System-Wide Profiling by Proteome Integral Solubility Alteration Assay of Drug Residence Times for Target Characterization

Most drugs are used in the clinic and drug candidate target multiple proteins, and thus detailed characterization of their efficacy targets is required. While current methods rely on quantitative measurements at thermodynamic equilibrium, kinetic parameters such as the residence time of a drug on its target provide a better proxy for efficacy in vivo. Here, we present a residence time proteome integral solubility alteration (ResT-PISA) assay, which facilitates monitoring temporal protein solubility profiles after drug removal (“off-curve”) in cell lysates or intact cells, quantifying the lifetime of drug–target interaction. A compressed version of the assay measures the integral under the off-curve enabling the multiplexing of binding affinity and residence time assessments into a single proteomic analysis. We introduce a combined scoring system for three parametric dimensions to improve prioritization of targets. By providing complementary information to other characteristics of drug–target interaction, the ResT-PISA approach will be useful in drug development and precision medicine.

Step by step description of the web interface.

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1) The user can upload own data here.
2) The user needs to specify the reference condition, in most cases the vehicle (DMSO here).
3) The user can specify the p-value cutoff for significance in the analysis.
4) The user can specify the ΔSm cutoff for significance in the analysis.
5) The user needs to specify which sample is to be compared to the reference sample in the analysis. PISA assay. Each condition was analysed in triplicate. For each replicate, 20 µl of cell suspension were aliquoted into 9 PCR tubes and each was heated for 3 min at 9 different temperatures 48, 49.3, 50.7, 52.2, 53.5, 54.9, 56.3, 57.7 and 59 °C. The samples were then kept for 3 min at RT before snap freezing in liquid nitrogen. After that, aliquots corresponding to each temperature point were combined. For ResT-PISA in cell, one sample designated for protein expression measurement was incubated at 37 °C (n=3 for each condition) and processed alongside the pooled samples. Cells were lysed using repeated freeze/thaw cycles and aliquots were combined as for the lysate experiment. Finally, all samples were transferred to ultracentrifuge tubes, placed into a Ti 42.2 rotor (Beckman-Coulter), and centrifuged at 100 000 x g for 20 min using an Optima XPN-80 Ultracentrifuge (Beckman-Coulter). 70 µl of the supernatant were collected and the same volume of lysis buffer (8 M urea, 20 mM EPPS pH 8.5) was added. The protein concentration was measured using Pierce bicinchoninic acid assay (BCA) protein assay kit (Thermo Fischer protein and the samples were processed for MS analysis as previously described 26, 27 (see below).

Protein sample preparation for expression proteomics and TMT labeling.
For all proteomics experiments, 20 µg of proteins were used in sample preparation. S-S bond reduction was performed using 5 mM DTT at RT for 1 h followed by alkylation using 15 mM IAA at RT in the dark. The reaction was quenched by adding 10 mM of DTT. Then methanol/chloroform precipitation was performed as follows: 3 sample volume of methanol were added, then 1 sample volume of chloroform and 3 volumes of water. Samples were vortexed between each step and then centrifuged at 20 000 x g for 10 min at 4 °C. The aqueous layer was removed, and the protein pellet was rinsed with one sample volume of methanol, vortexed and centrifuged using the same speed as in the previous step. Finally, all the liquid was removed, and the protein pellet was air-dried.
Air-dried protein pellets were resuspended in 8 M urea, 20 mM EPPS pH 8.5. The samples were diluted once by adding 20 mM EPPS pH 8.5 (4 M urea), and lysyl endopeptidase digestion was carried out at a 1:100 ratio (LysC/protein, w/w) overnight at RT. The following day, samples were diluted 4 times (1 M urea) with 20 mM EPPS pH 8.5, then tryptic digestion was composed of one tenth of each sample pooled together was prepared for normalization purpose. After that, TMT11, TMT16 or TMT18 labeling was performed for 2 h at RT by adding 0.2 mg of reagent dissolved in dry ACN according instructions. The ACN content in the samples was adjusted to a final concentration of 20%. The reaction was then quenched by adding triethylamine to a final 0.5% concentration. The samples were incubated for 15 min at RT and all temperature points were combined into one pooled sample per replicate. The pooled samples were acidified to pH < 3 using TFA, desalted using Sep Pack (Waters) and vacuum dried overnight using miVac DNA (Genevac).
High pH reversed-phase peptide fractionation. 150 µg of peptides were resuspended in 20 mM NH4OH. Then, samples were off-line high-pH reversed-phase fractionated as described previously 38,39 using an UltimateTM 3000 RSLCnano System (Dionex) equipped with a XBridge Peptide BEH 25 cm column of 2.1 mm internal diameter, packed with 3.5 µm C18 beads having 300 Å pores (Waters). The mobile phase consisted of buffer A (20 mM NH4OH) and buffer B (100% ACN). The gradient started from 1% B to 23.5% in 42 min, then moved to 54% B in 9 min, 63% B in 2 min and stayed at 63% B for 5 min, and finally moved back to 1% B and stayed at 1% B for 7 min. The fractions were collected for 0.7 min each resulting in 96 fractions that were concatenated into 24 fractions (fraction 1 was pooled with fractions 25, 49 and 73, fraction 2 -with fractions 26, 50 and 74 and so on) and dried using miVac DNA (GeneVac, England).

Bioinformatics analysis.
All further data processing was performed by a home-written algorithm in R (version 4.1.1). For MS2-based experiments, protein quantification was performed as follows: 1) PSMs mapping to a reverse sequence, known contaminants, with a precursor purity below 0.5 or a PeptideProphet probability below 0.9 were removed.
2) Reporter ion intensities were adjusted to correct for the isotopic impurities of the different TMT reagents by solving a system 3) If multiple PSMs were detected in the same fraction with the same charge state, the one with the highest purity was retained, while for PSMs with the same purity the one with the highest Hyperscore was selected. 4) Individual protein TMT reporter intensities were calculated as the sum of the individual PSMs. 5) Proteins with less than two unique peptides were removed. S9 6) To correct for pipetting error, protein TMT reporter intensities were normalized by median centering to the total intensity. 7) Batch effects between different TMT sets were corrected by dividing each protein's TMT intensity by their corresponding linker value.
For the Hyperplexed samples (SILAC-TMT) the protein quantification was performed as followed: 1) PSMs mapping to a reverse sequence, known contaminants, with a precursor purity below 0.5 or a PeptideProphet probability below 0.9 were removed.
2) In each replicate identified PSMs were filtered for matching pairs of light and heavy ones. These pairs were kept together for all further analysis.
3) Reporter ion intensities were adjusted to correct for the isotopic impurities of the different TMT reagents by solving a system 4) If multiple PSMs pairs were detected in the same fraction with the same charge state, the one with the highest individual purity was retained, while for PSMs pairs with the same purity the one with the highest individual Hyperscore was selected. 5) Individual protein quantification was calculated based on the sum of the TMT reporter channels of the individual PSMs for light and heavy labeled samples. 6) Proteins with less than two unique light and heavy peptides were removed. 7) To correct for pipetting error, protein intensities were normalized by median centering to the total intensity. 8) Batch effects between different TMT sets were corrected by dividing each protein's TMT intensity by their corresponding light or heavy labeled linker value.