RE - Proteomics and protein-protein interactions

Question 1

What 3 fields are proteomic techniques applicable to?

Answer 1

• Fundamental biological questions
• Health and disease
• Agriculture and environment

Question 2

What can proteomics can tell us about proteins? (4)

Answer 2

• Protein sequence
• Post-translational modifications
• Level of expression (abundance)
• Protein interactions

Question 3

What are the main processes that contribute to the dynamic and transient nature of protein interactions? (3)

Answer 3

• Translocation
• Modification
• Synthesis and degradation

Question 4

What is the Protein interactome?

Answer 4

The complete set of protein-protein interactions in a biological system

• Estimate from estimated number of proteins and existing protein-protein interactions

Question 5

How is the protein-protein interactome discovered?

Answer 5

Binary interactions + Affinity purification
–> Protein interaction network
–> Protein interactome

Question 6

What are 3 ways to identify protein-protein interactions?

Answer 6

a) Immunochemical purification
b) One-step affinity purification
c) Two-step affinity purification

–> Mass spectrometry proteomics

Question 7

What is the difference between one-step and two-step affinity purification?

Answer 7

One-step affinity purification:

• The target protein is purified in a single affinity chromatography step using an immobilized ligand that binds specifically to the target.
• Simpler and faster, but may have lower purity if the target protein binds non-specifically to the matrix.

Two-step affinity purification:

• The target protein is purified through two consecutive affinity chromatography steps using two different ligands that bind to distinct regions of the target.
• More efficient in removing contaminants and increasing purity

Question 8

What is involved in Affinity-purification mass-spectrometry? (2)

Answer 8

• Use antibody to purify “bait” protein and associated interactors
• Use mass-spectrometry techniques to identify and quantify the proteins

Question 9

What techniques are involved in protein separation and what are the challanges?

Answer 9

Separate the proteins into simpler ‘fractions’ before further analysis

Techniques include:

• I-dimensional SDS Polyacrylamide electrophoresis (separate proteins by size)
• 2-dimensional gels (separate by mass and charge)

Challenges include:

• Hydrophobic (membrane) proteins
• Low abundance proteins

Question 10

Why may proteins be digested into peptides before analysis?

Answer 10

Analysis of high molecular weight molecules (e.g. intact proteins) challenging

Analyze peptides derived from intact proteins

• Greater solubility, smaller size …
• Peptides serve as ‘tag’ for the proteins from which they are derived

Requires Protease

Question 11

How does peptide fragmentation occur?

Answer 11

The mass spectrometer imparts energy into the peptide causing it to fragment at the peptide bonds between amino acids

Question 12

What 2 pathways are involved in identifying peptide sequences?

Answer 12

1) Protein –> Peptides –> MS spectra of peptides

2) Protein database –> Peptides predicted from proteolysis –> in silico MS pattern from theoretical peptides

–> Matching –> Identified peptides/proteins

Question 13

How can protein networks be explored?

Answer 13

Using a database like STRING

• Integrates protein-protein interactions with indirect functional associations
• From large-scale e.g. proteomics experiments and small-scale studies
• Provides a combined score for each interaction
• Available for many different organisms