W - BIOL200 - 2.7 Proteomics and Protein/Protein Interactions

What is proteomics?

Study of all proteins in a sample—what, how much, where, and how they change/interact.

What questions does proteomics help answer?

 What proteins are present, their amount, location, variants, changes in state, disease links, and drug targets.

 What is LC-MS/MS?

High-throughput method to sequence proteins using liquid chromatography and tandem mass spectrometry.

Steps in LC-MS/MS?

Digest proteins → LC separates → ionize → MS/MS cycles determine peptide sequences.

Pros of LC-MS/MS?

Identifies hundreds of proteins and their organelle origin simultaneously.

Cons of LC-MS/MS?

Requires many cells (200k–700k) and has sensitivity limits.

 What is phosphoproteomics?

 Study of phosphorylation sites and their functional impact.

What does phosphorylation affect?

Protein function and interactions.

 Limitation of phosphoproteomics?

 Few proteins are phosphorylated → needs 50–100x more cells; affinity chromatography often required.

What is ChIP used for?

Studying protein-DNA interactions in live cells.

 Steps of ChIP?

 Crosslink DNA-protein → lyse → sonicate → immunoprecipitate → isolate DNA → analyze.

What makes ChIP different from IP

 ChIP studies protein-DNA, uses sonication and specific antibodies, can use live cells.

 Why might LC-MS/MS be hard to use on C. elegans neurons?

 Requires large number of cells—hard to isolate 10 million neurons.

Can ChIP identify unknown protein interactions?

No, it identifies known DNA-protein interactions only.