Fig. 4 ∣. C-terminal cyclic imides are PTMs that form readily in vitro.

a, Schematic of cyclic imide formation, which reveals a degron for CRBN, and the subsequent hydrolysis product. b, Unique proteins and peptides with at least one cN or cQ modification in CPTAC global proteomics datasets. c, Frequency chart of the sequence alignment flanking the amino acids in poitions −5 to +5 relative to cN and cQ sites observed in CPTAC datasets. Alignment generated by weblogo.berkeley.edu. d, Schematic of HBA and HBB sequences. Peptides with cQ or cN modifications observed with trypsin or chymotrypsin digestion and mass spectrometry analysis are underlined: global datasets, blue; RBC tryptic peptides, pink; RBC chymotryptic peptides, orange; recombinant HBB tryptic peptides, brown. Modification sites are shown in red. e, In vitro time course of the formation of ACTB(96–cN111) and ACTB(96–N111) from the synthetic peptide representing ACTB(96–113). f, In vitro time course of the formation of HBB(42–cN58) and HBB(42–N58) from full-length recombinant HBB protein. Quantification was performed by selected ion monitoring. Data are mean ± s.d. (n = 3 biologically independent samples). Unpaired two-tailed t-test; P-values are shown. g, Quantification of HBB(42–cN58) and HBB(42–N58) from HBB(42–60) peptide and recombinant HBB protein after 144 h of incubation. h, Quantification of HBB(42–cN58) and HBB(42–N58) in two RBC donors by selected ion monitoring.