Fig. 1: Antibody-based enrichment of CML-modified peptides. | Nature Communications

Fig. 1: Antibody-based enrichment of CML-modified peptides.

From: Mapping protein carboxymethylation sites provides insights into their role in proteostasis and cell proliferation

Fig. 1

a Workflow for the identification of CML-modified peptides. Ab: antibody b Absolute quantification of intracellular GO levels in HUVEC after treatment with GO (1 mM, 24 h). n = 5, mean + SD, * p < 0.01, paired t-test, two tailed. c Immunoblot for CML-modified proteins from HUVEC treated with GO for 48 h. Densitometric quantification is shown in Supplementary Fig. 1b. n = 3. d Quantification of total CML levels in MEF and HUVEC treated with GO for 8 h or 48 h, respectively. n = 6, mean + SD, * p < 0.05 vs. control, one-way ANOVA using Geisser-Greenhouse correction. e Percentage of peptide-spectrum matches (PSM) containing CML modification among different conditions. n = 3 (MEF), n = 2 (HUVEC), mean + SD, * p < 0.05. Multiple t-test corrected using Holm–Šidák method, two tailed. f Validation of CML-modified peptides by parallel reaction monitoring (PRM) using heavy spike-in peptides. g Quantification of a CML-modified peptide from histone H4 by PRM in MEF treated with GO for 24 h. h Venn diagram showing the overlap between unique CM sites identified in MEF and HUVEC. i Enrichment of Gene Ontology biological processes among CM sites identified in both HUVEC and MEF. The enrichment was performed using the Cytoscape App ClueGO. j, k Left, protein turnover of CM-modified proteins grouped according to their subcellular localization, as defined by Gene Ontology annotation. Turnover data were taken from Mathieson et al.32. C: cytoplasm; ER: endoplasmic reticulum; GA: Golgi apparatus; M: mitochondria; N: nucleus. Right, scatterplot comparing protein abundance (intensities, averages of n = 3 (MEF), n = 2 (HUVEC)) and protein turnover of CM-modified proteins. Data were fitted with a generalized additive model and the shades represent 95% confidence intervals. * p < 0.05, Wilcoxon Rank Sum test with continuity correction, two-sided. Source data are provided as a Source Data file. Specific p values are listed in Supplementary Data 6. Related to Supplementary Fig. 1 and Supplementary Data 1.

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