Bioinformatics 12: Gene regulation 2

Question 1

What is mass spectrometry? Advantages?

Answer 1

Technique which ionizes chemical species and sorts the ions into a spectrum based on their mass-to-charge ratio

Very accurate + sensitive, analysis is fast

De novo determination of protein post-translation modifications

Question 2

Method of sample extraction and preparation for mass spec?

Answer 2

Fractionation of mixed protein sample via 2D protein electrophoresis

Separated into spots (individual proteins) by mass and charge

Protein spots extracted -> digestion (trypsin) to make smaller + prep for MS

Mass spec fingerprint created -> comparison to fingerprint database

Question 3

What is cRAP?

Answer 3

Database of common contaminants often found in mass spec

Question 4

Applications of mass spec in genetics / genomics? How/why? Issues?

Answer 4

A>T differ by 9, C>G differ by 40

• Useful in SNP genotyping
• Detect epigenetic modification e.g. methylation changes
• Gene expression analysis (relative to internal standard)
• Non-invasive pre-natal diagnostics (Down’s, HD)

Issues:
Low number of assays at a time
Array prep slow

Question 5

What is the yeast 2-hybrid system?

Answer 5

Method of rapid identification of a protein’s interaction partners

Question 6

WHat is an interactome and what does it show?

Answer 6

Maps interactions of proteins with other proteins
- some interact with many partners, most with only a few

Explains redundancy in genetic networks

Question 7

Low throughput methods of looking for protein interactions?

Answer 7

Chemical crosslinking

Co-immunoprecipitation

FRET

SPR

BioID

Question 8

High throughput methods of looking for protein interactions?

Answer 8

ChIP-seq (enabled by Next Generation Sequencing, NGS)

Phage display

Question 9

Main protein interaction database?

Answer 9

BioGRID