Dynamic lysine acetylation and succinylation of platelet proteins regulates platelet storage lesion: mechanistic insights from multi-omics.

Figures

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  Figure 1.. Workflow and strategy for quantitative multi-omics during platelet storage. (A): Schematic of platelet sample collection and storage timeline. Peripheral venous blood was collected, and platelets were isolated using a blood component apheresis machine. Isolated platelets were stored in dedicated containers under standard conditions, with samples harvested at days 1, 3, 5, and 7 of storage for subsequent multi-omics analyses. B: The sample diagram illustrating PLT storage. C: The global proteome, lysine acetylome, and succinylome were performed using the same platelets in triplicate. There was no difference in preparing the platelet samples stored within the same storage time among the three proteomic groups. Note: D1 means platelet stored for 1 day.

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  Figure 2.. Evaluation of MS and MS/MS data quality, and reproductibility of 3 repeated trials by Pearson correlation coefficient. A. Mass errors distribution of all identified peptides in parts-per-million (ppm) for global proteome (A1), acetylome (A2) and succinylome (A3). B. Peptide length distribution for global proteome (B1), acetylome (B2) and succinylome (B3). C. Reproducibility of global proteome (C1) for the 4 groups with 3 biological repeats. Reproducibility of acetylome for the 4 groups with 3 biological repeats (C2). Reproducibility of succinylome for the 4 groups with 3 biological repeats (C3). D. Typical MS/MS spectra images of 3 Kac and 3 Ksucc peptides. Note: D1 means platelet stored for 1st day of storage.

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  Figure 3.. Differential expression of global proteins, Kac sites/proteins and Ksucc sites/proteins. A. up- and down-regulated proteins; B. up- and down-regulated Kac sites/proteins; C. up- and down-regulated Ksucc sites/proteins.

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  Figure 4.. Temporal expression dynamics of stored platelets at days 1, 3, 5, 7. A: Proteins SDHA, NDUFS5, RHOT2, COX4I1, COX6A1 exhibit progressive decline. B: Proteins SHC1, BCL2L1, GLB1, SELP, BAD show escalating expression relative to day 1 (D1). Significance vs D1 denoted by * (p < 0.05), n = 3 donors. Gene details in Table 3.

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  Figure 5.. Gene Ontology (GO) enrichment analysis of up- and down-regulated proteins during platelet storage. The x-axis denotes -log10(Fisher’s exact test P-value), and y-axes list enriched GO terms encompassing molecular function (MF), cellular component (CC), and biological process (BP) ontologies. Analyses utilized Fisher’s exact test, with n = 3 donors (consistent with prior experiments).

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  Figure 6.. Pathway enrichment of differential proteins in specific comparison groups. Log-transformed Fisher’s exact test was carried out to assess biochemical and physiological changes observed at different time points for up-regulated and down-regulated proteins. Note: Red indicates enrichment of up-regulated proteins. Green indicates enrichment of down-regulated proteins.

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  Figure 7.. Heatmaps from GO and KEGG pathway enrichment analysis of the differential protein expression based on Z scores. A: Enriched biological processes for global proteins. B: KEGG pathway-based enrichment analysis.

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  Figure 8.. Cross-analysis combined with random forest screened 20 core proteins for differential expression.A.The venn diagram presents the 350 candidate proteins obtained from the cross analysis. B.The bars show the 20 core proteins obtained by random forest.C.The heat map presents the results of differential analysis of the 20 core proteins.D.Scatter plots show the validation of key gene expression levels.