Extended Data Fig. 10 ∣. Analysis of substrates bearing C-terminal cQ/cN and Q/N modifications in vitro and in cells.

(a) Schematic of expressed protein ligation using HBB(1–129) thioester and Sec-YcQ or Sec-YcQMe to generate HBB(1–cQ132) or HBB(1–Me132). (b) Western blot of in vitro ubiquitination of HBB proteins in triplicate. Unfortunately neither the HBB[1–cQ132] nor recombinant HBB was amenable to electroporation, which precluded our ability to evaluate CRBN-dependent degradation in cells. (c) TMT-based quantification of K-ε-GG sites identified from the in vitro ubiquitination samples using mass spectrometry. Data are presented as mean ± SD (n = 4 technical replicates). Comparisons were performed using unpaired two-tailed t-tests and p-values are noted. (d) Volcano plots of peptide groups bearing C-terminal cyclic imides in WT HEK293T or CRBN CRISPR/Cas9 knockout HEK293T over 48 h. Upregulated proteins at 1% FDR = red, 5% FDR = pink. ACTB(96–cN111) peptide = blue. P-values for the abundance ratios were calculated by one-way ANOVA with TukeyHSD post-hoc test. (e) Volcano plots of peptide groups bearing C-terminal glutamine or asparagine (protein terminus excluded) in WT HEK293T, CRBN KO HEK293T, or HEK293T treated with 200 μM lenalidomide over 48 h. Upregulated peptide groups at 1% FDR = red, 5% FDR = pink, consistent with a model where blockade of degradation of substrates bearing the C-terminal imide allows the modifications to be hydrolyzed. P-values for the abundance ratios were calculated by one-way ANOVA with TukeyHSD post-hoc test. (f) Volcano plots of peptide groups bearing C-terminal glutamine or asparagine (protein terminus excluded) in MM.1S treated with DMSO or 200 μM lenalidomide over 48 h. Upregulated peptide groups at 1% FDR = red, 5% FDR = pink. P-values for the abundance ratios were calculated by one-way ANOVA with TukeyHSD post-hoc test. (g) Ion intensity chromatogram extracted for the masses of the corresponding species for HEK293T lysates and the mixture of isotopically labeled ACTB(96*–113) containing the modifications. The cell samples were spiked with the peptide mixture and ran on the selected ion monitoring mode to validate the overlap of retention times and amplify the signal of selected ions. For uncropped western blot images, see Supplementary Fig. 11.