The whole process consists of nine steps (Fig. 1A). A total of 1209 laidol acylation sites were identified, of which 1109 of 347 proteins were quantifiable (Table S1). As fold changes over 1.2 as an up regulation and below 1/1.2 as a down regulation. There was a differential expression of 260 protein loci in chronic renal failure and healthy controls, of which 772 were from up-regulated 260 proteins and 51 from 69 down-regulated proteins. Another result showed thatSignificantly differential expression lysine crotonylation between the CRF and NC(Table S2). In order to verify the validity of mass spectrum data, the mass errors of all identified peptides were evaluated. The quality error is centered at 0 and below 10 ppm, which shows that the quality accuracy of ms data meets the requirement (Fig. 1B). Among 347 crotonyl proteins, most proteins contain one or two crotonylation sites, while fewer proteins have 7 or more crotonylation sites (Fig. 1C). The length of most peptidesvaried from 8 to 20 amino acids, which was in accordance with the law of trypsin digestion peptide ( Fig. 1D) for one day.
Functional classification of GO and subcellular lysine crotonylation the subcellular localization characteristics of lysine crotonylation have been identified (Fig. 3A). It turned out that the up-regulated proteins were mainly distributed in cytoplasm (56%), nucleus (10%), mitochondria (10%) and extracellular (10%), while down-regulated proteins were mainly distributed in cytoplasm (57%), nucleus (7%), mitochondria (4%) and extracellular (17%) (Fig. 3B). It showed that there was no significant difference in protein localization between up-regulated and down-regulated proteins. In order to understand the general situation of crotonyl protein in chronic renal failure, on the basis of its biological process, molecular function and cell composition, the GO functional classification of all crotonyl proteins was studied (Table S4). In the category of biological process, most crotonyl proteins are associated with cellular processes, single organism processes, biological regulation and stimulation (Fig. 3C), and most down-regulated proteins are associated with cellular processes, single organism processes, biological regulation and stimulation (Fig. 3D). In cell components, most crotonyl proteins are associated with cells, organelles, extracellular domains, and the membrane of up-regulated proteins (Fig. 3E), while most of the down-regulated proteins are associated with cells, organelles, extracellular domains and membranes (Fig. 3F). In the molecular functional category, most crotonyl proteins are associated with up-regulated protein binding, catalytic activity, structural molecular activity and molecular function modulators (Fig. 3G), while most down-regulated proteins are concerned with binding, catalytic activity, molecular function modulators and structural molecular activity (Fig. 3H). There was no significant difference in GO functional classification between up-regulated and down-regulated proteins, suggesting that crotonylation of lysine might have a large scale of biological functions.
3.4 Functional enrichment of Kcr in GO, KEGG, and protein domain
The functional enrichment of GO, KEGG and protein domain lysine crotonylation based on GO was studied(Table S5). Highly expressed Croton protein is highly enriched on platelets and erythrocytes CD36 (Fig. 5A). However, there is no up-regulated crotonyl protein in GO, but down-regulation of crotonyl protein mainly includes (Fig. 4A). At the same time, the function enrichment analysis based on KEGG was carried out (Table S6). It was found, however, that there was no up-regulated Croton acylation protein in KEGG (Fig. 4B). In addition, the down-regulated crotonylation domain includes the S100/CaBP9K calcium binding subdomain, globin, globin-like, Globin/Protoglobin. The EF-hand domain pair and EF-hand domain (Table S7, Fig. 4C) suggest an important part for crotonylation during these processes. Similarly, there was no up-regulated crotonylation of the protein domain in this study. Pentose phosphate pathway has an important relationship with complications of CRF. Search for crotonyl proteins involved in carbon metabolism, including dense protein interaction networks (Fig. 5B).
3.5 Cluster analyses in GO, KEGG, and protein domain
For the sake of understanding the function of lysine crotonylation in more detail, we performed GO, KEGG, and protein domain enrichment-based clustering analyses. All quantized crotonylation sites were separated into four quartiles according to the multiple changes of the Lysine crotonylation sites: Q1 (0<−<0.77), Q2 (0.77 < 0.77), Q3 (1.2<−<1.3), and Q4 (ratio > 1.3), P value < 0.05. Q1, Q2, Q3 and Q4 have 54, 15, 98 and 647 crotonylation, respectively. Then, the four kinds of quantifiable proteins were analyzed by cluster analysis. Q1 and Q2 are considered down-regulated, while Q3 and Q4 are considered up-regulated (Fig. 5C).
For GO analysis,It was found that the crotonylated protein in Q1 was mainly enriched in cell secretion, and the crotonylated protein in Q2 was mainly enriched in cell substrates, neurons and cytoskeleton, the crotonylated protein in Q3 was mainly enriched in cell junctions, while the crotonylated protein in Q4 was mainly abundant in cell microcrystals composed of cell membranes and cells (Fig. 6A). In addition, the biological enrichment process of crotonylation was also carried out. It was found that the Croton acylated protein in Q1 was mainly concentrated in the cell response to pathophysiology, and that the crotoacylated protein in Q2 was located in cell function, such as migration and development. However, the crotonyl protein in Q3 is mainly enriched in cell metabolism. In addition, the crotonyl protein in Q4 is mainly concentrated in the cell response to the outside world (Fig. 6B). For the molecular functional crotonyl proteins, they were found to be highly enriched in the activity of Q1 cells and highly increased in the cell-binding processes of Q3 and Q4 (Fig. 6C).
The enrichment of KEGG suggests that certain pathways of Q3 and Q2 are associated with diseases, for instance, pancreatic cancer, type II diabetes, cellular metabolism, and salmonella infection, while Q1 crotonylated acylated protein is abundant in cell signaling pathways and measles and influenza viruses, and Q4 crotonylated acylated protein is enriched in cell signaling pathways and germ cell division (Fig. 6D).
At the same time, the protein domain of Croton protein was studied. They were found to be highly enriched in Q2: Calponin homology domain, aldolase-Tim type barrel, globin/proton protein, globin, globin-like (Fig. 6E).
3.6 Protein–protein interaction network of the acylation of Croton acylation proteins
The protein-interaction network of acylation of Croton acylation protein was established by further identifying the cellular processes regulated by the acylation of Croton acylation protein (Fig. 7). A total of 888 pairs of protein-protein mapping to the protein interaction database, showing the different cellular function of Croton protein in CRF. The physiological interaction between these protein complexes may contribute to their synergy and coordination in CRF.