Proteomics

Question 1

Aims

Answer 1

• examine protein expression levels (spatio-temporal)
• examine post-translational modifications
• examine protein interactions

Question 2

2D Electrophoresis

Answer 2

• separation technology for proteomics (1st stage)

- however, just tells us there is a protein present not the identity

Question 3

Pros of Gels

Answer 3

1. quick and cheap
2. works with heterogenous or crude samples
3. visual representation/comparison of entire sample
4. reproducible

Question 4

Proteomics Mapping Strategy

Answer 4

1. 2D gel
2. excise spots identified as different (vs control)
3. in gel tryptic digest
4. MALDI MS mixture of tryptic peptides
5. database search to find protein / further analysis
6. if unknown compare to computer generated tryptic peptide for identification
   - can fragment selected peaks to determine sequence (if unknown i think)

Question 5

Cons of Proteomic Mapping

Answer 5

• need complete genome to find protein identity
• relying on digestion being complete and correct
• PTM can affect this

Question 6

Top Down

Answer 6

• sequence intact complexes
• eg. spectra of ribosomal subunits using an ES-Q TOF mass spectrometer
• difficult and specialist
• uses multiply charged molecular ions that bring the m/z ratio down into a usable spectral range