Module 2- Mass Spec

Question 1

General principle of mass spec and how this led to the finding of isotopes

Answer 1

Ions pass through ion source and ion optics, then a mass analyser (magnetic field) and detector
Defelction of the magnetic field is dependent on the mass and charge of the ion
Mass spec showed different signals for one ion, reflecting that there are different isotopes differing slightly by mass

Question 2

How does a time of flight analyser work

Answer 2

Ions sit on anode, then gains charge/ is exhilarated to the same kinetic energy through uniform electric field with uniform electric potential energy which then pass through a cathode grid
Then travel through free field and high vacuum region with no electric field. Travel by mass:charge ratio so measures m/z
m/z= t^2 x k

Question 3

Matrix assisted laser desorption/ionisation (MALDI) ionisation

Answer 3

Samples are co-crystalised with matrix, then activated by laser energy
Molecules transition into gas in activated matrix and the matrix accepts or transfers protons from or to analytes
Then molecules all have a charge

Question 4

Electrospray ionisation (ESI)

Answer 4

Analyte solution (has water and organic solvent and some acid for positive charge) sprayed into electric field
Droplets generated which evaporate through field, leading to increase in surface charge density
Repulsion between gas molecules occurs and gas explodes, transferring charge to analyte molecules during liquid to gas transition

Question 5

Methods for protein indentification

Answer 5

Protein electrophoresis
Immunoblotting
Edman sequencing
Mass spec

Question 6

Protein identification by mass spec/ tandem mass spec

Answer 6

Site specific proteases used to generate different protein fragments
Pass fragments through mass spec once and identify a single peptide sequence of interest
Fragmentation of peptide by collision induced dissociation (collides with gas which causes vibrations which overcome bond energy and break up peptide)
Measure fragment masses by mass spec, gives fragment sequence as know mass between different fragments to tell certain amino acid in the sequence one at a time

Question 7

Affinity purification

Answer 7

Tag onto target protein
Pass through system with immobilised bead connected to affinity partner- get binding to specific protein

Question 8

Affinity purification with immunoprecipitation

Answer 8

Control with immobilised control antibodies to get negative control- unbound proteins washed away and negative control proteins remain
Target protein binds to immobilised antibodies and non-specific interaction proteins are washed away, target protein eluted out
Can use to see changes in protein amount under different cell conditions eg stress

Question 9

Labelling of interacting partners with BioID

Answer 9

Expression of fusion protein of E.coli biotin ligase with bait protein
Addition of biotin with proteins
BirA biotinylates bait proteins and interaction partners
Biotinylated proteins (interaction partners/ proteins that are close to or interact with target) purified from all other cell proteins by Streptavidin-biotin affinity purification

Question 10

What is the 14-3-3 system and what was the experiment done with it

Answer 10

14-3-3 recognises P on ser and thr on target proteins
Binding specificity and downstream signalling depends on P of either or both P sites, or one of the two
Binding to target causes conf change= downstream signalling
Wanted to see what proteins it would recognise after ATK P events

Question 11

Error model- estimation of false discovery rate (identify significant ones)

Answer 11

Number of false discoveries/ total number of discoveries
Data searched against decoy database with sequences in reverse or randomised (nonsense)
Significant hits in decoy mean false positives

Question 12

Looking for specific interactions

Answer 12

Volcano plot of log2(fold change) versus -log10(p-value)
1% prob samples are same is minimum significant threshold for quantification (2 on -log10)
1 is lowest value for fold change threshold (2 on log2)
T test done to see if null is correct (protein abundance of sample isnt different to protein abundance in control)
Low p-value shows significant difference

Question 13

Which interactions are involved in IGF1 signalling?

Answer 13

Clustering proteins over the time course measurements based on abundance in response to IGF1 stimulation
Need inhibition to occur at beginning to show it is IGF1 stimulation. Then need either fast or slow response to IGF1 stimulation

Question 14

What is degree of connectivity

Answer 14

Number of edges connecting a particular node within the network

Question 15

What is betweenness centrality

Answer 15

Global position of a protein in the network
Protein node connected with highest total number of shortest paths has highest betweenness centrality

Question 16

What is network hub

Answer 16

Node with a high degree of connectivity and high betweenness centrality

Question 17

Aspects of protein interaction networks- heirarchy

Answer 17

Biological process
Molecular function (parent term)
Cellular component (child term)- more specific functions given
Each aspect describes heirarchial structure of GO-terms
Single gene can be assigned to several child and parent terms within heirarchial strucutre

Question 18

Sample frequency in protein interaction network

Answer 18

Number of genes assigned to the same GO-term in entire sample set

Question 19

Background frequency in protein interaction network

Answer 19

Number of genes assigned to the same GO term in entire genome (background genome)

Question 20

What is a functional GO term

Answer 20

Enriched in a set of input genes/ proteins when the sample frequency is high in relation to the background frequency

Question 21

Liquid chromatography coupled mass spec

Answer 21

Mass spec measures peptides that come out of chromatography at a certain phase
Hydrophobic proteins on beads in stationary phase covered with hydrophobic C18 and run over organic solvent which interferes with hydrophobic interactions- peptides separated by increasing hydrophobicity
Mass spec takes 10-20 strongest for CID and then more mass spec

Question 22

What is proteome dynamic complexity further increased by

Answer 22

Proteins
Protein stability (half life time)
Subcellular localisation and different functions in these different locations
Protein/protein interactions
Differential protein abundances
PTMs

Question 23

PTM phosphorylation and complexity

Answer 23

Can be reversible or irreversible
On serine, threonine and tyrosine side chains
Leads to different outcomes (signalling, function and activity regulation, subcellular localisation, complex assembly)
Adds negative charge so hard to ionise and do mass spec, need to remove

Question 24

Ways to remove phosphorylation PTM for mass spec

Answer 24

Immunoprocipitation with antibodies against phosphorylated amino acid residues
Metal oxide affinity chromatography that binds phosphorylated amino acid side chains- eg titanium dioxide

Question 25

Kinases and cancer

Answer 25

Kinase inhibitors are often used as potential cancer drugs because kinases can phosphorylate and cause upregulation of cell activity and are upregulated in cancer. So drugs that inhibit would prevent cancer from targeting

Question 26

Difference between mass spectrometer and time of flight analyzer

Answer 26

Both mass analysis methods- both separate ions based on mass/charge ratio, mass spec uses magnetic field while time of flight uses electric field to separate ions