Intro To Proteomics Flashcards
0
Q
Problems with 2DE/MS
A
- limited by the bias of the method against certain protein groups
- very laborious
- many steps
- limited ability to resolve proteins in complex mixture
- low dynamic range of detection
1
Q
Challenges in proteomics
A
- diverse chemical composition if proteins
- extreme differences in protein abundance
- change of abundance over time
- different localizations
- post translational modifications
- interaction partners
2
Q
General Ms- based proteomics workflow
A
- sample fractionation
- proteins digested with trypsin to peptide mixture
- peptide chromatography and ESI
- Ms
MS/MS
3
Q
Examples of gel-free proteomics analysis
A
Metabolic labeling (SILAC) Chemical labeling (iTRAQ)
4
Q
SILAC
A
Stable isotope labeling with amino acids in cell culture
Proteins labelled by growing cells in media containing isotopic ally labeled amino acids
5
Q
Advantages of SILAC
A
Labeling allows mixing of differentials labelled samples soon after cell lysis
- no sample variation
- accurate quantitation
- reagents are cheap
6
Q
Disadvantages of SILAC
A
- amino acids used for labeling might not be present in all proteins
- minimal medium
- some SILAC organisms are not practical for samples
- 2-3 conditions at one time
7
Q
ITRAQ
A
Isobaric tags for relative and absolute quantification
In Vitro labeling involves incorporation is stable isotopic tags at selective sites on peptides
8
Q
Advantages if iTRAQ
A
- isobaric tags lead to reduced complexity
- can handle up to 8samples
- all peptides are labelled
9
Q
Disadvantages of iTRAQ
A
- very expensive
- not all MS machines can be use for iTRAQ
