proteaMatt

I asked this question on another forum, one of the posters showed me a solution that works perfectly for what I need.

STRAP - http://www.ncbi.nlm....les/PMC2787672/
http://www.bumc.bu.e...cpctools/strap/

Just to clarify my work flow a little -

• Digested a whole cell lysate
  
• SCX MuDPit
  
• C18 LC-ESI
  
• Paragon search of collected masses from each of the MuDPit fractions in ProteinPilot

This yielded a long list of protein IDs. It would be possible to go down the list of IDs and look them up individually using UniprotKb (or something like that on expasy), but this process would be quite time consuming. I know that ProteinCenter can do this for a data set at once, but we don't currently have a license for that software.

Maybe I misunderstood your first post. But the difference between what I took as your suggested workflow and what I suggested is the point at which you do the IP. It seemed like you are concerned if Co-IP will still occur if you do the USP5 digestion on the whole lysate, so take the guess out of it and let the MALDI sort it out.

I guess the point I am making is this - if you aren't sure how the about the above workflow then just do 1. the Co-IP on the whole lysate (I am assuming you already know that your POIs Co-IP in their "intact" state), 2. apply treatments to the POIs (use whatever petidase you want here), 3. let the MALDI sort it out.

If you want to have a set of data to cross reference with the intact analysis I would suggest doing an in-solution tryptic digestion on the Co-IP samples and then analyze that by LC-MS, this could give you the exact modification sites of your POIs.

It seemed like you wanted to treat the Co-IP as sort of like an assay, and I am suggesting to treat it as a sample prep method and analyze with MALDI.

This seems a like an interesting experiment that I could learn from, since I haven't done IP before. I don't know if you have access to MALDI-TOF, but that seems like the perfect way to analyze these proteins, depending on the condition of the sample (how "clean" it is) and the molecular weights of your POIs. It seems to me like you could perform IP on the your whole lysate, which would leave you with purified POIs. You could then follow that up with MALDI analysis (likely using Sinapic Acid as the matrix) with three samples - 1. the intact associated protiens, 2. disassociate the proteins but leave the ubiquitination intact (would be convenient if DTT could do this for you), 3. Isopeptitase enzymatically treated POIs.

If your hypothesis is true that ubiquitination contributes to the association, after MALDI you should know the masses of: 1. each POI without ubiquitin, 2. the ubiquitinated intact associated POIs, 3. the two POIs disassociated, which should allow you to determine which one retains the PTM due to the mass shift from #1

Maybe I'm totally off base with this, but that seems like it could work, if you don't have MALDI available you could try that with 2DE and Western Blot.

Do you have access to a Gel Doc or some other gel imaging system? I know with the system we have you can perform densitometry on coomassie stained gels. If you don't have access to a machine like that, you might be able to find another method to perfom densitometry using equipment you do have access to. Perhaps a google (scholar) search for densitometry and SDS-PAGE could get you started.

An easy approach might be to :

• run your sample in duplicate on a gel
  
• divide the gel in half
  
• coomassie stain one half, do your transfer with the other half
  
• do densitometry on your coomassie stained half to know how much of your POI is there
  
• proceed with edman degradation with the transferred half based on your findings from the coomassie stained half

I don't have the details on all of this, because I don't have much experience with it, but hopefully this can at least point you in the right direction to start looking.