Intro To Proteomics Flashcards Preview

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Flashcards in Intro To Proteomics Deck (10):
0

Challenges in proteomics

- diverse chemical composition if proteins
- extreme differences in protein abundance
- change of abundance over time
- different localizations
- post translational modifications
- interaction partners

1

Problems with 2DE/MS

- 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

2

General Ms- based proteomics workflow

- sample fractionation
- proteins digested with trypsin to peptide mixture
- peptide chromatography and ESI
- Ms
MS/MS

3

Examples of gel-free proteomics analysis

Metabolic labeling (SILAC)
Chemical labeling (iTRAQ)

4

SILAC

Stable isotope labeling with amino acids in cell culture

Proteins labelled by growing cells in media containing isotopic ally labeled amino acids

5

Advantages of SILAC

Labeling allows mixing of differentials labelled samples soon after cell lysis

- no sample variation
- accurate quantitation
- reagents are cheap

6

Disadvantages of SILAC

- 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

ITRAQ

Isobaric tags for relative and absolute quantification

In Vitro labeling involves incorporation is stable isotopic tags at selective sites on peptides

8

Advantages if iTRAQ

- isobaric tags lead to reduced complexity
- can handle up to 8samples
- all peptides are labelled

9

Disadvantages of iTRAQ

- very expensive
- not all MS machines can be use for iTRAQ