Lecture 1: proteomics Flashcards
What is a mass spectrometer?
A device that measures the mass of the analytes (e.g. proteins, peptides, carbohydrates, fat, small molecule, chemicals).
Explain the general principle of mass spectrometry (in regard to proteomics).
A molecule is injected into the mass spectrometer that eventually arrives at the detector. Here, the detector measures 2 parameters: mass and intensity/concentration.
Why do we mostly measure peptides and not proteins themselves?
Proteins have a low resolution and poor mass accuracy (due to post-translational modifications like acetylation). Amino acids and thus peptides all have their own unique mass which can be used to identify the peptide (identify the peptides = identify the protein)
What enzyme is used to digest proteins into proteolytic peptides? How does it work?
Trypsin, it cleaves the peptide bond between carboxyl group of arginine or the carboxyl group of lysine and the amino group of the adjacent amino acid.
So easy enough you can use and identify the amino acids/peptides to determine the protein. A problem is that different amino acid sequences can have the same mass. How do we solve this problem?
We need to determine the sequence to identify the peptides and protein.
So how can you determine the sequence of peptides?
By using tandem mass spectrometry. Here, the molecules are ionized and these ions are seperated by the first spectrometer (MS1) based on their mass-to-charge ratio (m/z). Ions of a particular m/z from MS1 are fragmented into smaller ions. These ions are then introduced intro the second spectrometer (MS2), which also separates the fragments by their m/z-ratio and detects them.
So in tandem spectrometry a protein is digested into smaller fragments of peptides and these peptides are again fragmented into smaller fragments. How can the peptide sequence be deduced from this?
Each amino acid has a mass and by substracting the masses of the smaller fragments, the sequence of the peptide can be deduced.
A sample can contain up to (or more than) 3000 proteins. A protein contains approximately 30 peptides. 90000 peptides is too complex to analyse. What is typically used to lower this sample complexity?
High pressure liquid chromatography (LC-MS)
How is the complexity of a sample reduced when high pressure liquid chromatography is used?
By separation of peptides according to their hydrophobicity.
Analysis of a sample during mass spectrometry can only occur in the gas phase of the sample. Name two techniques that ensure the gas phase of the sample.
- Electrospray ionization
- MALDI (not discussed)
What is electrospray ionization?
The result of this technique is the formation of charged droplets in the gas phase. This is done by repeated evaporation of the sample with appliance of high voltage. This creates small droplets of charged peptides in gas phase, so that they can enter the mass spectrometer.
A very important measurement in mass spectrometry is time of flight, What is this?
A mass spectrometer is composed of different chambers. You’ve got the sample inlet, the ionisation area, the acceleration area, the flight path and the ion detector. When ions are accelerated in the acceleration area they enter the flight path. Due to the difference in mass, there’s a difference in velocity. Lighter ions arrive earlier at the ion detector than heavy ions. The time ions travel in the flight path is called the time of flight.
Why not transcriptomics only (so why are we using proteomics)?
Post-translational modifications are not revealed by mRNA and there’s no correlation between the amount of mRNA and protein produced.
What are symptoms of Alzheimer’s disease?
Memory impairment, disorientation, personality changes, cognitive decline and eventually complete dependence.
What mutations (probably) lay base to Alzheimer’s and what are risk factors?
Mutations in APP, PSEN1 and PSEN2. Risk factors are age and the apoE4 genotype.
What is the connection between AD pathology and clinical symptoms?
That these two highly correlate with each other.
Why is it actually already too late when someone gets diagnosed with Alzheimer’s ?
Because there’s a threshold for when clinical diagnosis is possible, but before this threshold AD pathology has already developed. This can also be seen in this picture.
The following questions are about a proteomics analysis of CA1 and the subiculum regions of the hippocampus from human postmortem brains of AD patients and controls. What was the goal of this study?
- To get insights in the disease mechanisms
- To identify potential early biomarkers and drug targets for diagnosis and treatment of AD
This is a picture of isolated CA1 and subiculum regions from the hippocampus. What is seen here?
There are clear differences in the tissue of the hippocampus, the tissue that is deviant is outlined. This is also the tissue that you will isolate to analyse in a spectrometer.
What is seen in control and AD patients in regard to tau- and GFAP levels?
The amount of tau-tangles and GFAP (important in astrocytes) increases with Braak stage.
What does this figure conclude in regard to Alzheimer’s and gene activation?
That in certain genes/proteins there’s a turnover → in early Braak stage one group of genes is inactive while other genes are active. In progression to later Braak stages, these two groups turn around, where the first is activated and the other group of genes are inactivated.
Study these graphs and choose and explain two graphs that display potential biomarkers.
Graphs E and H are the most interesting. In graph E you can see that all microtubule-associated proteins stay at a stable level during Braak stages, but only one increases in the development of AD. This is exactly the same for graph H where all ionotropic glutamate receptors stay stable, except one which increases.
What can be concluded from this figure where they isolated tissue from control, AD and AD+CAA?
Certain proteins are only present when a patients has AD+CAA. For example: NDP and COL6A2 are only present in patients with AD+CAA.
