Systematic Profiling of Lysine Acetylation in Soybean Leaves
In this study, to globally investigate the acetylome in soybean leaves, we coupled with immune affinity enrichment and high-resolution LC-MS/MS methods (Figure 1A). To roughly detect lysine acetylome in soybean, we examined total proteins were isolated from soybean leaves by western blot using a pan acetyl-Lysine antibody (Micrometer Biotech Company, Hangzhou, China). In consequence, we detected many major protein bands which molecular weight higher than histones (Figure 1B).
So as to verify the MS/MS data, we detected the distribution of mass errors and lengths of the identified peptides. As the results shown, the distribution of most identified peptides mass errors was close to 0, and less than 2 ppm. Furthermore, the lengths of identified peptides were in the range of 7 to 15 amino acids, coincide with the properties of trypsin. proving the precision of MS/MS data (Figure 1C,D).Totally we identified 3148 acetylation sites in 1538 proteins from three biological replicates with a false discovery rate that were below 1%. Which includes 59 lysine acetylation sites in core histones, among 28 Kac sites in H2A, 15 Kac sites in H2B, 16 Kac sites in the two most conserved histones, H3 and H4 (Supplementary. Table1). Expected consistent with previously reported in other plant samples (Li et al., 2008; Lu et al., 2015; Smith-Hammond et al., 2014; Xiong et al., 2016) As far as we know, it is the largest dataset of lysine acetylation in plants to date.
Functional classification of acetylated proteins in Soybean Leaves
To better elucidate the potential roles of lysine acetylome in soybean leaves. We detected the Gene Ontology (GO) functional classification of all identified acetylated proteins based on their biological process, molecular function and cellular component (Figure 2A). According to the biological process classification there are 798 acetylated proteins involved in metabolic processes (52%) and 619 acetylated proteins involved in cellular processes (40%). The classification results for molecular function showed that 703 acetylated proteins were related to catalytic activity (43%), followed by 667 acetylated proteins binding of various targets (41%), and 128 acetylated proteins involved in structural molecule activity (8%) (Supplementary. Table2). Which highly consistent with acetylated proteins identified in other organisms(Li et al., 2016; Xue et al., 2018).
Within the cluster of subcellular localization, a total of 42% of the acetylated proteins was predicted in chloroplast and 29% was in cytoplasm (Figure 2B). The results showed that the largest proportion acetylated proteins were assigned to photosynthesis (Supplementary. Table3).
Functional enrichment analysis of acetylated proteins
To further elucidate the biological functions of acetylated proteins in soybean leaves, we performed functional enrichment of GO (biological process, molecular function and cellular component) and KEGG pathway analyses (Figure 3). In the biological process category, most of the acetylated proteins were shown to be involved in metabolic processes, includes carboxylic acid metabolic process (p value = 1.23e-34), oxoacid metabolic process (p value = 6.67e-33), nucleoside metabolic process (p value =9.78e-13), nucleoside phosphate metabolic process (p value =2.93e-12), ribose phosphate metabolic process (p value =3.03e-12). This result was also in agreement with the Functional classification analysis that most acetylated proteins were involved in cellular metabolism. Besides, we also found that a large number of acetylated proteins were significantly enriched in the glycosyl compound biosynthetic process (p value =1.19e-11) and amide biosynthetic process (p value =5.18e-12). The results of KEGG pathway enrichment analysis indicated that most of the acetylated proteins were related to Photosynthesis, Carbon fixation in photosynthetic organisms and Citrate cycle (TCA cycle).
Taken together, wide distribution of lysine acetylated proteins invovled in diverse pathways suggests lysine acetylation plays an important regulatory role in almost every aspects of cell metabolism in soybean leaves.
Analysis of acetylated-lysine peptide motifs
To evaluate the possible sequence motifs surrounding the acetylated lysine sites, we analyzed 3148 of the identified acetylated-lysine peptides from the −10 to + 10 positions around the Kac by motif-x algorithm(Schwartz and Gygi, 2005). 2840 acetylated peptides matched to 17 conserved motifs. Especially KacK, KacR, Kac*K, KacS, KacH and KacN motifs occupied the highest proportion, the acetylated peptides with these motifs were 478, 428, 264, 261, 238 and 196 (Kac represents acetylated lysine and * represents a random other amino acid residue) (Figure 4). In line with our observations, most acetylation motifs identified in soybean were also found in Fragaria ananassa (Fang et al., 2015), Oryza Sativa(Li et al., 2008) and Camellia sinensis (Xu et al., 2017). It was confirmed that lysine acetylation is a highly conservative modification between different organisms. According to the heat map of the amino acid composition around the acetylation site, Alanine (A), Glycine (G) and Valine (V) have the highest frequency at positions -1 to -4, while the occurrence of Serine (S), arginine (R) and Lysine (K) was lowest. According to these results, we can draw conclusions that proteins with A, G and V but without S, R and K around lysine residues will be preferred targets of lysine acetyltransferases in soybean.
Secondary structure analysis of acetylated lysine
In order to evaluate the relationship between protein structure and lysine acetylation in soybean. We used NetSurfP (v1.1) to analyze protein secondary structures of all acetylated proteins. The results show that, nearly 30% acetylated lysine and 27.5% all lysine were located in α-helices, besides 6.0% acetylated lysine and 6.4% all lysine were located in β-strands, approximately 63% acetylated lysine and 66% all lysine were located in disordered coils. 38.7% acetylated lysine and 66% all lysine were exposed to the protein surface (Figure 5). According to the results of distribution pattern between acetylated lysine and all lysine didn’t have obvious difference, acetylated lysine likely does not affect protein secondary structure.
Protein-protein interaction network analysis.
Interactions between proteins within cells are very important, since many molecular processes involve large amounts of protein components(Subramaniam et al., 2013). To further evaluate the acetylated protein-protein interaction how to regulates multiple metabolic processes and cellular functions. To generate the Protein-protein interaction network, all of the identified acetylated proteins were queried against to the STRING database (version 11.0), CytoScape softwear (v3.7.0) used to visualize the interaction network. Totally 1503 acetylated proteins were mapped to the STRING database, interaction network form STRING was visualized in Cytoscape. A graph theoretical clustering algorithm, molecular complex detection (MCODE) was utilized to analyz densely connected regions. As shown in (Figure 6), the top five clusters identified were the Ribosome (Cluster I), Oxidative phosphorylation (Cluster II), Proteasome (Cluster III), TCA cycle (Cluster IV) and Photosynthesis (Cluster V).