Proteomics

Question 1

Bottom up proteomics (4 steps)

Answer 1

isolate proteins

reduction of disulfide briges (DTT) and free thiol groups

alkylation (IAA) - maintain disrupted bounds - add cysteine groups
(disrupt 3D structure)

digestion (proteins to peptides) by trypsin C-terminus of R or K - cuts at lysine and arginine (except when followed by proline)

Question 2

Separation of peptides (how) and focus on the common tecniques

Answer 2

pI, hydrophobicity (common) and size
MS (m/z) - vaporisation, ionisation and detection
LC-MS/MS - aa sequencing - separation of peptides prior by decreasing polarity - detection - selected for fragmention
fragmentation at N-terminal (B-series) or C-terminal (y-series)

Question 3

FDR

Answer 3

false discovery rate - proportion of predicted positives that are false
FDR= decoy count (FP) / Target Count (FP+TP)

Question 4

Quantification

Answer 4

Relative (ratio) or absolute (mole/volume)
chemical tags (MS/MS - isobaric) or label free