Fig. 1: The molecular glue degrader (MGD) proteomics screening workflow.

a Schematic of the proteomics screening workflow. A subset of 100 CRBN-based molecular glue degraders (MGDs) from a 5000-compound library was screened in Huh-7 and NB-4 cells (10 µM, 24 h) in 96-well plates. Each plate included 25 MGDs in triplicate. Proteins were quantified using Bruker timsTOF MS and processed with DIA-NN¹⁴. Significantly downregulated proteins (>25%) were selected for 6-h re-testing with or without MLN4924, a neddylation inhibitor. Ubiquitinomics (K-GG peptide profiling) was used to validate MLN4924-dependent neosubstrates. Created in BioRender. BioRender.com/h02z778. b Count of significant downregulation events (LIMMA⁶⁹, FDR = 1%) for known neosubstrates in Huh-7 or NB-4 cells after 24-h compound treatment (see Supplementary Data 2) for details. c Scatter plot showing suppression of MGD-induced downregulation (6 h) of known CRBN neosubstrates by MLN4924 (5 µM). d Volcano plots showing protein-level effects of SJ11322 (10 µM), from left to right: (i) 24 h, (ii) 6 h, and (iii) 6 h with MLN4924 (5 µM). Log₂ fold-change (x-axis) and log₁₀ standard error (y-axis) are shown. The chemical structure of SJ11322 is shown on the left side. Up- and downregulated proteins or ubiquitination sites (LIMMA⁶⁹, FDR = 1%/5%) are colored in blue and red. P-value adjustments were made for multiple comparisons using the Benjamini-Hochberg procedure. n.s. not significant. The chemical structure of SJ11322 is shown. e Left: Volcano plot of SJ11322-induced ubiquitinated peptides (30 min). Upregulated sites (LIMMA⁶⁹, FDR = 5%) are in blue, including four FIZ1 sites. Right: Correlation of proteome (x-axis) and ubiquitinome (y-axis) t-statistics. Significantly regulated proteins/sites are colored; turquoise highlights proteins downregulated at 24 h with increased ubiquitination at 30 min. n.s. not significant. f Heatmap of log₂ fold-changes in ubiquitination for CSNK1A1 and ZFP91. Asterisks (*) indicate statistically significant regulations (LIMMA⁶⁹, FDR = 5%). g Correlation between site-specific ubiquitination—CSNK1A1-K51 (left; Spearman’s ρ = −1, p-value = 0.02) and ZFP91-K357 (right; Spearman’s ρ = −1, p-value = 0.08)—with corresponding 6-hour protein downregulation. Asterisks (*) indicate significant protein downregulation (LIMMA⁶⁹, FDR = 1%); linear regression line and Spearman coefficient are shown. Source Data are provided as a Source Data file.