DT2216 is a specific BCL-XL PROTAC
To validate the specificity of DT2216, we used the stable isotope labeling with amino acids in cell culture (SILAC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics to analyze the changes in proteins in WI-38 normal human diploid fibroblasts after DT2216 and DT2216NC treatment. The results show that DT2216, but not DT2216NC, reduced the levels of BCL-XL, but none of other proteins were significantly affected by either agent, demonstrating that DT2216 is a specific BCL-XL PROTAC.
Sample Processing Protocol
To label cells with stable isotopic amino acids, WI38 cells were propagated in DMEM SILAC media deficient in both L-lysine and L-arginine (Cat #88364; Thermo Fisher Scientific) and supplemented with light lysine (12C614N2-K) and arginine (12C614 N4-R) for light state (Cat # 89987 and #89989; Thermo Fisher Scientific), and 13C615N2-K and 13C615N4-R for heavy state labeling (Cat # 88209 and #89990; Thermo Fisher Scientific). Cells were cultured for at least six doubling times for complete incorporation. The light-labeled WI38 cells were untreated (DMSO) and the heavy-labeled WI38 cells were treated with 1 M DT2216 or DT2216NC for 6 hrs, respectively. Reverse labeling was used in the second biological replicate. The untreated and DT2216 or DT2216NC-treated cells were harvested by centrifugation at 500 g for 5 min. Pellets were washed twice by resuspending in 1 ml of prechilled PBS (Cat #20012027; Gibco). The cell pellets were resuspended in 20 mL freshly prepared Lysis Buffer (2% SDS, 100 mM Tris/HCl pH 7.6) containing MS-SAFE Protease and Phosphatase Inhibitor (Cat #MSSAFE-5VL, Sigma) for sonication. The lysate was centrifuged at 15,000 g for 10 min at 18°C. The supernatant was stored at −80°C for proteomic analysis. Protein concentration was measured by BCA assay (Cat #23227; Pierce) and SILAC pairs were mixed in equimolar amounts. Purified proteins were reduced, alkylated, and digested using filter-aided sample preparation 1. Tryptic peptides were separated into 36 fractions on a 100 x 1.0 mm Acquity BEH C18 column (Waters) using an UltiMate 3000 UHPLC system (Thermo) with a 40 min gradient from 99:1 to 60:40 buffer A (0.1% formic acid, 0.5% acetonitrile):B (0.1% formic acid, 99.9% acetonitrile) ratio under basic pH conditions, and then consolidated into 12 super-fractions. Each super-fraction was then further separated by reverse phase XSelect CSH C18 2.5 um resin (Waters) on an in-line 150 x 0.075 mm column using an UltiMate 3000 RSLCnano system (Thermo). Peptides were eluted using a 60 min gradient from 97:3 to 60:40 buffer A:B ratio. Eluted peptides were ionized by electrospray (2.15 kV) followed by MS/MS analysis using higher-energy collisional dissociation (HCD) on an Orbitrap Fusion Lumos mass spectrometer (Thermo) in top-speed data-dependent mode. MS data were acquired using the FTMS analyzer in profile mode at a resolution of 240,000 over a range of 375 to 1500 m/z. Following HCD activation, MS/MS data were acquired using the ion trap analyzer in centroid mode and normal mass range with precursor mass-dependent normalized collision energy between 28.0 and 31.0.
Data Processing Protocol
Proteins were identified and SILAC ratios determined using MaxQuant with a parent ion tolerance of 3 ppm and a fragment ion tolerance of 0.5 Da. The derived peak list was searched with the built-in Andromeda search engine against the reference human proteome downloaded from Uniprot (http://www.uniprot.org/) on 03-13-2018. The search parameters for both algorithms included carbamidomethylation of cysteine residues as a fixed modification and N-terminal acetylation, oxidation at methionine, and SILAC labeling 13C615N2-K and 13C615N4-R as variable modifications. Trypsin was specified as the protease and a maximum of two missed cleavages were allowed. The data were screened against a target decoy database and the false discovery rate (FDR) was set to 1% at the peptide level and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm 2. Proteins with ratios greater than 1.5-fold change in each biological replicate were used for further analysis.