* Proteins cannot be amplified experimentally, in contrast to DNA (with PCR).
Proteomics Technology
a. Targeted Analysis
- Immunochemical (antibody based) methods.
- Restricted to antibody availability.
- Needs preconception of proteins that could be involved in the question being addressed.
- Normally limited to the detection of a few hundred proteins.
- Reversed Phase Protein Arrays:
1. Cells/tissues are lysed and protein extracted.
2. Cell lysates are printed in
appropriate surface (nitrocellulose
paper).
3. Antibodies against desired proteins
are used.
4. Secondary antibody derivatised
with luminescent or fluorescent
probes applied.
5. Array scanned and images
analysed.
6. Extent of signal is proportional to
amount of protein in cell
lysate/tissue.
b. Untargeted Analysis
- Gel based methods
- Madd spectrometry
, Peptide fragmentation patterns (MS/MS spectra) are used to infer peptide sequence.
It can analyse 10 peptide per second.
With mass spectrometry we can identify the relative quantification to compare peptide/protein
abundance.
Global analysis of proteins (Proteomics) is more challenging than DNA sequencing- proteomics lags
behind genomics in terms of technology.
LC-MS/MS technology developing fast.
Motivation:
- Proteins are the ultimate mediators of biological function and the targets of all drugs.
- mRNA levels do not always correlate with protein levels.
- Need to consider proteins for a better understanding of cell biology and mechanism
of disease.
Protein (enzyme copy numbers do not necessarily correlate wit how active such enzyme may be in
cells.
Regulatory proteins and drug targets are enzymes.
Protein copy numbers in cells is only one factor in determining enzyme activity.
Mechanisms of Enzyme Regulation
Expression
Covalent modifications, such as:
Phosphorylation, Acetylation, Lipidation
Allosteric regulation. E.g. feedback
inhibition. End product of metabolic pathway
inhibits enzyme that catalyses the rate
limiting step in the pathway.
Cofactors. Holoenzymes (inactive) may
require cofactors to become active
(apoenzyme).
Proteolytic cleavage. E.g. some enzymes
(zymogens) need to be active.
Proteins-protein interactions.
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