Peer review of: Split Intein-Mediated Protein Ligation for detecting protein-protein interactions and their (2020)


 
Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
I thank the authors for the thorough explanations that have dissipated my concerns. I think the 
manuscript is much improved and deserves publication. A side note: recently, a different method, 
also based on split inteins, has been published that has practically the same name, albeit being 
written differently: SiMPl (Palanisamy et al., Nature communications, 2019, 10:4967). It could be 
confusing for the scientific community to have two methods that are used for very different 
purposes, with practically the same name. I am sure the authors are attached to “SIMPL” as name 
for their method. However, if they could imagine changing it, it may help. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
I was equivocal in my view of the suitability of the original version of this manuscript. My concerns 
centered on two major points. First, the authors demonstrate no challenging example of how their 
method works, instead showing data only for cases in which the answer was already known. 
Demonstrating a novel protein-protein interaction method without using it to detect a novel 
interaction is troublesome, as much can happen when the answer is not known, most of it not 
good. Second, the lack of any useful quantification, especially by Western blot but even in terms of 
how to interpret ELISA results, is extremely problematic. I believe that my concern here is similar 
to the other referee’s concerns about issues like expression levels, foldability of the fusions and 
effects of the orientation of the tags, and can be partly captured in the questions: How does a user 
of the method know if a signal for interaction is real? How much prior testing of fusions, use of 
standards, comparison to previous ELISA results, reversal of tag orientations, expression of the 
fusions at variable levels, etc. must be done to be confident that one is either detecting or not 
detecting an interaction? 
 
In response to my concerns, the authors provide no substantive new experiment to address them. 
Instead, they seem to argue, at considerable length, that my concerns are unwarranted. 
Regarding their carrying out a single screen to demonstrate that the SIMPL method is able to 
detect a novel interaction, they write that this is beyond the scope of this manuscript. I 
respectfully disagree. If it takes three years of a new grant to demonstrate this application, then 
perhaps the method at this time is still early enough in its development that publication is 
premature. 
 
Regarding the quantification of Western analysis, they write that “it is very difficult to set universal 
standards for Western analysis-based assays.” This seems tantamount to saying that their assay 
results in Figures 1c, 1d, 1e, 1f, 2b, 4a, 4b, 4c and Supplementary Figures should be viewed with 
caution because they are not quantifiable. 
 
The text still contains numerous worrisome rationales or qualitative comparisons, such as: 
(l. 176) It should be noted that a basal splicing signal appeared….However, the corresponding 
rapamycin-treated sample shows a dramatically increased signal… 
(l. 199) Although two combinations…showed relatively elevated basal splicing levels, these 
background signals did not interfere… 
(l. 251) …while no obvious signal was observed. Assay with the…construct exhibited a more 
marked splicing signal.…We speculate that the signal enhancement is caused by…N-terminal 
tagging… 
(l. 265) We reason that the high basal signal…derived…when its local concentration is 
high….Accumulated background splicing protein masked the signal… 
 
I am sorry that my previous comment about Figure 3f was unclear, and see how this lack of clarity 

might have led the authors to explain how ROC analysis works. I was asking if the authors could 
quantify, e.g. in a heatmap, how each of the interactions of the Positive Reference Set behaved in 
the SIMPL assay, rather than only indicating by a red box that the signal was “positive.” However, 
they do now provide these data in Supplementary Figure 3. 
 
My request for the ELISA results to be at least roughly calibrated to signals on the Western blots 
elicited the response that I should compare Figure 2b with 3C, but I do not believe that viewing 
these two panels adequately addresses this request. 
 
My request for a clear statement of what constitutes “detection” of an interaction was addressed 
with the comment that I should see the response to Comment 3 (where I could not find the 
answer) and that “Additionally, like all screening methods, use of sophisticated statistical 
algorithms can greatly aid in hit identification and analysis.” 
 
Given that my major concerns from the original manuscript have not been resolved, I do not find 
that the revised manuscript is acceptable.