When performing ChIP-proteomics/ChIP-MS based techniques, if one chooses to do X-ChIP using standard 1% formaldehyde cross-linking is it necessary to reverse the cross-links before performing mass-spec based proteomics techniques?
Many studies tend to use SDS PAGE prior to mass-spec which would lead to cross-link reversal when samples are boiled. Is it possible to send the beads or eluate directly for analysis?
Hi Lee, I don't know much about proteomic analysis, but I do know that formaldehyde mostly crosslinks -NH2 functional groups.
This formaldehyde crosslink can be partially reversed by adding excess free -NH2 compounds. Tris in SDS PAGE buffers has a free -NH2 group. Boiling would help the reversion.
Dear Lee, it is a known fact that formaldehyde fixation results in extensive cross linking of proteins to themselves and with the nucleic acids, which overall reduces protein extraction efficiency qualitative and quantitative in both ways. So there is an immense web of protein-protein and protein-nucleic acid cross links side by side within each cell. Intranuclear DNA-histone complex, which although is quite dense in nature, but is more reactive to formaldehyde. So performing downstream without any satisfactory formaldehyde reversal, I won't go for it at all.
That is why, often researchers prefer to do deparaffinization with methanol or hapten, which is more effective instead of conventional xylene. And it is also established now that the use of SDS (2 %) and 2-mercapto (4 %) along with high temperature exposure (99 C for 20-30 minutes) in a buffer solution like Laemmli to xylene-treated pellets is doing the most efficient reversal of protein cross links either by dry heat or boiling.
With regards.....
Dear Lee,
It is possible to carry out "on bead proteolysis". Eg., such as the "Click-iT protein enrichment kit" from Invitrogen : protocol which is designed to look for newly synthesized proteins utilizes on bead trypsin digestion of proteins. After extensive washings, the protein bound on the bead is digested with trypsin. I have obtained good data with the approach in the protocol.
On the other hand, if you can indeed reverse the cross-link and carry out SDS-PAGE, in gel digestion of the purified proteins should also result in successful proteomics experiemnts.
Good luck,
Hediye.
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