Proteomics Flashcards
(26 cards)
What is isoelectric focusing?
Happens when the protein is folded!
A method for separating proteins based on their charge .The charge depends on the amino acids, so we will get an electric field that separates based on pH. Once it reaches the isoelectric point it will stop in the gel
What are the 4 separation methods?
- Iso electric focusing
- SDS-PAGE
- 2D-PAGE
- DIGE
What is the isoelectric point?
At some point we will reach a pH where charge = 0. There is a positive charge below and a negative charge above. Once the protein reaches it’s isoelectric point it will stop in the gel
What is SDS-PAGE?
A gel electrophoresis separation technique that separates based on molecular weight. This is done ONLY AFTER isoelectric focusing.
What are the steps in SDS page?
- Add mercaptoethanol to the sample to denature the protein
- Add SDS what will bind to each amino acid residue. SDS has a negative charge that it adds to each amino acid to make them similar in charge.
- Add to gel for electrophoresis where smaller proteins will migrate quicker than bigger ones.
How does gel electrophoresis work?
We have a gel made of agarose gel in a waterbased buffer solution. At one end of the gel we have different welds for different samples. It has electrodes in each end. The end with the welds have the cathode (-), and the other have the anode (+). When turning on the current the cathode will repel the proteins and larger molecules will travel to the anode quicker than small ones.
What is 2D-PAGE?
We do both isoelectric focusing and SDS page in one!
So first dimension we do pH based separation and in the second dimension based on weight. We therefore get a 3d model with information about the pH and charge.
What is DIGE
Differential in gel electrophoresis.
We want to measure the differential expression between two samples.
What are the steps in DIGE?
We want to compare differential expression in 2 conditions.
1. We make 3 samples: condition A, condition B, condition A+B (control).
2. Link each sample with different fluorescent dyes that excites at different wave lengths
3. Apply all samples to a single gel
4. 2D PAGE separation
5. Gel fluorescent imaging superposition
What requirements do we have to the dyes in DIGE?
- photostability
- pH stability
- no spectral overlaps between dyes
What do we get out of DIGE?
A relative ratio between two different samples
What is peptide mass fingerprinting?
Identification method where the mass of an unknown protein can be determined. It is usually performed with MALDI-TOF.
What is MALDI-TOF?
Type of mass spec that measures the mass of compounds.
What is a peptide?
A protein fragment
How do we fragment proteins?
Using trypsin to cleave
What are the steps in peptide mass fingerprinting?
- Protein is separated using gel electrophoresis
- Protein fragmentation with trypsin
- MALDI-TOF used to find the real mass of peptides in our sample
- Simulated digestion on database proteins to generate a theoretical peak list
- Compare real mass found in MALDI-TOF to the theoretical found by simulation
What are the steps in MALDI-TOF?
- Ionization by MALDI to ensure the molecules becomes electrically charged
- Analyzer separates them based on their mass to charge ratio (MZ ratio)
- Detector is like a tube. Molecules start at one end and will fly through the tube. Smaller fly faster. The time it takes them to get from one end to another is their time of flight (TOF)
What is time of flight (TOF)?
Used to describe the amount of time a moelcule flies through a detector tube in MALDI-TOF. Molecules start at one end and will fly through the tube. Smaller fly faster. The time it takes them to get from one end to another is their time of flight (TOF)
What is MALDI?
Matrix assisted Laser desorption ionization.
It consists of a matrix that absorbs laser light and transfers it to the sample molecules since direct exposure would cause fragmentation. The mixture is put onto stainless steel and dryes into crystals. It turns into a gas phase that can now travel through the tunnel in MALDI-TOF
What is MS-MS identification? Also called tandem mass spec
A protein identification method using a database that can identify the sequence for a specific protein. It can do this by undergoing 2 MS procedures, separated by a collision cell where a neutral gas will further fragment the ions to be analyzed in the next MS. From this we get a peak list that we can compare to a database to identify the protein.
What is de novo peptide sequencing?
Identification technique where we can get the amino acid sequence without using a database. We can then do BLAST to find possible proteins and find the sequence from there.
What are the steps in de novo peptide sequencing?
- Fragment sample with trypsin
- Ionize with MALDI/ESI
- First MS getting peaks for the peptides
- Select peptides and add to collision cell
- Collision cell uses a neutral gas to further fragment the peptides of our choice.
- Second MS to get peaks of the ions in the selected peptides
- De novo mass spec where we get rid of all B type ions. This is done by guanidation (blocking the epsilon amino groups in lysines) and sulfonylation of the N terminal (creates a negative charge at the N terminal part).
- Ionize and dissociate
- Introduce positive charge to the B fragments meaning it now has a positive and negative charge. This means the ion will not fly anymore in the MALDI-TOF
- Do MS on now only the y type fragments
- We get a peak list where we can measure the mass difference between peaks and translate this into amino acids
- Possible to do BLAST to find proteins and their sequences.
What are the steps in MS/MS?
- Fragment sample with trypsin
- Ionize with MALDI/ESI
- First MS getting peaks for the peptides
- Select peptides and add to collision cell
- Collision cell uses a neutral gas to further fragment the peptides of our choice.
- Second MS to get peaks of the ions in the selected peptides
- Compare peak lists to database peaks and identify protein.
What is quantitative proteomics using MS (iCAT)?
Identification technique that does not rely on gels. The point of this is to find difference in abundance (for example cancer cells), so we look at differentially expressed proteins.
