I am crosslinking cells with 0.1% formaldehyde and performing an IP in order to isolate protein-protein complexes. The downstream analysis for this experiment is mass spectrometry. I am interested in finding protein interacting partners. Is it necessary to reverse the formaldehyde cross link prior to mass spec? Does anyone have experience with this?
I've tried heat to reverse the crosslink, but am having trouble with protein precipitating out of the solution. So if I can skip the reversal step, I will no longer have to worry about protein precipitating out. Thanks for your advice!
Hi Samantha,
Although I don't have experience with the technique, I'd say that keeping the cross-link will actually aid identification of the interaction partners of your protein (complex) of interest using MS, so I wouldn't bother reversing the cross-link!
Hope this helps.
The problem with formaldehyde is the non-specificity of the X-link. Formaldehyde will react with any amine. Contrary to the literature on the mechanism it is only partially reversible with heat and pressure. I think the "Schiff base", which supposedly can't progress to a more permanent bond, actually does. The duration of the crosslinking and the age of the linkage post-crosslink both matter for the degree of reversal. We spent several years doing this for proteomics analysis, but sorry never published anything since it was all negative results. There is a patent by O'Leary et al (US8481283) that does work partially. It is the best approach to reversal that we've used, but it is only partial and then only really works for cytosolic proteins. You can keep the proteins in solution at the high temperatures using the Pressure Biosystems TD buffers. It's the only way (see the attached citation).
I would look at Jonathan Amster's work on protein-protein crosslinking for MS analysis. He extended some of the work that we pioneered. He's published. We haven't, sorry.
Article Method for Recovery and Immunoaffinity Enrichment of Membran...
Do you run a gel before mass spec or do you run directly what you eluted from the beads? Have you ever run your samples (IP eluates) onto a gel? I tried this method without reversion, my sample looked like a single smear after WB staining for my protein and the proteomic core facility told me they couldn't run the sample on mass spec. So reversion sounds pretty important to me.
Hi Henri -- we are sending our samples to a core facility for in-solution digestion. Therefore, they do not need to be run on the gel.
Hi,
I am going to do crosslinking for CO-IP. I will use formaldehyde could you please help me with the protocol. Thank you Samantha Jeschonek
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