How are humans more complex than a nematode worm?
In terms of pure gene numbers, we are not that much more complex. However, where we are more complex is what we do with those genes (out of our 20,000 - 30, 000 genes, many more transcripts/ mRNA molecules (around 34,000) are created). Thus, we make/ have more transcripts than we have genes. From these transcripts we make proteins.
Why does the amount of proteins we have not equal number of genes?
This is due to alternative splicing. The cell does not have to undergo this process the SAME way every time. It cuts the gene together in different ways depending on the types of tissue it finds itself in. So, many proteins can be made from the SAME gene, simply by combining different exons together.
What is alternative splicing?
This is a process by which introns are cut out of the long premature mRNA to make into mature mRNA.
What is an intron?
The part of the genes that do not give rise to proteins.
Difference between transcriptomics and proteomics.
Transcriptomics allows us to see if a gene is transcribed more in a diseased cell or in a healthy cell. Changes in protein function (due to a medical condition) are NOT detected by a DNA microarray (transcriptomics screen) because these detect the amount of mRNA/proteins; which even if it undergoes mutation,it (may) still produce protein --> even if it is non functional. Proteomics looks directly at the fate of proteins i.e. the actual biological agent --> more useful (kinda)
What are the ways in which medical conditions can affect protein function?
1. mutation which results in a non functioning protein 2. proteins that are sequestered away from where they need to work 3. proteins that are degraded before they do their job 4. alternate mRNA processing (intron splicing) 5. chemical modification regulates function
What substance separates proteins?
What substance separates DNA?
Why is network/ protein pathway analysis beneficial?
It gives rise to more drug targets.
What are the 3 steps to proteomics?
1. Sample preparation 2. Sample separation (2D gel electrophoresis or Ion Exchange chromatography) 3. Analysis and Identification ( - Trypsin digestion - Mass spectrometry - Identifying proteins from a mass spectrum)
Brief description of first step of proteomics.
Sample isolation. Pairwise comparison made. Choose appropriate sample tissue or fluid. Extract all cell PROTEINS.
Brief description of second step of proteomics.
Sample separation. (a) Isolectric focussing. - put proteins on a gel strip that has a pH gradient - apply an electric field/charge - proteins will start migrating to a point where they have no net charge (isoelectric point) (b) SDS-PAGE - isoelectric strip placed on one end of another rectangular gel - gel contains SDS - electrical field applied - amino acids move towards +ve end. Larger proteins take longer to move through pores. - once protein is removed from gel, separation process is completed. (c) Spot picking - compare gel to a reference/ comparative gel and look for differences in protein expression (proteomics) - remove interesting spots from gel (can get 10,000 spots in one gel) - Proteomics allows us to see changes in protein e.g. position/ absence/ general change - we do NOT know what protein this is at this stage
Difference between isoelectric focussing and SDS-PAGE?
Isoelectric focussing is the separation of proteins in FIRST dimension. SDS-PAGE is separation of proteins in SECOND dimension (according to size).
What is the fate of a protein with a +ve charge on a gel strip with an electrical field applied?
It will move to the -ve electrode, giving H+ to surrounding, until net charge = 0, where it stops moving. (isoelectric point)
What is the fate of a protein with a -ve charge on a gel strip with an electrical field applied?
It will move to the +ve electrode, taking H+ from surrounding, until net charge = 0, where it stops moving. (isoelectric point)
What is SDS?
A detergent that unfolds proteins into an amino acid chain and codes them with a uniform distribution of -ve charge. The amino acid moves to the +ve end. Larger proteins take longer.
What are the three steps of analysis and Identification in proteomics?
1. Digestion 2. Mass spectroscopy 3. Identifying proteins from mass spectra
Brief description of first step of analysis and identification in proteomics.
Digestion. - Digest picked sports with trypsin to yield fragments --> cut the protein into much smaller pieces. This will make it easier to identify than a full length protein. - Trypsin digestion yields random fragments of all sizes --> it breaks down peptide bonds of the amino acids. - Fragments are then analysed by mass spectrometry (measures molecular weight by measuring mass:charge ratio) - Any protein will have a distinctive set of fragment sizes when digested in this way.
Brief description of second step of analysis and identification in proteomics.
Mass spectroscopy. - Substance is bombarded with an electron beam having enough energy to fragment the molecule (ionisation) - The +ve fragments which are produced (cations and radical cations) are accelerated in a vacuum through a magnetic field and are sorted on the basis of mass: charge ratio - Ionisation methods which are used in proteomics are (a) Electrospray ionisation - generates ions by creating a fine liquid aerosol (b) MALDI (sample embedded in a metric which is ionised by a laser) - Analysers used in proteomics (a) Quadrupole analyser (more complicated) (b) Time of Flight (more simple)
What are the ionisation methods used in proteomics?
1. Electrospray ionisation - generates ions by creating a fine liquid aerosol 2. MALDI (sample embedded in a metric which is ionised by a laser)
What is the more complicated analyser method used in proteomics?
What is the more simple analyser method used in proteomics?
Time of Flight
Brief description of third step of analysis and identification in proteomics.
Identifying proteins from a mass spectra - Each peak represents and ion of a particular mass/ charge - The analysis of mass spectroscopy information involves the reassembling of fragments, working backwards to generate the original molecule.
What are the two ways in which mass spectra can be applied to identify proteins?
1. Peptide mass fingerprinting 2. Direct amino acid sequencing Both depend heavily on computer analysis or bioinformatics
What are some technical challenges of proteomics?
- What tissue to sample? which will give the best clinical feature? - How do you fractionate the interesting from the highly abundant (e.g. the 98% of erythrocyte protein that is haemoglobin --> would have to deplete this protein in order to see the interesting proteins) - How to fractionate the difficult to extract (membrane proteins) It is harder to get receptor proteins that are embedded to the membranes so it is easier just to get the proteins from the cytoplasm. - How to identify low abundant proteins? - How to separate those with overlapping profiles? - How to identify unknowns? Maybe because it novel.
What are some applications of proteomics?
- Discovery of drug targets - Protein based diagnostic tests - Toxicoproteomics e.g. expose liver cells to a drug and see if anything happens to them e.g. death of CYP450 enzyme - Pharmacoproteomics
Are pairwise comparisons from use of proteomics causative or correlative?
Which step are -omics in target identification?
First step (typically)