Today's quiz section involves interpretation of an antibody pull-down assay using mass spectrometry, and validation of the results using existing publicly available databases.

DTASelect

We will be using a new tool for the visualization of mass spectrometry results. This tool is called DTASelect. It takes mass spec results and filters the peptide identifications against thresholds that determine if the identification was real or not. The peptide identifications that are real are then grouped according to the protein they belong to, and listed in an HTML file. Below is an image of one protein from a DTASelect analysis.

A = protein accession number
B = percent of protein identified by peptides in the sample
C = name of protein and description
D = mass spectra of peptides related to this protein
E = number of times this peptide was observed in the sample
F = peptide sequence
G = statistics and scores that were used to qualify this peptide as "real"

Yeast Resource Center: Public Data Repository

This is a public, online database of experimental results that can be used by any researcher. The database can be found HERE.

The YRC: PDR is very well documented. So as you navigate through the pages, be sure to read their explanations of in the information being presented to you!


To get started with the YRC: PDB, just type in the gene abbreviation you are interested in. Above, we are going to query ADH1, or alcohol dehydrogenase 1, a very, very important gene.


You will get a list of results similar to what is shown above. The results include your gene of interest, plus similarly described genes, or homologous genes in other organisms. However, the right most column, "Data Available", will tell you if the YRC: PDB has a novel contribution to your gene of interest. As you can see, our gene, ADH1, is listed first and contains mass spectrometry (MS) and protein structure (PSP) data. Click on ADH1 to access this information.

The next page (image too big to reproduce here - so pay attention in quiz section) contains a summary of the experiments done on ADH1 in relation to protein-protein interactions. There are six major sections, but I will only go over the four that are relevant to today's homework.
Protein Overview (blue section) - This section gives background information about the protein, such as its function, gene ontology annotation, and amino acid sequence.
Protein Complex Data (yellow section) - This section gives bubble-and-stick diagrams of how your protein interacts with other known proteins. There are usually multiple diagrams, each one based on the results of different experiments. Notice how some experiments can be fairly non-specific, and may predict more interactions than really exist. Others may miss real interactions.
Mass Spectrometry Data (green section) - This section shows interactions that have been identified by antibody pull-downs analyzed by mass spectrometry. Essentially, a bait protein was used to enrich for other proteins that interact with it. The green table shows all the bait proteins that were used in which ADH1 was associated with it.
Yeast Two-Hybrid Data (purple-ish section) - This section is similar to the mass spec section, but instead of antibody pull-downs, yeast two-hybrid experiments were done. This section lists any proteins that were used as bait to identify interactions to your protein of interest.

IMPORTANT: The database only contains DATA THAT INDICATES A POSSIBLE INTERACTION, not proof that the interaction is real. Many of the conditions are artificial to how they would normally exist in the organism. However, you can view any of the results that were submitted and choose whether or not you think the data indicate a valid interaction.

Homework, Due Monday 5pm

You are interested in a particular yeast protein, Tub4. In particular, you would like to know what proteins interact with Tub4. So you perform an antibody pull-down of Tub4, and presumably, any proteins that interact with it. Thus, you now have a sample that is enriched for Tub4 and any proteins associated with it. You decide to analyze this enriched sample using mass spectrometry. You digest the sample into peptides, and use reverse-phase liquid chromatography coupled with ion trap fragmentation to identify the proteins in the sample.

Here are your experimental results: Enriched Sample DTASelect File

Being a thorough scientist, you also run a negative control experiment. This is essentially the same as the protocol above, however, this time you enrich for nothing instead of Tub4. (Think for a moment... what does it mean to enrich for nothing? How is this different from not enriching at all?) Any proteins identified in this control can therefore be considered background.

Here are your control results: Control DTASelect File

Use the DTASelect files and the YRC: PDB to answer the following questions:

Question #1: Why is there a rabbit protein in your experimental results?
Question #2A: What proteins are in your control results, but not in your experimental results?
Question #2B: Give an explanation of what you are seeing and why this might happen?
Question #3: What proteins might be complexed with Tub4?
Question #4: For each protein listed in question #3, answer: (A) Do you think they are real? and (B) why?

Submit your answers by email to maxboeck@u.washington.edu
Please just type your answer in the body of the text (do not send word documents or attachments).