Proteomics 1

Question 1

What is proteomics?

Answer 1

Qualitative and quantitative comparison of proteomes under different conditions

Question 2

Discuss PAGE

Answer 2

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

Question 3

Discuss 2D PAGE

Answer 3

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

Question 4

What is the benefit of liquid chromatography to PAGE in proteomic analyses?

Answer 4

Liquid chromatography can be performed in line with specific growth condition
Better purification of proteins

Question 5

What are the three different types of column used in liquid chromatography?

Answer 5

Anion/Cation column
Affinity tag column
Reverse Phase column

Question 6

What must follow all of the purification techniques in order to quantify the proteins?

Answer 6

Mass spectrometry

Question 7

How does mass spec separate proteins

Answer 7

based on mass charge ratio

Question 8

What are the three parts that make up a mass spectrometer?

Answer 8

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)

Question 9

How can ionisation be induced to a molecular sample that isn’t naturally ionised (in general)

Answer 9

ionisation can be induced by molecular collisions (adding or minusing protons)

Question 10

what is MALDI?

Answer 10

Matrix assisted laser desorption ionisation.

Question 11

How does MALDI work?

Answer 11

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

Question 12

What is ESI?

Answer 12

Electrospray ionization

Question 13

How does ESI work?

Answer 13

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

Question 14

How does Time of Flight mass analysing work?

Answer 14

How long until the charged particles reach the detector is measured (ie how big or small it is)

Question 15

How can you achieve higher resolution in Time of Flight mass spec?

Answer 15

Increased resolution comes from increase flight distance or more convoluted paths

Question 16

How does quadrupole mass analysing work?

Answer 16

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

Question 17

How does Ion Trap mass analyser work?

Answer 17

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

Question 18

What are the electrodes called in Ion Trap mass analyser?

Answer 18

toroidal endcap electrodes

Question 19

How does FT-ICR work?

Answer 19

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

Question 20

What is the purpose of all detectors in mass spec?

Answer 20

To convert the impact of ions on their surface in to an electric voltage signal that can be amplified

Question 21

Which mass analyser matches to which detectors?

Answer 21

Quadrupole/Ion Trap = CEM detector
Time of Flight = MCP detector
FT-ICR has detectors in built

Question 22

What is the raw data called that comes off a mass spec and what does it represent?

Answer 22

Spectra

x axis is mass charge ratio and y is relative abundance

Question 23

What is an isotopic mass envelope?

Answer 23

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

Question 24

Why is good fractionation of sample upstream of detection important?

Answer 24

Needed to produce simple spectra

otherwise can have complex spectra with overlapping peaks

Question 25

What is the average mass quoted for a peptide or protein from a mass spec referring to?

Answer 25

Calculated from the centroid of distribution not just the middle value of the distribution

Question 26

What are problems related to using simple identification of proteins from mass spec on a database?

Answer 26

Protein degradation - fragment created in handling could produce a match with a known protein

Question 27

What is the point in using peptide mass spec over protein?

Answer 27

Aids mass identification

Multiple matches give confidence to assignment

Question 28

How are proteins fragmented for peptide mass spec?

Answer 28

Chemically (eg cyanogen bromide) or enzymatically (trypsin)

Question 29

What is the point of MS/MS?

Answer 29

Use two mass analyser steps to separate fragments as far as possible
Can identify peptide fragments within an already identified protein
Can give in depth analysis of protein structure

Question 30

What happens in the collision cell of MS/MS

Answer 30

Fragments break favourably at peptide bonds by resonant excitation