On 2017 Jul 18, Youhe Gao commented:
The strategy I posted here last month was published three years ago on Proteome Sci. 2014 Feb 1;12(1):6. doi: 10.1186/1477-5956-12-6. Fast fixing and comprehensive identification to help improve real-time ligands discovery based on formaldehyde crosslinking, immunoprecipitation an... - PubMed - NCBI https://www.ncbi.nlm.nih.gov/pubmed/24484773
On 2017 Jun 14, Youhe Gao commented:
I found two "overexpression"s in the paper. "Although the extent of bait overexpression is difficult to judge and varies across IP's, previous experimentation has shown that over-expression has little effect on identification of true interacting partners (Sowa et al., 2009)" "VAPBWT overexpression strongly increased the association of EGFP-LSG1 and OSBP with the ER (Figure 7E,G)" Personally, I am not sure if those are enough. In a system, increasing [A] or [B] will lead to more [AB]. As we know more about protein interaction now, this kind of systematic false positive should not be ignored any more. In cells, overexpression with tag may even change the location of the protein. That is why I think the next generation of massive protein interaction studies should start from in vivo crosslinking. I do not want to overemphasize the problem. Most of the protein interactions identified are probably true in cells. The amount of work done is very impressive and respected. I hope users who is using a particular interaction data as the only clue for their future experiment design, maybe they should start with an in vivo crosslinking as a conformation of that interaction. It may make them more confident to proceed.
On 2017 Jun 13, J Wade Harper commented:
These issues have been addressed and discussed in many prior publications (see for example - Cell. 2015 Jul 16;162(2):425-40) and are widely known
On 2017 Jun 09, Youhe Gao commented:
I hope that authors can discuss the impact and possibility of false positive and negative made by tagging, overexpression and washing conditions on protein interactions.
On 2017 May 31, Youhe Gao commented:
This is a great master piece in the field of protein interaction. It is the largest network so far. It is extremely valuable for all biologists. I guess for the authors to reach such a great throughput, using tagging, overexpression and affinity purification was almost inevitable. These procedures could produce some false positives and false negatives. A strategy named 4F-acts was proposed a few years ago trying to minimize false positives and false negatives. Fast Fixation is necessary to study real-time protein-protein interactions under physiological conditions. Fast formaldehyde crosslinking can fix transient and weak protein interactions. With brief exposure to a high concentration of formaldehyde during the crosslinking, the complex is crosslinked only partially, so that the complex is small enough to be resolved by SDS-PAGE, and the uncrosslinked parts of the proteins can be used for identification by shotgun proteomics. Immunoaffinity purification can Fish out complexes that include the proteins of interest. Because the complex is covalently bound, it can be washed as harshly as the antibody-antigen reaction can stand; the weak interactions will remain. Even if the nonspecific binding can persist on the beads or antibody, it will be eliminated at the next step. To Filter out these complexes, SDS-PAGE is used to disrupt non-covalent bonds, thereby eliminating uncrosslinked complexes and simultaneously providing molecular weight information for identification of the complex. The SDS-polyacrylamide gel can then be sliced on the basis of the molecular weight without staining. All the protein complexes can be identified with the sensitivity of mass spectrometry rather than sensitivity of the staining method. The advantages are the following: (i) The method does not involve tagging. (ii) It does not include overexpression. (iii) A weak interaction can be detected because the complexes can be washed as hard as the antigen-antibody reaction can stand as the complexes are crosslinked covalently. No new covalent bond can form as a false positive result. (iv) The formaldehyde crosslinking can be performed at the cellular, tissue, or organ level fast enough so that the protein complexes are fixed in situ in real time. The throughput of this strategy is probably not high enough. But I hope one day a large-scale study can be conducted with it. No matter what, this is a great milestone!
On 2017 Jun 03, J Wade Harper commented:
The results are being loaded now into biogrid. IntAct I believe will take data from biogrid to upload directly.
On 2017 May 29, Christopher Southan commented:
Will these valuable results be loaded into the EBI IntAct Molecular Interaction Database? (http://www.ebi.ac.uk/intact/)
On 2017 May 29, Christopher Southan commented:
Will these valuable results be loaded into the EBI IntAct Molecular Interaction Database? (http://www.ebi.ac.uk/intact/)
On 2017 May 31, Youhe Gao commented:
This is a great master piece in the field of protein interaction. It is the largest network so far. It is extremely valuable for all biologists. I guess for the authors to reach such a great throughput, using tagging, overexpression and affinity purification was almost inevitable. These procedures could produce some false positives and false negatives. A strategy named 4F-acts was proposed a few years ago trying to minimize false positives and false negatives. Fast Fixation is necessary to study real-time protein-protein interactions under physiological conditions. Fast formaldehyde crosslinking can fix transient and weak protein interactions. With brief exposure to a high concentration of formaldehyde during the crosslinking, the complex is crosslinked only partially, so that the complex is small enough to be resolved by SDS-PAGE, and the uncrosslinked parts of the proteins can be used for identification by shotgun proteomics. Immunoaffinity purification can Fish out complexes that include the proteins of interest. Because the complex is covalently bound, it can be washed as harshly as the antibody-antigen reaction can stand; the weak interactions will remain. Even if the nonspecific binding can persist on the beads or antibody, it will be eliminated at the next step. To Filter out these complexes, SDS-PAGE is used to disrupt non-covalent bonds, thereby eliminating uncrosslinked complexes and simultaneously providing molecular weight information for identification of the complex. The SDS-polyacrylamide gel can then be sliced on the basis of the molecular weight without staining. All the protein complexes can be identified with the sensitivity of mass spectrometry rather than sensitivity of the staining method. The advantages are the following: (i) The method does not involve tagging. (ii) It does not include overexpression. (iii) A weak interaction can be detected because the complexes can be washed as hard as the antigen-antibody reaction can stand as the complexes are crosslinked covalently. No new covalent bond can form as a false positive result. (iv) The formaldehyde crosslinking can be performed at the cellular, tissue, or organ level fast enough so that the protein complexes are fixed in situ in real time. The throughput of this strategy is probably not high enough. But I hope one day a large-scale study can be conducted with it. No matter what, this is a great milestone!
On 2017 Jun 09, Youhe Gao commented:
I hope that authors can discuss the impact and possibility of false positive and negative made by tagging, overexpression and washing conditions on protein interactions.
On 2017 Jun 13, J Wade Harper commented:
These issues have been addressed and discussed in many prior publications (see for example - Cell. 2015 Jul 16;162(2):425-40) and are widely known
On 2017 Jul 18, Youhe Gao commented:
The strategy I posted here last month was published three years ago on Proteome Sci. 2014 Feb 1;12(1):6. doi: 10.1186/1477-5956-12-6. Fast fixing and comprehensive identification to help improve real-time ligands discovery based on formaldehyde crosslinking, immunoprecipitation an... - PubMed - NCBI https://www.ncbi.nlm.nih.gov/pubmed/24484773