Extended Data Fig. 8 ∣. Analysis of C-terminal cQ/cN modifications on hemoglobin derived from red blood cells and beta-crystallin derived from bovine lens.

(a) Unique proteins and peptides that carry a semi-tryptic terminal N or Q from global proteomics datasets. (b) Western blot of red blood cell (RBC) lysates from two donors in comparison to HEK293T lysate. RBCs do not express CRBN. (c) Representative MS2 spectra of HBB(42–cN58) and HBA(63–cN79) detected in RBC lysates. Western blot is representative of 2 independent replicates. (d) Comparison of peptide spectral matches (PSMs) for selected hemoglobin subunits and actin observed in global proteomics datasets, recombinant hemoglobin beta (HBB) protein, and red blood cell (RBC) lysates. HBA(63–cN69) was observed by extracted ion chromatogram in the MS1. Sites selected for further analysis are highlighted in red or blue. (e) Quantification of the three major peptide groups bearing a C-terminal cyclic imide from RBC samples with or without base treatment. Data are presented as mean ± SD (n = 3 biologically independent samples). The noted p-values were obtained by one-way ANOVA with TukeyHSD post-hoc test from Proteome Discoverer. (f) Quantification of the two major chymotryptic peptide groups bearing C-terminal cyclic imides in RBC samples with or without base treatment. Data are presented as mean ± SD (n = 5 biologically independent samples). The noted p-values were obtained by one-way ANOVA with TukeyHSD post-hoc test from Proteome Discoverer. (g) Quantification of the absolute masses and percentages of HBB(42–60), HBB(42–cN58), and HBB(42–N58) in RBC samples using selected ion monitoring. (h) Ion intensity chromatograms extracted for the masses of the cyclic imide fragment and the corresponding tryptic peptide for three cyclic imide-bearing peptide groups identified in bovine lens. (i) Quantification of these peptide groups validates the sensitivity of the cyclic imide modifications to base treatment. Data are presented as mean ± SD (n = 4 biologically independent samples). The noted p-values were obtained by one-way ANOVA with TukeyHSD post-hoc test from Proteome Discoverer. For uncropped western blot images, see Supplementary Fig. 11.