Proteomics

The proteome is all proteins in a cell encoded by the genome; proteomics is the study of these proteins and their activities.

Before we can study them, we need to get the proteins out of the cell – so, break the cells either manually (blender, sonicator, glass beads), with detergents (these remove cell membranes) or cycles of freeze/thaw. Then, …

Proteins can be separated based size and/or charge (polyacrilimide gels and column chromatography). Note that unlike nucleic acids, amino acids do not have the same charge because the amino acids have the different R groups. Therefore, SDS is used to help to denature proteins and to give them a net negative charge.

How can we pick out a specific protein after separating samples on a gel? Antibodies and western blots -- note that this will give us the protein's location on the blot and if it's in a sample. Understand how an antibody recognizes a specific protein, and how Western blots are the protein equivalent of a Southern blot (DNA) or a Northern blot (RNA).

Mass spectroscopy (MALDI-TOF and electrospray) allows for the precise measurement of the molecular weight of the protein, and from the molecular weight, we can determine the identity of the protein itself. 

Protein tags (MBP, His-tag, FLAG-tag, Strep) can be added to proteins by cloning the coding sequence with the tag sequence. The two get expressed linked together, and now there’s a handy “tag” on your protein of interest that allows you to isolate it easily.

Phage display allows us to screen for particular proteins based on function – does the protein bind another specific protein, does it interact with RNA?. The power of this technique comes from being able to screen for a function, and then have the coding sequence for proteins with that function.

Yeast 2-Hybrid is a large scale screening methods that can identify interactions between proteins. Understand the basic premise of this method -- that if two proteins interact, they form an intact transcription factor and turn on transcription of a reporter gene. The gene coding sequence of expressed proteins are cloned into a vector with the DNA binding domain of a transcription factor; this cloning is repeated for the vector with the transcription factor's activator domain. Yeast strains carrying the two separate vectors are mated (this creates cells with both vectors in them), and if the two proteins interact and form the intact transcription factor, the mated cells will transcribe, and then translate, the reporter gene. How is this reporter gene detected? How can the results of yeast 2-hybrid be confirmed?

Co-Immunoprecipitation (or co-IP). First understand how immunoprecipitation works, in that an antibody is added to a cell lysate (or other sample with proteins in it) and recognizes/binds to its epitope on a protein. This interaction makes for a 'heavy' complex that can be isolated by centrifuging the sample (hence, precipitate. Beads with protein A can also be added to help with the isolation). Anything that interacts with the protein that the antibody binds will get precipitated, too. Understand how we see what is precipitated or pulled-down. For the Co-IP part, this confirms that two proteins are in the same precipitated complex by running immunoprecipitations in parallel, and then checking if the other protein was also in the precipitate.