For unbiased degradation profiling of compounds at the proteome level, HEK293 cells were seeded onto 10 cm plates at a density of 2-2.5×10 6 cells per plate. When cells were confluent, culturing media was refreshed and treated with 10 nM final concentration of test compounds KH1 (24) and KH2 (26) and non-degrading negative control compounds (27 and 28) and DMSO as vehicle control for 2 hours in n=3 biological replicates. After treatment, cells were washed twice with PBS (Gibco), collected in Lobinding tubes (Merck) and lysed in RIPA buffer supplemented with Protease (Roche) and Phosphatase (Roche) inhibitors as previously described for 20 min on ice. Lysates were cleared and protein concentrations were determined by using Pierce BCA assay (Thermo Scientific). 150 µg of protein for each condition was aliquoted into LoBind tubes and the STRAP (Protifi) protocol was followed according to manufacturers' instructions with slight modifications. Briefly, liquid protein samples mixed with 2×Lysis buffer (10%SDS, 100 mM TEAB pH 8.5), protein was reduced with 5 mM DTT final concentration at 55 ºC for 15 mins, alkylated (final concentration 20 mM IAA or CAA) at room temperature for 10 mins and then acidified (12% Phosphoric acid). Protein was then trapped into S-trap columns and washed with S-trap binding/wash buffer (100 mM TEAB in 90% methanol) via centrifugation at 4000×g for 30 sec. Cleaned protein then went through tryptic digestion (Trypsin (Thermo Scientific) in 50 mM TEAB) overnight at 37 ºC. Digested peptides were sequentially eluted in 80 µL of 50 mM TEAB (Sigma-Aldrich), 0.2 % formic acid (Sigma-Aldrich) and 50 % acetonitrile (Sigma-Aldrich). Elutions were pooled and dried down. Dried and cleaned peptides were reconstituted in 110 µL of 100 mM TEAB. TMT-16-plex reagent (Thermo Scientific) was brought into room temperature and reconstituted in 40 µL of anhydrous acetonitrile. TMT reagent solution was transferred to the peptides and incubated on thermoshaker (Eppendorf) for at 400 rpm at room temperature for 1 hour in the dark. Peptides were quenched by the addition of 8 µL of 5% Hydroxylamine for 1 hour at room temperature. TMT-labelled peptides pooled together and dried down in the speedVac for downstream processing. Pooled peptides were separated by basic reverse phase chromatography fractionation on a C18, column with flow rate at 200 μL/min with two buffers: buffer A (10 mM ammonium formate, pH 10) and buffer B (80% ACN, 10 mM ammonium formate, pH 10). Peptides were resuspended in 100 μL of buffer A (10 mM ammonium formate, pH10) and resolved on a C18 reverse phase column by applying a non-linear gradient of 7-40%. A total of 80 fractions were collected and concentrated into 30 fractions. The dried peptides were reconstituted in 0.1% formic acid and analyzed on an Orbitrap Ascend mass spectrometer coupled to a Thermo Fisher Scientific Vanquish Neo UHPLC. The peptides were separated on an analytical column (Acclaim PepMap RSLC C18, 75 μm × 50 cm, 2 μm, 100 Å) at a flow rate of 300 nL/min, using a step gradient of 2-7% solvent B (90% ACN/0.1% FA) for the first 6 min, followed by 7-18% up to 89 min, 18-27% up to 89-114 min and 27-35% to 114-134 min. The total run time was set to 155 min. The mass spectrometer was operated in a data-dependent acquisition mode in SPS MS3 (FT-IT-HCD-FT-HCD) method. A survey full scan MS (from m/z 350-1500) was acquired in the Orbitrap at a resolution of 120,000 at 200 m/z. The AGC target for MS1 was set as 4 × 105 and ion filling time as 50 ms. The precursor ions for MS2 were isolated using a Quadrupole mass filter at a 0.7 Da isolation width, fragmented using a normalized 30% HCD of ion routing multipole and analyzed using ion trap. The top 10 MS2 fragment ions in a subsequent scan were isolated and fragmented using HCD at a 55% normalized collision energy and analyzed using an Orbitrap mass analyser at a 60,000 resolution, in the scan range of 100-500 m/z. The proteomics raw data were searched using SEQUEST HT search engines with Proteome Discoverer 3.0 (Thermo Fisher Scientific). The following parameters were used for searches: Precursor mass tolerance 10 ppm, Fragment mass tolerance 0.1, Enzyme: trypsin, Mis-cleavage: -2, Fixed modification: carbamidomethylation of cysteine residues and TMT of lysine and N-terminal, Dynamic modification: oxidation of methionine. The data were filtered for 1% PSM, peptide and protein level FDR. Only unique peptides were selected for the quantification.