NMR/MS

Question 1

MS types

Answer 1

Intact Mass spectrometry (IMA)
- Top down

Peptide Mass spectrometry (PMA)
- Bottom up

Question 2

MS types

Answer 2

Intact Mass spectrometry (IMA)
- Top down

Peptide Mass spectrometry (PMA)
- Bottom up

Question 3

IMA MS what it does

Answer 3

Monitor protein-ligand interaction
Through changes in protein mass
- Traditional MS: covalent ligand
- Native MS: non covalent ligands

Question 4

IMA MS What you get

Answer 4

As a fuction of ligand concentration: gets KD
Stoichiometry
With time as variable: Kinetics

Question 5

PMA MS what it does

Answer 5

Map ligand binding sitr
Crosslink ligand to protein

Question 6

NMR labels used

Answer 6

19F
1h 15N: HSQC

Question 7

19 F NMR

Answer 7

• Protein labelled at 3 sites with fluorine 19F
• Protein ligand binding results in protein conformational changes
• Monitor change in chemical shift ( binding )
  Depends:
  –environment
  –chemucal change assoxiated with fluorine
• See intermediate states

Get
KD

Question 8

HSQC NMR

Answer 8

Label protein 1H 15N
Titrate ligand
Monitor change in chemical shift

Get KD of each aa

(Hard to discern change ligand binding vs conformational changes)

Question 9

Pro MS

Answer 9

KD
real time through put
Ligand binding location
Low sample consumptions
Label free

Question 10

Pro NMR

Answer 10

KD (uM to mM)
KD of each aa
Ligand binding location
Protein conformational changes
Reuse sample

Question 11

Contra MS

Answer 11

Need validate KD with others
Non specific interactions
Interaction need to be strong

Question 12

Contra NMR

Answer 12

Expensive
Time consuming
Large sample requiremeng