Changes of ubiquitylated proteins in atrial fibrillation associated with heart valve disease: proteomics in human left atrial appendage tissue

Figures

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  Figure 1. Flow chart of the study and QC validation. (A) Flow chart showing the proteomic ubiquitination analysis of SR and AF samples. (B) Consistency among 3 mixed tissue sample replicates was indicated by Pearson's correlation coefficient. (C) QC validation of the MS data: the accuracy of the mass data was in line with that needed, and most mass errors were <5 ppm. (D) The length of most peptides ranged from 7 to 20 AAs.

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  Figure 2. Ubiquitination analysis of SR and AF tissue samples. (A) Changes were observed at 4,788 quantifiable sites and in 1,631 quantifiable proteins. (B) Identified protein site number distribution: most proteins harbored <5 modification sites. (C) Differential ubiquitination sites and protein numbers: A total of 271 sites in 162 proteins exhibiting upregulated ubiquitination and 467 sites in 156 proteins exhibiting downregulated ubiquitination were identified. (D) Volcano plot showing differentially ubiquitylated proteins and sites.

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  Figure 3. GO enrichment-based functional classification of differentially ubiquitylated proteins. (A) We classified the sites with different modification abundances into 4 quartiles, called Q1–Q4, according to their different modification multiples. (B) Biological process analysis based on Q1–Q4 quantiles. (C) Cellular component analysis based on Q1–Q4 quantiles. (D) Molecular function analysis based on Q1–Q4 quantiles.

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  Figure 4. Protein domain and KEGG pathway analysis of differentially expressed ubiquitylated proteins. (A) KEGG pathway analysis based on Q1-Q4 quantiles. (B) Top 11 pathways of all KEGG pathways. The proteins involved in all KEGG pathways were most highly enriched in hsa05410 hypertrophic cardiomyopathy (HCM). (C) Protein complex analysis. (D) Summary of the 17 proteins associated with hsa05410 HCM: TTN had the most (348) modifications in all enriched KEGG pathways, followed by MYH6 (266).

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  Figure 5. Motif analysis of differentially altered ubiquitylated proteins. (A) Heatmap of the 10 bilateral AAs down- and upstream of the ubiquitylated lysine. Enrichments of valine (V), glutamic acid (E), aspartic acid (D) and alanine (A) residues were found downstream of the ubiquitylated lysine, whereas enrichments of valine (V) and alanine (A) were found upstream of the ubiquitylated lysine. (B) Sequence logo of acetylation motifs. The results revealed nine significantly enriched ubiquitination site motifs from the quantifiable ubiquitylated sites, where x stands for a random AA and Kac stands for a ubiquitylated lysine.

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  Figure 6. Ubiquitylated PPI network analysis. (A) PPI of glycolysis-related proteins. (B) PPI of hypertrophic cardiomyopathy-related proteins. (C) PPI of ribosome-related proteins. (D) PPI of endocytosis-related proteins.

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  Figure 7. Validation results. Validation of ubiquitylated MYOM1 and ubiquitylated MYOM3 as determined by coimmunoprecipitation (co-IP) combined with Western blotting (WB). (A,B) Ubiquitinated MYOM1. (C,D) Ubiquitinated MYOM3. IP, immunoprecipitation; IB, immunoblotting; MYOM1, myomesin 1; MYOM3, myomesin 3; ub, ubiquitylated. *P < 0.05.