Mass Spectometry Flashcards
What does PTM stand for?
Post-Translational Modification
What does MS or PMS stand for?
(Protein) Mass Spectrometry
What are the 4 advantages of using MS compared to other methods?
- Provides protein ID and PTMs with high specificity
- High Sensitivity
- Multiplexing: Can identify multiple proteins in a single sample and can processes multiple samples at the same time
- Can even discover novel proteins
What does mass spec measure?
Measures the mass-to-charge ratio of ions. (Therefore, molecular or atomic ions). Molecules must be charged before being analyzed by MS.
Describe the mass spectrometry system.
- Ion Source:
ESI and MALDI - Mass Analyzer:
Quadruple and TOF - Detection Device:
CEM and MCP - Database system:
Excalibur, SEQUEST or Mascot
What is the role of Ion source in MS?
Converts molecules from the sample into gas-phase ions. It is done because only ions can be manipulated by electric and magnetic fields inside the MS.
What does ESI stand for?
How does it work?
Electrospray Ionization
Produce ions from large molecules so they can be analyzed based on their mass-to-charge ratio.
What are the advantages of ESI?
- Carried out at atmospheric pressure.
- Carried out from bulk solution. (No need to mix.)
- Very good at producing multiple charged ions (all sizes and weights can be analyzed.)
- Gentle.
- Not tolerant to non-volatile salts.
What does MALDI stand for?
SELDI?
Matrix Assisted Laser Desorption Ionization
Surface Enhanced Laser Desorption Ionization
What are the main differences between ESI and MALDI?
Sample type: Liquid VS Solid
Ionization source: UV laser VS High Voltage
Ionization type: Evaporation + coulombic explosion VS Proton transfer from matrix
Charge state: Multiply charged VS Singly charged
Mass range: Any MW VS High MW
What is the role of mass analyzers in MS?
They separate ions based on their mass-to-charge ratio.
Simply put, how does quadropole work?
Draw a diagram
4 rods in parallel with RF and DC voltage applied causing oscillating electric fields that effect ion trajectories. Only ions with specific mass-to-charge ratios will have a stable path through the quadrupole.
What does TOF stand for?
How does it work?
Time of Flight
Ions are accelerated down a long flight tube via a brief pulse electric field (called a pusher).
Could be linear or reflector.
What does CEM stand for?
Chanel electron multiplier
What does MCP stand for?
microchannel plate
Name the Limits and Advantages of TOF.
Advantage:
- Great for analyzing large proteins because the m/z can go up to about 15000.
- Very high duty cycle from MCO detector.
Limitations:
- Low sensitivity due to MCP detector
- Resolution depends on the length of the tube. The tube length can be doubled at the price of sensitivity.
What is the difference between bottom-up and Top-down approach.
Bottom-up involves enzymatic digestion that results in higher sensitivty but info about PTM is lost. Larger proteins are able to be analyzed.
Top-down approach involves keeping the proteins intact, allowing for PTM info to be preserved, but has a lower sensitivity and cannot handle large proteins.
What does LC-ESI-TOF stand for?
Liquid Chromatography – Electrospray Ionization - Time of flight
What are the steps involved in PMF?
- Cut out protein spot from 2D gel (show what a 2D gel looks like)
- Trypsin digestion to create a protein mixture.
- Protein mixture undergoes either MALDI-TOF or LC-ESI-TOF
- Results of the MS is a peptide mass fingerprint graph (show the graph as %Intensity VS Mass (m/z))
- Perform a data base search on the results
- Identify the protein.
Draw what the bottom-up and top-down MS approach looks like.
y’know
What does SILAC stand for?
Stable Isotope Labelling by Amino acids in Cell culture
What does ICAT stand for?
Isotope-Coded Affinity Tags
Draw a diagram for proteolytic 10O labeling.
What are the advantages and limitation of enzymatic labeling?
Advantages:
- Applicable to all types of biological samples.
- Effective with very low sample amounts.
- No side reactions or by-products
Limitations:
- Incomplete incorporation of 2nd O18 atom is a common challenge. Rate of exchange will differ depending on peptide size, type of amino acid, etc.
- C-terminus peptides are not labeled.
- Losses during sample prep
