The study reports that 18% of the essential genes tagged with a TAP tag resulted in haploid cells that were not viable. Do you think that 1/5 of studied proteins being left inactive from this large tag causes serious doubt for this study's efficacy? For the 4/5 of proteins that were active, isn't it still likely that the tag distorted the protein's abilities for interaction causing questionable results?
I had two questions concerning the primary article. First, in the protein complex interaction map, there were a lot of lone islands of proteins. What was the motivation behind including these in a protein interaction map where there is no interaction for those particular complexes?
Second, the article keeps mentioning "essential" proteins but this was never consistently defined. The section talking about orthologues and their significance, the researchers termed the orthologues to be the "essential" proteins. But then they go on to separate "essential" vs "nonessential" differently. I guess I just don't understand the definition according to this paper.
In the primary article, their experiments are done in yeast and comparisons are made to human orthologues. They then make conclusions about the eukaryotic 'core proteome' in general based off of these two organisms? Is two a sufficient sample base of eukaryotic organisms to validate this claim? Also, how do they distinguish essential from non-essential genes/proteins?
The authors found that orthologous proteins preferentially interact with complexes enriched with other orthologues, but they began with many more orthologues than non-orthologous proteins. Do you think their support of the 'orthologous proteome' is warranted, or do you think it is an experimental artifact?
My first question is similar to Victoria's in that one of the drawbacks of this method seems to be that adding the tag to the 3' end can disrupt the ability of proteins to interact. Since this paper was published over 10 years ago, has a better tagging method been developed as to not disrupt the C-terminus?
My second question is that throughout the paper they use the phrase "entry point" multiple times. Is this referring to the protein that is used to identify the complex (so you could identify the same complex through all of its components)?
In regards to the tags used in the primary article, they mentioned a number of issues that affect the viability of the cell and detection of the proteins. Is it also possible that the tag could cause unnatural interactions as opposed to inhibiting them?
The primary article mentions multiple ways that this method could lead to false positives and abnormal results due to their experimental strategy. It's been 12 years since these findings were published. What proteomic techniques today do you think should be used to confirm these results?
The following sentence is in the "Sensitivity, specificity, and reliability of the approach" section: "Because we used haploid cells, we were able to score for viability." I don't understand what they mean by it. Also, figure 4 is a crazy association map like the one we talked about last week. What is your critique of this diagram? How could the authors have illustrated their findings in a clearer manner?
The primary article indicates that 102 proteins "showed no detectable association with other proteins when purified directly, or as part of other complexes." Do you think that changing surrounding biological conditions (heat, temperature, pH) could have significantly altered this degree of association, and could it have lead to alternate formation or function of protein complexes?
The primary article states that the "...TAP/MS method does not provide information on the orientation of complex components..." identified within the yeast proteome. Is there a method that they could have used in this study to look at the various identified proteins orientation within the cell or a method that has been developed today (since the paper is 12 years old)?
In the primary article they stated that ribosomal proteins were identified when the mock-transformed control strains were purfied. From what I understand they determined a cut off point for the expression of these proteins but I am confused on how they determined this point and if it was sufficient enough to have pure data.
In the primary article, it stated "we used only one set of experimental parameters here to grow and maintain cells for the evaluation of complex composition". Do you think this affects the results, especially since protein expression can be altered by environmental factors? Maybe protein interactions change as well.
How does TAP allow for the efficient identification of low-abundance proteins that would not be detectable by approaches involving expression proteomics and the purification of very large complexes? Why is it that other approaches fail to achieve these?
It said only one set of parameters were used to grow cells. Proteins are complex and change shape over time. How do you think the different environments would effect the proteins and their compositions?
In the review paper "Analysis of protein complexes using mass spectrometry”, the author demonstrates Mass spectrometry to identify protein interactions. It is sure that Mass spec is more accurate than Y2H. However, it costs a lot more to Mass spec than Y2H. Other then cost issue, what could be drawbacks of mass spec?
In the primary paper the authors made the claim that their method was useful for human multiprotein complexes. Has this method be used a lot in human cells yet? What kinds of cells can they use? Can these authors really make this claim even though they only tested a few proteins?
The paper mentions that a 78% association demonstrated that this was a very efficient method. What is the cutoff for 'efficiency' in the context of this paper? It also mentions that they looked at previous literature to gauge the quality of their results, but could comparing their results with older data pose more problems depending on how up-to-date this older data is?
The paper states that the 1,143 eukaryotic orthologues studied are thought to have evolved through vertical descent. However, the paper cited in the study, when discussing vertical descent from a common ancestor is from 1970, when methods of horizontal gene transfer were significantly less accurate. What changes could horizontal gene transfer within the yeast strains cause in experimental analysis and results?