KJB - Proteomics Flashcards
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?
- 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?
Acrylamide
What substance separates DNA?
Agarose gel
Why is network/ protein pathway analysis beneficial?
It gives rise to more drug targets.
What are the 3 steps to proteomics?
- 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)
