Proteomics 1 Flashcards
What is proteomics?
Qualitative and quantitative comparison of proteomes under different conditions
Discuss PAGE
PAGE = 1d polyacrylamide gel electrophoresis
quick and easy but poor resolution
each band can carry 10s-100s of proteins so thick band does not necessarily mean lots of protein
Discuss 2D PAGE
separate proteins by PI values in 1D gel then Molecular Weight in 2D gel (SDS)
each spot may not be exactly 1 protein but much more resolved than 1D.
More intense spot = more protein
What is the benefit of liquid chromatography to PAGE in proteomic analyses?
Liquid chromatography can be performed in line with specific growth condition
Better purification of proteins
What are the three different types of column used in liquid chromatography?
Anion/Cation column
Affinity tag column
Reverse Phase column
What must follow all of the purification techniques in order to quantify the proteins?
Mass spectrometry
How does mass spec separate proteins
based on mass charge ratio
What are the three parts that make up a mass spectrometer?
1 = part that makes ionized forms of sample 2 = part that separates according to mass charge ratio (mass analyser) 3 = computer result production part (detector)
How can ionisation be induced to a molecular sample that isn’t naturally ionised (in general)
ionisation can be induced by molecular collisions (adding or minusing protons)
what is MALDI?
Matrix assisted laser desorption ionisation.
How does MALDI work?
Matrix contains a specific compound which is added to assist ionisation
Hit the sample/matrix mixture with laser beams, causing proteins to dissociate from the mixture carrying a charge from the matrix
What is ESI?
Electrospray ionization
How does ESI work?
Sample of proteins is sprayed out of a capillary which is charged at the end, causes release of charged droplets from end of capillary
Sent in to an evaporation chamber until the droplets become so small that repulsion between the newly charged particles causes them to burst into gas phase
How does Time of Flight mass analysing work?
How long until the charged particles reach the detector is measured (ie how big or small it is)
How can you achieve higher resolution in Time of Flight mass spec?
Increased resolution comes from increase flight distance or more convoluted paths
How does quadrupole mass analysing work?
Apply a radio frequency field between the sample ejection and detector.
Using a chosen radio frequency, you can divert off charged particles of a certain size.
Allows only certain molecules to get to the detector (good for 1st stage of MS/MS - this is just note to self not part of answer)
can compare results of which particles made it to the detector in different radio frequencies
How does Ion Trap mass analyser work?
Electrodes in the flight chamber hold certain particles in a bunch.
Electrodes at entrance and exit of chamber can have their charge changed to pull out or select for different particles trapped in the chamber
What are the electrodes called in Ion Trap mass analyser?
toroidal endcap electrodes
How does FT-ICR work?
You hold your pulse of sample in the chamber, then cause it to circulate due to a field spread across it, changing charge.
Sample passes by detectors in the chamber which can measure the signal of charge
What is the purpose of all detectors in mass spec?
To convert the impact of ions on their surface in to an electric voltage signal that can be amplified
Which mass analyser matches to which detectors?
Quadrupole/Ion Trap = CEM detector
Time of Flight = MCP detector
FT-ICR has detectors in built
What is the raw data called that comes off a mass spec and what does it represent?
Spectra
x axis is mass charge ratio and y is relative abundance
What is an isotopic mass envelope?
If you zoom in on a peak on the spectra you see lots of little peaks, due to abundance of heavy isotopes in biological sample
Why is good fractionation of sample upstream of detection important?
Needed to produce simple spectra
otherwise can have complex spectra with overlapping peaks
