Mass Spectrometry Flashcards
Give a basic outline of mass spectrometry
- Gaseous sample is loaded
- The ionization chamber generates ions
- Ion are separated in some way
- Ion detector detects ions
How does hard ionisation occur?
Electron ionisation
What techniques can be used to produce soft ionisation of a sample?
- MALDI
- ESI
What is MALDI?
Matrix assisted laser desoption ionisation (place in a matrix with a laser)
Compare soft and hard ionisation
- Hard ionisation is high energy but soft ionisation is low energy
- Hard ionisation tends to fragment molecules whilst soft does not
How are ions usually created?
By protonation or deprotonation but may also be charged by adding Na+
How can molecular weight be selected for?
Strength of magnetic field
How does ESI work?
Sample is sprayed through a very fine capillary tube and put under a high voltage
What is the detector detecting?
Mass to charge ratio
Give 2 examples of detectors used in mass spec
- Time of flight
- Quadruple
How does a time of flight detector work?
- Ions pass through a straight (not curved) tube
- Measures the time it take for ions to ‘fly’
How can a peptide be sequenced using mass spec?
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
What are the problems with sequencing a peptide using mass spec?
- Only works for relatively small peptide
- Some amino acids have the same Mr
How does peptide mass fingerprinting work?
- Digest the protein into smaller peptides using trypsin
- Compare the resulting peptide masses to a database
- This can be used to idenity a protein
What is a tandem mass spec?
2 mass specs are linked together, one separates ions by m/z whilst the other analyses fragmentation of the ions
What is coupled mass spec?
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
What is proteomics?
Study of all the proteins expressed in an organism
What is native mass spec?
A mass spec technqiue whereby modifications are made which allow large biomolecules and complexes of to be run in a mass spec
Give some modifications for native mass spec
- 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
What is the oligomeric state?
Whether a protein is a monomer, dimer, trimer etc.
How does a high energy collision dissociation cell work?
- 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
How can the mass of a single polypeptide subunit of a polymeric protein be calculated?
(The charge of the ion x the mass)/the number of subunits
