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 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 treatment and control sample

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 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 peptide intensities obtained across temperatures in the presence of 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. A fold difference significance cut off of +2 and -2 with a p-value cutoff of p ≤ 0.05 is applied. Hollow blue dots indicate ATP-binding soluble proteins (SP) and solid blue dots indicate ATP-binding IMPs. Data represents the mean from three replicates (n = 3).

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

(A) Global protein intensities derived from label-free quantification (LFQ) values of peptidisc-reconstituted liver extract. The plot displays soluble proteins, peripherally bound and IMPs 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 at 51°C based on a fold difference cutoff of > 2 or < -2 and p-value of ≤ 0.05. The ATP-binding soluble proteins (SP) are represented as hollow blue circles, and the ATP-binding IMPs 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) 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

. At least two unique peptides were detected per protein at each temperature. Stabilization was defined using a fold change > 2 between treatment and control samples, with a significance set at p ≤ 0.05, calculated from triplicate samples (n = 3).

Off-target ATP ligand effect.

(A) GO-term analysis of molecular functions and distribution for all IMPs significantly stabilized at all temperatures tested with the mouse liver library (n = 178). (B) 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) 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 or with 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 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. 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. 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.

Stabilized and destabilized IMPs are represented by hollow blue circles and IMPs annotated as ATP-binding are represented by solid blue circles with label on Mao-B. The mean value is obtained from three replicates at the temperature exposure assay (n = 3).

Number of proteins identified in the mouse liver peptidisc library at the indicated temperature to assess loss of IMPs compared to global protein abundance. At least two unique peptides were identified for each protein (n = 2).