Lecture 1 mass spectronomy Flashcards
What does a mass spectronomer do?
– A device/apparatus that measures the mass of the
analyte(s)
• Proteins, peptides
• Carbohydrate, fat, small molecules, chemicals
How to use a Mass Spectrometer for peptide/protein quantitation?
1) Inject molecule into MS
2) Detector gives a peak: mass & intensity
3) Peak intensities correlate to peptide concentration = Quantitation
But, mass is not enough for protein identification
Each amino acid has a specific mass
A peptide has a mass of amino acids masses combined
A protein is cut into many peptides, each with a specific mass.
MS measurement of peptides : excellent
MS measurement of proteins : possible but with low resolution and poor mass accuracy. Why?
Low resolution with MS of proteins because of modifications (acetylation, phosphorylation..) that change the mass.
What is the first step for protein indentification with MS?
Digest protein into peptides by Trypsin: this enzyme cleaves specifically the peptide bond
between the carboxyl group of arginine, or the carboxyl group of lysine, and the amino group
of the adjacent amino acid.
These peptides are derived from the protein: identify the peptides = identify the protein
But proteins can have a different amino acid sequence and the same mass. What is a solution? How does it work?
Sequence determination: MSMS (2 mass sp. Joined together): to determine the sequence. Also called tandem mass spectronomy.
1) Protein: digestion
2) Peptides: ionization
3) MS (m/z = mass to charge ratio)
4) Isolation of peptides
5) Fragmentation
6) Peptide fragments: mass analysis (MS/MS)
Peptide bonds are broken (weakest bonding) -> peptide fragments are measured.
Looking at mass difference of the fragments will reveal the amino acid sequence
What are ways to reduce sample complexity?
High pressure liquid chromatography-coupled (scheidingsmethode)
–an hour run to reduce the sample complexity
•LC-MS/MS is the most commonly used methodology for proteomics analysis
•Pre-fractionation by SDS-PAGE
–Cut gel into several slices and perform MS analysis separately to further reduce sample complexity
BRAAK stages: what increases?
Tau and GFAP increase with Braakstages
(tau = tangled neurons). GFAP = protein
AD pathology: what happens?/what causes the neurodegeneration?
Amyloid-beta (Aβ)Plaques (extracellular deposition)
Aβaccumulation in brain vasculature: Cerebral Amyloid Angiopathy (CAA)
Neurofibrillary Tangles (intracellular accumulation of phospho-tau)
Granulovacuolardegeneration (GVD)
What is the amyloid cascade hypothesis?
The amyloid cascade hypothesis posits that the deposition (accumulation/afzetting) of the amyloid-β peptide in the brain (main component of amyloid plaques) parenchyma is a crucial step in Alzheimer’s disease (AD).
85% of AD patiënts are late onsets. What does that mean and what is another type of AD?
Familial AD
•Young onset 50-60year
•Mutations in APP/PSEN1/2
Sporadic AD
•Old onset
•30-40% occurrence for 80+ population
Mutations/major risk factors?
•Ethiologyis largely unknown –Mutations: APP, PSEN1 and PSEN2 •Major risk factors' –Age –APOE4
Alzheimer’s Disease (AD): symptoms? Diagnosis? Cure?
•Most common symptoms of dementia•Memory impairment, disorientation, personality changes, cognitive decline •Complete dependence•Definitive diagnosis of AD only possible post-mortem•No cure available
Why is transcriptomics not enough for protein identification?
Methylation, phosphorylation, sulfation, etc. modifications are not shown by transcriptomics. More needed than mRNA.
How can mass be detected in a MS?
Sample inlet -> ionisation area -> acceleration: in their flight path, light ions take less time to travel to the detector. Therefore, mass can be determined by time measurement.
2 types of desorption/ionization techniques?
1) Electrospray. Electrospray: extremely high voltage, temperature. Evaporation of solvent -> molecule enters the mass spectrometer
2) MALDI
