The Membrane Mimetic-Thermal Proteome Profiling (MM-TPP) experimental workflow.

1) Crude membranes are prepared from the liver organ. 2) Integral membrane proteins (IMPs) are solubilized with detergent and reconstituted in the Peptidisc library. The water-soluble library is exposed to the ligand of interest (treatment) or the corresponding vehicle (control). 3) Protein samples are heated at specific temperatures to induce precipitation, followed by ultracentrifugation. The soluble fraction is analyzed by mass spectrometry to detect changes in protein abundances between the treatment and control samples.

MM-TPP of integral membrane proteins (IMPs) prepared from E. coli.

(A) Stability of purified MsbA in Peptidisc in the presence of the indicated ligands. Samples are heat treated and centrifuged before analysis by 12% SDS-PAGE and Coomassie blue staining. (B) Grouped scatterplot representation of mean log2-transformed IBAQ value obtained for all identified proteins in the E. coli library at the indicated temperatures. The location of MsbA on the plot is shown as a red dot. The mean value is obtained from three replicates of the temperature exposure assay (n = 3). (C) Relative abundance of MsbA based on the LFQ peptide intensities obtained across temperatures in the presence of ATP + orthovanadate (ATP-VO4; orange) compared to a control sample (gray). Data is a mean ± standard deviation from triplicate assays (n = 3). (D) Volcano plot analysis of stabilized and destabilized proteins following ATP-VO4 exposure at 61°C based on log2-transformed LFQ peptide intensities. A log2 fold difference significance cut-off of +1 and -1 with a -log10 p-value cut-off of p > 1.3 is applied. Hollow blue dots indicate annotated ATP-binding soluble proteins (SP) and solid blue dots indicate ATP-binding integral membrane proteins (IMPs). Data represent the mean from three replicates (n = 3).

MM-TPP of integral membrane proteins (IMPs) prepared from the mouse liver tissue.

(A) Global protein intensities derived from log2-transformed LFQ values of peptidisc-reconstituted liver extract. The plot displays soluble proteins (blue), membrane-associated (green), and IMPs (red) identified at the indicated temperatures compared to room temperature (RT). The dashed line is the identical value line. (B) Volcano plot of stabilized and destabilized proteins based on log2-transformed LFQ values at 51 °C based on a fold difference cutoff of > 1 or < -1 and -log10 p-value of > 1.3. The soluble proteins (SP) annotated as ATP-binding are represented as hollow blue circles, and the IMPs annotated as ATP-binding are represented as solid blue circles. The mean value is obtained from three replicates at the temperature exposure assay (n = 3). (C) GO-term enrichment analysis of molecular functions of stabilized IMPs identified in B. The presented top 10 significant terms are based on adjusted p-value (FDR = 5% after Benjamini-Hochberg correction) (D) Number of ATP-binding cassette (ABC) transporters identified and stabilized by ATP-VO4 at the indicated temperatures. (E) Log2 transformed LFQ Peptide intensities of BCS1L in the presence of ATP-VO₄ (green), AMP-PNP (orange), and vehicle control (gray) across the temperature range. Data is a mean ± standard deviation from three replicates (n = 3).

ATP-binding cassette transporters detected in the mouse liver Peptidisc library.

A minimum of two unique peptides was required to identify the protein at a given temperature. The exact number of unique peptides identified for each protein under each ligand and temperature condition is provided in Supplementary Table 2. Stabilization was defined using a log2 fold change > 1 between treatment and control samples, with a -log10 p > 1.3, calculated from replicate samples (n = 3).

Protein Stabilization Profiles at 51 °C with ATP–VO₄ in DB-TPP and 2-MeS-ADP in MM-TPP.

(A) Volcano plot of stabilized and destabilized proteins at 51 °C in detergent-based TPP (DB-TPP) with ATP-VO₄. Log₂-transformed LFQ peptide values were used, with thresholds set at a fold-change > 1 or < –1 and a –log₁₀ p-value > 1.3. Soluble proteins (SP) annotated as ATP-binding are shown as hollow blue circles, and integral membrane proteins (IMPs) annotated as ADP-binding are shown as solid blue circles. Data represent the mean of three biological replicates (n = 3). (B) Volcano plot of stabilized and destabilized proteins at 51 °C in MM-TPP with 2-methylthio-ADP (2-MeS-ADP), displayed using the same thresholds and annotations as in panel A.

Comparison of ATP-Binding Protein Stabilization in MM-TPP and DB-TPP.

Reported values at each temperature represent the total number of proteins meeting the inclusion criteria (at least 2 unique peptides) across triplicate control and treatment conditions. As protein counts are determined post-analysis, they are reported as single values rather than as means with standard deviations. Each protein was identified based on at least two unique peptides (n = 3).

ATP and Analogue-Induced Off-Target Stabilization of Liver Membrane Proteins.

(A) GO-term analysis of molecular functions and distribution for all IMPs (n= 178) significantly stabilized with ATP-VO4 at temperatures tested with the mouse liver library. (B) Log2 transformed LFQ Peptide intensity variations of P2RY6 and P2RY12 over the temperature range with AMP-PNP (orange), ATP-VO4 (green), or none (gray). Data is a mean ± standard deviation from three replicates (n = 3). (C) Relative LFQ Peptide intensity variations of P2RX4 at the indicated temperature in the presence of ATP-VO4 (left panel) or AMP-PNP (right panel). Data from treatment samples (orange) and control samples (blue) is from triplicates (n = 3). * Represents p-value ≤ 0.05. **Protein not detected. (D) Structural model of homodimeric Mao-B with the predicted binding of FAD, ATP, ADP, and AMP ligands within the FAD binding pocket, indicated as red dots. Each ligand is presented individually in the FAD binding pocket or as an all-ligand overlap generated by AlphaFold3. The respective Predicted Local Distance Difference Test (pLDDT) score for each ligand is shown, with higher scores representing more favourable ligand fitting. The colour gradient represents a high pLDDT score as blue and a low pLDDT score as orange.

Thermal stability of MsbA in detergent and in Peptidisc with different ligands.

(A) The E. coli membrane fraction enriched for MsbA was solubilized with 1% DDM or reconstituted in Peptidisc. The detergent extract and Peptidisc library were incubated at 45°C for the indicated times. After ultra-centrifugation, the supernatants were analyzed on 15% SDS-PAGE and visualized with Coomassie blue staining. (B) The Peptidisc library prepared in (A) was incubated with ATP-VO4 or AMP-PNP at the indicated temperature. After ultra-centrifugation, the supernatants were analyzed on 15% SDS-PAGE and visualized with Coomassie blue staining.

Volcano plot of stabilized and destabilized proteins at 51 °C in the presence of AMP-PNP.

Soluble proteins (SP) annotated as ATP-binding proteins are presented as hollow blue circles, and IMPs annotated as ATP-binding are presented as solid blue circles. Values taken from log2-transformed LFQ peptide intensities. The mean value is obtained from three replicates at the temperature exposure assay (n = 3).

Volcano plot of stabilized and destabilized proteins at 64 °C in the presence of ATP-VO4.

IMPs are represented by hollow blue circles and IMPs annotated as ATP-binding are represented by solid blue circles. Values taken from log2-transformed LFQ peptide intensities. The mean value is obtained from three replicates at the temperature exposure assay (n = 3).

Volcano plot of stabilized and destabilized proteins at 57 °C in the presence of 2-MeS-ADP.

The soluble proteins (SP) annotated as ADP-binding are represented by hollow blue circles and the IMPs annotated as ADP-binding are represented by solid blue circles. Values taken from log2-transformed LFQ peptide intensities. The mean value is obtained from three replicates at the temperature exposure assay (n = 3).

Correlation plot of protein differential stabilization at 51 °C in MM-TPP, performed with DIA data acquisition from a whole mouse liver library.

ATP-binding proteins with |log₂ fold change| ≥ 0.5 are highlighted. Soluble proteins (SP) annotated as ATP-binding are shown as hollow blue circles, while IMPs annotated as ATP-binding are shown as solid blue circles. ABC transporters that pass the significance threshold are labeled on the plot.

Total and Integral Membrane Protein Counts Across Temperatures in Mouse Liver Peptidisc Libraries.

The % value is used to assess IMP loss relative to total protein count. Each protein was identified based on at least two unique peptides (n = 2)

Unique peptide counts for proteins highlighted in this study.

MsbA, ABCA6, ABCB1, ABCB6, ABCC2, ABCC3, ABCC9, ABCG2, ABCG5, BCS1L, P2RX4, P2RY6, P2RY12, and MAO-B. Counts are from MaxQuant analyses using a data-dependent acquisition (DDA) workflow across the temperatures tested in MM-TPP (with ATP– VO₄, 2-MeS-ADP, and AMP-PNP) and in DB-TPP (with ATP–VO₄).