Membrane mimetic thermal proteome profiling (MM-TPP) towards mapping membrane protein-ligand dynamics

Reviewer #1 (Public review):

Summary:

The idea is appealing, but the authors have not sufficiently demonstrated the utility of this approach.

Strengths:

Novelty of the approach, potential implications for discovering novel interactions

Weaknesses:

The Duong had introduced their highly elegant peptidisc approach several years ago. In this present work, they combine it with thermal proteome profiling (TPP) and attempt to demonstrate the utility of this combination for identifying novel membrane protein-ligand interactions.
While I find this idea intriguing, and the approach potentially useful, I do not feel that the authors had sufficiently demonstrated the utility of this approach.
My main concern is that no novel interactions are identified and validated. For the presentation of any new methodology, I think this is quite necessary.
In addition, except for MsbA, no orthogonal methods are used to support the conclusions, and the authors rely entirely of quantifying rather small differences in abundances using either iBAQ or LFQ.
Furthermore, the reported changes in abundances are solely based on iBAQ or LFQ analysis. This must be supported by a more quantitative approach such as SILAC or labeled peptides
In summary, I think this story requires a stronger and broader demonstration of the ability of peptidisc-TPP to identify novel physiologically/pharmacologically relevant interactions.

Reviewer #2 (Public review):

Summary:

The membrane mimetic thermal proteome profiling (MM-TPP) presented by Jandu et al. seems to be a useful way to minimize the interference of detergents in efficient mass spectrometry analysis of membrane proteins. Thermal proteome profiling is a mass spectrometric method that measures binding of a drug to different proteins in a cell lysate by monitoring thermal stabilization of the proteins because of the interaction with the ligands that are being studied. This method has been underexplored for membrane proteome because of the inefficient mass spectrometric detection of membrane proteins and because of the interference from detergents that are used often for membrane protein solubilization.

Strengths:

In this report the binding of ligands to membrane protein targets has been monitored in crude membrane lysates or tissue homogenates exalting the efficacy of the method to detect both intended and off-target binding events in a complex physiologically relevant sample setting.

The manuscript is lucidly written and the data presented seems clear. The only insignificant grammatical error I found was that the 'P' in the word peptidisc is not capitalized in the beginning of the methods section "MM-TPP profiling on membrane proteomes". The clear writing made it easy to understand and evaluate what has been presented. Kudos to the authors.

Weaknesses:

While this is a solid report and a promising tool for analyzing membrane protein drug interactions, addressing some of the minor caveats listed below could make it much more impactful.

The authors claim that MM-TPP is done by "completely circumventing structural perturbations invoked by detergents". This may not be entirely accurate, because before reconstitution of the membrane proteins in peptidisc, the membrane fractions are solubilized by 1% DDM. The solubilization and following centrifugation step lasts at least for 45 min. It is less likely that all the structural perturbations caused by DDM to various membrane proteins and their transient interactions become completely reversed or rescued by peptidisc reconstitution. In the introduction, the authors make statements such as "..it is widely acknowledged that even mild detergents can disrupt protein structures and activities, leading to challenges in accurately identifying drug targets.." and "[peptidisc] libraries are instrumental in capturing and stabilizing IMPs in their functional states while preserving their interactomes and lipid allosteric modulators...'. These need to be rephrased, as it has been shown by countless studies that even with membrane protein suspended in micelles robust ligand binding assays and binding kinetics have been performed leading to physiologically relevant conclusions and identification of protein-protein and protein-ligand interactions.

If the method involves detergent solubilization, for example using 1% DDM, it is a bit disingenuous to argue that 'interactomes and lipid allosteric modulators' characterized by low-affinity interactions will remain intact or can be rescued upon detergent removal. Authors should discuss this or at least highlight the primary caveat of the peptidisc method of membrane protein reconstitution - which is that it begins with detergent solubilization of the proteome and does not completely circumvent structural perturbations invoked by detergents.

It would also be important to test detergents that are even milder than 1% DDM and ones which are harsher than 1% DDM to show that this method of reconstitution can indeed rescue the perturbations to the structure and interactions of the membrane protein done by detergents during solubilization step. Based on the methods provided, it appears that the final amount of detergent in peptidisc membrane protein library was 0.008%, which is ~150 uM. The CMC of DDM depending on the amount of NaCl could be between 120-170 uM. Perhaps, to completely circumvent the perturbations from detergents other methods of detergent-free solubilization such as using SMA polymers and SMALP reconstitution could be explored for a comparison. Moreover, a comparison of the peptidisc reconstitution with detergent-free extraction strategies, such as SMA copolymers, could lend more strength to the presented method.

Cross-verification of the identified interactions, and subsequent stabilization or destabilizations, should be demonstrated by other in vitro methods of thermal stability and ligand binding analysis using purified protein to support the efficacy of the MM-TPP method. An example cross-verification using SDS-PAGE, of the well-studied MsbA, is shown in Figure 2. In a similar fashion, other discussed targets such as, BCS1L, P2RX4, DgkA, Mao-B, and some un-annotated IMPs shown in supplementary figure 3 that display substantial stabilization or destabilization should be cross-verified.