Fig. 2: The CM-modified proteome in mouse organs.

a Workflow for the analysis performed on mouse organs from young (Y) and old (O) mice. Image was created with BioRender.com. Tandem Mass Tag (TMT) was used for monitoring protein abundance, CMLpepIP for CM site identification and PRM for quantification. b Venn diagram of CM-modified proteins (left) and sites (right) identified in heart, kidney and liver from young and old mice combined. For each tissue, only unique entries were considered for the overlap. P-value was calculated by Fisher’s Exact Test. c Enrichment of Gene Ontology biological processes among CM sites identified from heart, kidney and liver. Enrichment was performed using the Cytoscape App ClueGO. d Left, percentage of surface exposed CML sites. A 25% threshold for relative surface area (RSA) score was used to define sites as surface exposed. Right, percentage of CML sites located in different protein secondary structures. e Overlap between CML sites and other known PTMs. Gray scale pie charts indicate the number of overlapping CML sites and colored pie charts the overlapping PTM classes (shown as percentages). Known PTM were obtained from Minguez et al.⁹² and Hornbeck et al.⁹³. f Effect of aging on the abundance of proteins of the glyoxalase 1 (GLO1) network. The network was extracted from https://string-db.org using GLO1 as input. n = 5, * adj. p < 0.05, unpaired t-test, two tailed with Benjamini–Hochberg correction for multiple testing, as implemented in limma. g. GLO1 activity tested on heart, kidney and liver lysates from young (Y) and old (O) mice. n = 7, mean + SD, * p < 0.05, unpaired t-test, parametric, two tailed. h Immunoblot (left) and densitometry-based quantification (right) for CML-modified proteins in mouse organs from young and old mice. n = 6 (kidney, heart), n = 8 (liver), mean + SD, * p < 0.01, unpaired t-test, two tailed. i, j Normalized protein intensity of histone H4, ADP/ATP translocase type 1 and ATP synthase subunit γ from TMT experiment (protein—left panel) and intensity of CML-modified peptides from PRM (right panel). n = 5 for protein abundance, n = 8 for CML-modified peptides, mean + SD, * p < 0.05, unpaired t-test, two tailed. Source data are provided as a Source Data file. Specific p values are listed in Supplementary Data 6. Related to Supplementary Fig. 2 and Supplementary Data 2.