Assessing target engagement using proteome-wide solvent shift assays
- Department of Cell Biology, Harvard Medical School, United States
Figures
Figure 1 with 3 supplements
Solvent profiling of the HCT116 proteome.
(A) Schematic diagram of solvent-induced precipitation. (B) Count of quantified proteins in each replicate and those to which sigmoidal curves were fit well (R2 > 0.8 and plateau < 0.3). Each …
-
Figure 1—source data 1
Protein quantifications in the solvent-induced precipitation assay with 16 AEA concentrations (N = 2).
- https://cdn.elifesciences.org/articles/70784/elife-70784-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Solvent-induced denaturation of the HCT116 proteome.
Upper panel: native HCT116 lysates were treated with 14 increasing concentrations of AEA (0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 32.5%). Soluble fractions were resolved by …
Figure 1—figure supplement 2
Solvent profiling of the HCT116 proteome.
Heatmap representation of all proteins quantified in replicate 1 before (left panel) and after (right panel) normalization with thermal proteome profiling (TPP) package. For each protein, its …
Figure 1—figure supplement 3
Solvent profiling of the HCT116 proteome.
(A) Heatmap representation of all proteins quantified in replicate 2. For each protein, its relative abundance (fold change) at the indicated %AEA compared to 0% AEA is presented. The proteins to …
Figure 2 with 3 supplements
Solvent proteome profiling (SPP) can be used to determine compound target engagement.
(A) Schematic diagram summarizing the SPP workflow. (B) Native HCT116 lysates were treated with 100 μM SCIO-469. SPP melting curves for MAPK14 and MAPK12—known target proteins of SCIO-469—are …
-
Figure 2—source data 1
Solvent proteome profiling (SPP) protein quantifications following treatment of HCT116 lysates with 100 µM SCIO-469 or vehicle.
N = 2.
- https://cdn.elifesciences.org/articles/70784/elife-70784-fig2-data1-v1.xlsx
-
Figure 2—source data 2
Solvent proteome profiling (SPP) protein quantifications following treatment of HCT116 lysates with 25 µM Alisertib or vehicle.
N = 2.
- https://cdn.elifesciences.org/articles/70784/elife-70784-fig2-data2-v1.xlsx
-
Figure 2—source data 3
Solvent proteome profiling (SPP) protein quantifications following treatment of HCT116 lysates with 25 µM MK-2206 or vehicle.
N = 2.
- https://cdn.elifesciences.org/articles/70784/elife-70784-fig2-data3-v1.xlsx
Figure 2—figure supplement 1
Schematic diagram of solvent proteome profiling (SPP).
Native cell lysates are prepared and divided into two aliquots. One aliquot is treated with drug and the other reserved for a vehicle-treated control. Samples are incubated with drug or vehicle for …
Figure 2—figure supplement 2
Solvent proteome profiling (SPP) of the HCT116 proteome.
(A–D) Heatmap representation of all proteins quantified in each replicate in the solvent proteome profiling (SPP) experiment with SCIO-469 treatment. For each protein, its relative abundance (fold …
Figure 2—figure supplement 3
Solvent proteome profiling (SPP) can be used to determine compound target engagement.
Native HCT116 lysates were treated with 100 μM SCIO-469. Solvent melting curve for MAPK13 is displayed for two replicate experiments. Asterisks indicate statistically significant hits (see Materials …
Figure 3 with 3 supplements
Solvent-PISA can resolve compound target engagement with increased efficiency.
(A) Native cell lysates are prepared and divided into multiple aliquots. Half the aliquots are treated with drug and the other half reserved for a vehicle-treated control. Samples are incubated for …
-
Figure 3—source data 1
Solvent-PISA and thermal-PISA protein quantifications following treatment of HCT116 lysates with 100 µM SCIO-469 or vehicle (N = 4).
- https://cdn.elifesciences.org/articles/70784/elife-70784-fig3-data1-v1.xlsx
-
Figure 3—source data 2
Solvent-PISA and thermal-PISA protein quantifications following treatment of HCT116 lysates with 25 µM vorinostat or vehicle (N = 4).
- https://cdn.elifesciences.org/articles/70784/elife-70784-fig3-data2-v1.xlsx
Figure 3—figure supplement 1
Solvent-PISA can resolve compound target engagement with increased efficiency.
(A) Solvent melting curves for MAPK9. (B) MAPK9/JNK2 in vitro kinase assay. Bars represent the mean relative kinase activity of four replicate measurements with (blue) and without (gray) 10 µM …
Figure 3—figure supplement 2
Solvent-PISA can resolve compound target engagement with increased efficiency.
(A) Count of total proteins quantified and proteins quantified with at least two peptides and with a %CV < 15% across replicates. (B, C) Distribution of CV values from each experimental group for …
Figure 3—figure supplement 3
Solvent-PISA can resolve compound target engagement with increased efficiency.
(A, B) HCT116 lysates were treated with 100 μM SCIO-469 and analyzed by solvent-PISA (A) or thermal-PISA (B). Proteins quantified with at least two peptides and having a %CV < 15 across replicates …
Figure 4 with 2 supplements
The range of AEA concentrations used in a solvent-PISA experiment impacts the ultimate fold change measurements.
(A) HCT116 lysates were treated with 25 μM MK2206 and analyzed by solvent-PISA. Data are presented as a volcano plot to highlight significant changes in abundance. Significant changes were …
-
Figure 4—source data 1
Solvent-PISA protein quantifications following treatment of HCT116 lysates with 25 µM Alisertib or vehicle (N = 4).
- https://cdn.elifesciences.org/articles/70784/elife-70784-fig4-data1-v1.xlsx
-
Figure 4—source data 2
Solvent-PISA protein quantifications following treatment of HCT116 lysates with 25 µM MK2206 or vehicle (N = 4).
- https://cdn.elifesciences.org/articles/70784/elife-70784-fig4-data2-v1.xlsx
-
Figure 4—source data 3
Solvent-PISA protein quantifications following treatment of HCT116 lysates with 25 µM MK2206 or vehicle across four solvent-PISA windows (see Materials and methods) (N = 2).
- https://cdn.elifesciences.org/articles/70784/elife-70784-fig4-data3-v1.xlsx
Figure 4—figure supplement 1
The range of AEA concentrations used in a solvent-PISA experiment impacts the ultimate fold change measurements.
(A) HCT116 lysates were treated with 25 μM MK2206 and analyzed by solvent-PISA. Proteins quantified with at least two peptides and having a %CV < 15 across replicates were sorted by the mean log2 …
Figure 4—figure supplement 2
The range of AEA concentrations used in a solvent-PISA experiment impacts the ultimate fold change measurements.
(A) Solvent proteome profiling (SPP) melting curves for CHEK1. Replicate experiments were separated into two different panels for visualization. (B) HCT116 lysates were treated with 125 μM MK2206 …
Figure 5 with 1 supplement
Combining solvent proteome profiling (SPP) with thermal proteome profiling (TPP) to maximize proteome coverage.
(A) Fractions of high-quality curves (R2 > 0.8 and plateau < 0.3) in SPP and TPP datasets. * indicates datasets taken from the Meltome Atlas (Jarzab et al., 2020). A similar fraction (~70%) of …
-
Figure 5—source data 1
Solvent proteome profiling (SPP) and thermal proteome profiling (TPP) protein quantifications (eight AEA concentrations or eight temperatures) (N = 2).
- https://cdn.elifesciences.org/articles/70784/elife-70784-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Combining SPP with TPP to maximize proteome coverage.
(A) Total number of high-quality curves (R2 > 0.8 and plateau < 0.3) in solvent proteome profiling (SPP) and thermal proteome profiling (TPP) datasets. * indicates datasets taken from the Meltome …
Author response image 1
HCT116 lysates were treated with 25 µM vorinostat and assays by thermal-PISA (N=2).
We included this explanation in the Results section of the main text (page 10).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HCT116 (adult, colorectal cancer) | ATCC | Cat# CCL-247 | Male |
| Antibody | Anti-Akt (rabbit monoclonal) | Cell Signaling Technology | Cat# 4691 | WB (1:1000) |
| Antibody | Anti-p38 MAPK (rabbit polyclonal) | Cell Signaling Technology | Cat# 9212 | WB (1:1000) |
| Antibody | Anti-Aurora A (rabbit monoclonal) | Cell Signaling Technology | Cat# 4718 | WB (1:1000) |
| Antibody | Goat anti-rabbit IgG-HRP | Santa Cruz | Cat# sc-2004 | WB (1:10,000) |
| Chemical compound, drug | SCIO-469 | Cayman Chemical | Cat# 29484;batch: 0575761-1 | 10 µM stock in DMSO |
| Chemical compound, drug | Alisertib(MLN8237) | Cayman Chemical | Cat# 13602;batch: 0565558-18 | 10 µM stock in DMSO |
| Chemical compound, drug | MK2206 (hydrochloride) | Cayman Chemical | Cat# 11593;batch: 0586491-5 | 10 µM stock in DMSO |
| Chemical compound, drug | Vorinostat(SAHA) | Cayman Chemical | Cat# 10009929;batch: 0512249-52 | 10 µM stock in DMSO |
| Commercial assay or kit | TMTpro 16plex Label Reagent Set | Thermo Fisher | Cat# A44520;batch: VI313212 | Solubilized in anhydrous acetonitrile |
| Commercial assay or kit | JNK2 kinase enzyme | Promega | Cat# VA7210 | |
| Commercial assay or kit | ADP-Glo kinase assay | Promega | Cat# V9101 | |
| Software, algorithm | Perseus | ttp://maxquant.net/perseusTyanova et al., 2016 | Version 1.6.15.0 | |
| Software, algorithm | Prism | GraphPad | Version 9.0.0 | |
| Software, algorithm | R | https://www.r-project.org/ | Version 4.0.2 | |
| Software, algorithm | TPP package | https://github.com/DoroChilds/TPP; Franken et al., 2015 | Version 3.17.6 | |
| Other | Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (Hydrophobic) | Cytiva | Cat# 44152105050250 | |
| Other | Sera-Mag Carboxylate-Modified Magnetic Particles (Hydrophylic) | Cytiva | Cat#45152105050250 |
