Mass Spectrometry

Question 1

Give a basic outline of mass spectrometry

Answer 1

1. Gaseous sample is loaded
2. The ionization chamber generates ions
3. Ion are separated in some way
4. Ion detector detects ions

Question 2

How does hard ionisation occur?

Answer 2

Electron ionisation

Question 3

What techniques can be used to produce soft ionisation of a sample?

Answer 3

• MALDI
• ESI

Question 4

What is MALDI?

Answer 4

Matrix assisted laser desoption ionisation (place in a matrix with a laser)

Question 5

Compare soft and hard ionisation

Answer 5

• Hard ionisation is high energy but soft ionisation is low energy
• Hard ionisation tends to fragment molecules whilst soft does not

Question 6

How are ions usually created?

Answer 6

By protonation or deprotonation but may also be charged by adding Na⁺

Question 7

How can molecular weight be selected for?

Answer 7

Strength of magnetic field

Question 8

How does ESI work?

Answer 8

Sample is sprayed through a very fine capillary tube and put under a high voltage

Question 9

What is the detector detecting?

Answer 9

Mass to charge ratio

Question 10

Give 2 examples of detectors used in mass spec

Answer 10

• Time of flight
• Quadruple

Question 11

How does a time of flight detector work?

Answer 11

• Ions pass through a straight (not curved) tube
• Measures the time it take for ions to ‘fly’

Question 12

How can a peptide be sequenced using mass spec?

Answer 12

The peptide is put through a mass spec and the peptide is sequentially fragmented producing one amino acid each time, the m/z can be read out to find out the sequence

Question 13

What are the problems with sequencing a peptide using mass spec?

Answer 13

• Only works for relatively small peptide
• Some amino acids have the same Mr

Question 14

How does peptide mass fingerprinting work?

Answer 14

• Digest the protein into smaller peptides using trypsin
• Compare the resulting peptide masses to a database
• This can be used to idenity a protein

Question 15

What is a tandem mass spec?

Answer 15

2 mass specs are linked together, one separates ions by m/z whilst the other analyses fragmentation of the ions

Question 16

What is coupled mass spec?

Answer 16

The mass spec is attached to a method of chromatography (either liquid or gas) which separates the molecules and then the m/z is analysed

Question 17

What is proteomics?

Answer 17

Study of all the proteins expressed in an organism

Question 18

What is native mass spec?

Answer 18

A mass spec technqiue whereby modifications are made which allow large biomolecules and complexes of to be run in a mass spec

Question 19

Give some modifications for native mass spec

Answer 19

• Nano-electrospray, very gentleso maintains non-covalent interactions and does not fragment the sample
• Quadruple adapted with a lower radiofrequency, allows higher m/z
• Orbitrap detector which is very high resolution

Question 20

What is the oligomeric state?

Answer 20

Whether a protein is a monomer, dimer, trimer etc.

Question 21

How does a high energy collision dissociation cell work?

Answer 21

• Ions are acceleratedthrougha cell containing inert gas molecules
• Collision with molecules causes collision-induced dissociation
• Can specifically dissociate some molecules

e.g. can strip detergent from purified membrane protein

Question 22

How can the mass of a single polypeptide subunit of a polymeric protein be calculated?

Answer 22

(The charge of the ion x the mass)/the number of subunits