Mapping Dysfunctional Protein-Protein Interactions in Disease

Our work maps dysfunctional protein-protein interactions directly from native cells and tissues, revealing how disease rewires cellular networks. Unlike most interatomic methods, dfPPI requires no genetic engineering and skills to patient cohorts, using one multiplex capture per sample. To begin, prepare a protein extraction buffer containing 20 millimolar Tris, 20 millimolar potassium chloride, 5 millimolar magnesium chloride, and 0.01%Nb-40.

Add protease and phosphatase inhibitors immediately before use and keep the buffer on ice. Place the frozen tissue sample into a micro tissue homogenizer tube fitted with a pestle. Add 500-700 microliters of the native lysis buffer, adjusting the volume based on tissue compactness and ease of homogenization.

Then, homogenize the sample on ice by gently moving the pestle up and down and against the abrasive walls until a uniform suspension is obtained. Incubate the lysates at 4 degrees Celsius for 30 minutes by placing the vial on a rotation unit. Gently mix the samples during incubation through rotation.

Using a benchtop centrifuge, centrifuge the samples at 13, 000 G for 10 minutes at 4 degrees Celsius to remove cellular debris. Carefully collect the supernatants and transfer them into clear 1.5-milliliter microcentrifuge tubes. Determine the total protein concentration in the supernatant using the BCA Assay Kit according to the manufacturer's instructions.

Next, take an aliquot of polyurethane beads directly from the isopropanol stock. Allow the beads to settle to remove the storage solvent. Carefully aspirate the isopropanol, then add native lysis buffer and fully re-suspend the beads by gentle pipetting or inversion.

To wash and equilibrate the beads, vortex the tube then centrifuge it. After that, aspirate the supernatant with a vacuum line fitted with a pipette tip, taking care not to disturb the pellet. Add binding buffer to the washed beads in an equal ratio to create a uniform working bead slurry.

Aliquot 40 microliters of polyurethane bead slurry into 1.5-milliliter microcentrifuge tubes, using a cut pipette tip for smooth dispensing. Then, wash the beads three times with native lysis buffer by adding 1 milliliter of buffer to each tube. Vortex the tube to re-suspend the beads, then centrifuge at 10, 000 G for 1 minute and discard the supernatant by aspiration.

After the final wash, remove most of the remaining liquid from the tubes, ensuring that the bead pellet remains undisturbed. Add the normalized protein extracts to individual 1.5-milliliter microcentrifuge tubes containing 40 microliters of control bead slurry. Adjust the final volume to 250 microliters by adding the appropriate amount of native lysis buffer.

Incubate the samples at 4 degrees Celsius for 30 minutes with rotation on an end-over-end rotator operating at 10-15 revolutions per minute. Centrifuge the tubes at 10, 000 G for 1 minute at 4 degrees Celsius to pellet the control beads along with aggregated or insoluble proteins. Carefully collect the supernatant from the control beads using a 1 milliliter pipette and transfer it into fresh 1.5-milliliter tubes containing 40 microliters of washed polyurethane bead slurry.

Incubate the samples at 4 degrees Celsius for 3 hours with rotation on an end-over-end rotator. After centrifuging the tubes carefully, aspirate the supernatant and wash the beads four times, as demonstrated earlier. Remove any residual PBS from the tube.

Re-suspend the washed beads in 80 microliters of 2 molar urea freshly prepared in 50 millimolar ammonium bicarbonate at pH 8.5 by pipetting or brief vortexing. Then, add dithiothreitol to achieve a final concentration of 1 millimolar. After capping the tube, incubate at 37 degrees Celsius for 30 minutes with shaking at 1, 100 revolutions per minute on a heated orbital shaker.

Add iodoacetamide to reach a final concentration of 3.67 millimolar. Incubate the tubes in the dark at room temperature for 45 minutes with shaking at 1, 100 revolutions per minute. Now, add additional dithiothreitol to quench any unreacted iodoacetamide, ensuring a final concentration of 3.67 millimolar and mix gently by pipetting.

Add 750 nanograms of 0.5 milligrams per milliliter mass spectrometry-grade Lys-C protease to the sample. Incubate the mixture at 37 degrees Celsius for 1 hour with shaking at 1, 150 revolutions per minute. Next, add 750 nanograms of freshly prepared 0.5 milligrams per milliliter sequencing-grade tripsin to the sample.

Incubate the mixture overnight at 37 degrees Celsius with shaking at 1, 150 revolutions per minute. On the following day, centrifuge the sample at 1, 000-5, 000 G for 1-5 minutes at room temperature. Carefully transfer the supernatant into a fresh 1.5-milliliter microcentrifuge tube using a pipette, and discard the beads.

Adjust the pH of the digest to below 3 by adding 50%trifluoroacetic acid dropwise. Verify the pH using indicator strips. PU-beads captured HSP90, HSC70, and hot proteins strongly from the epichaperome high lysate with minimal signal from the epichaperome low lysate, confirming biological specificity of the probe.

The PU-bead's cargo profile showed a rich, high molecular weight signal in the PU-beads lane and minimal background in the control bead lane, confirming successful probe activity. Coomassie-stained SDS-PAGE gels showed consistent band distribution across four in-gel processed samples, confirming successful enrichment of protein complexes from native lysates before mass spectrometry. Technical reproducibility was confirmed by principal component analysis, where replicate samples clustered tightly while different samples separated cleanly.

Precursor ion intensity distributions showed that most features had coefficients of variation below 20%with median values between 9.7%and 11.9%across samples, confirming consistent peptide detection and recovery. Hierarchical clustering of log-transformed protein abundances revealed strong sample separation and preserved inter-sample variation with protein intensities spanning from low abundance to highly enriched proteins. Pathway enrichment analysis demonstrated broad annotation coverage across multiple ontologies, including gene ontology categories and curated databases, such as Reactome, KEGG and WikiPathways.

dfPPI allowed us to uncover mechanistic and therapeutic insights that other approaches simply cannot reach. dfPPI brings interomics to the level of real-world disease cohorts and the native conditions, enabling precise mechanistic and therapeutic hypothesis. We're now expanding dfPPI to map network level changes in neurodegenerative diseases, such as Alzheimer's and Parkinson's.