Sequential IP's before mass spec analysis - searching for co-ip proteins (Feb/17/2006 )

Hi-

I am using CO-IP's run on 1D SDS-PAGES, and trying to identify novel binding proteins using Mass Spec (LC-tandem MS) of unique gel bands (ie in IP of my protein but not the control IgG IP). I had planned to do 2 immunoprecipitations using two different antibodies in order to limit the background, as I have heard good things from people using the TAP system (which is a vector system that uses 2 sequential IP's), but am unsure how exactly to do it.

My stable cells express my protein with a FLAG tag and I also have a good IP antibody for my protein as well. I was going to do a FLAG IP, elute with synthetic FLAG epitope and then Re-IP with the antibody to my protein. However, I am unsure if using a lot of FLAG epitope will mess with my mass spec data. Right now I get A LOT of hits for bovine serum proteins, from the FBS I use to grow the cells.

Does anyone do mass spec here? and know if this would be a problem? Would the 1D gel take care of removing enough of the peptides? it seems that the seperation is never perfect as the mass spec is incredibly sensitive. Also, I recently discovered you can cleave FLAG tags with enterokinase. Has anyone ever used either of these two methods with or without mass spec? If one of them works better I'll definitely opt for that method. I am worried that the enterokinase would give me the same problem as the flag tag though.

Thanks for any and all help,

Mountainman

PS can a mass spec ignore a certain peptide?

hi,

i wouldn't worry about eluting w/ a flag tag, especially if this step is the 1st IP you're performing.

by binding/washing your proteins of interest to the 2nd IP column, you'll get rid of most, if not all of the flag peptide. after that, running it on a PAGE gel should definitely get rid of the remaining flag peptide. as for cleaving the epitope tag prior to MS analysis, don't waste your time w/ it, unless you're doing something like seldi tof, which doesn't seem likely, given your intentions. if you're looking to do some sort of LC-MS/MS, leaving the flag epitope on your recombinant protein won't matter, especially if your focus is to identify interactors of your tagged protein.

usually, the only time you have to worry about a peptide tag in mass spec sequencing is when performing MALDI-TOF MS w/ His tagged proteins. the His tags ionize very well in a MALDI, and produce a very strong peak that can often swamp out other important peaks. this isn't the case w/ ES ionization. basically, don't worry about it.

i hope this helps.

hi
i think that you're mentionning currently the TAP tag system
you have a very good explanation on it at http://www-db.embl-heidelberg.de/jss/servl...raphin/TAP.html

i've done four years ago tap tag followed by MSMS and that was quite good.
Having a flag tag is not a problem as you know that hits done with this peptide shouldn't be taken in count as first candidates to test.
enterokinase is an enzyme that removes a tag, but i'm not sure it's the falg one

Hello-

Thanks for your replies. My supervisor is questioning the utility of doing two sequential IP's fearing we may lose interacting proteins, as we're hoping to set this up as a blind screen for multiple interacting proteins should we get lucky.

As the two of you seem to have done this type of thing before. Did you ever compare background and/or number of interacting proteins with one versus two IP's? And/or how much better was the TAP system versus a single IP if you did the comparison?

I would love to get at least a vague feeling of how much background I'll lose and/or interacting proteins I'll lose using two sequential IP's. I might have better luck convincing my supervisor of using two IP's if I said I talked to a couple experienced people who said it was a good idea.

Thanks again,

Mountainman

"Did you ever compare background and/or number of interacting proteins with one versus two IP's?"
hem... actually not. But i remember we did relative low stringency washes in second step, in order to avoid the diminishing interactants.