Identification and analysis of lysine crotonylation sites and crotonylated proteins in peanut leaves.
The experimental procedure is presented diagrammatically in Fig. 1a. In order to identify the crotonylated proteins and crotonylation sites, we used sensitive immunoaffinity purification and high-resolution LC–MS/MS. Altogether, 6051 crotonylation sites were identified in 2508 proteins. The statistics of the data are presented in Additional file 1: Table S1. Data are available via ProteomeXchange (https://www.ebi.ac.uk/pride), with the identifier PXD017675. Among the 2508 crotonylated proteins, the majority of the proteins had fewer than three crotonylation sites, with only a few proteins having seven or more crotonylation sites. The length of most of the peptides was between 8 and 28 residues, which agrees with the size distribution of tryptic peptides (Fig. 1c), indicating that sample preparation reached the standard required. The mass errors of the proteins was almost zero, meaning that the mass spectrometry data met the required standard (Fig. 1b).
Analysis of crotonylation site motifs
To investigate any patterns of amino acids adjacent to the Kcr sites, we used the Motif-X program. A total of six clearly conserved motifs (with a motif score >25) were identified (Fig. 2a), namely EKcrG, AKcrE, EKcrA, DKcrI, EKcrV and KcrE……K; a substantial bias in amino acid distribution was observed from the -10 to +10 positions around the Kcr sites in peanut (Fig. 2b). As shown in Fig. 2a, the first five motifs contained a residue with acidic groups (E, G, A, D, I or V) at the -1 or +1 position. The sixth motif contained a residue with acidic groups (E or K) at the +1 or +5 position.
Structural analysis of all the crotonylated proteins
To understand the relationship between Kcr and protein structure, the structures of all the crotonylated proteins were analyzed. A total of 46.5% of the crotonylated sites were located in α-helices, 10.6% of the sites were in β-strands and the rest of the sites were in disordered coils. The distribution pattern between the crotonylated lysines and all lysines was very similar (Fig. 2c). In addition, analysis of surface accessibility of crotonylation sites of peanut proteins showed that Kcr may not affect the surface properties of the modified proteins, because the enrichment of crotonylated sites on the protein surface is very similar to that of all lysine residues (Fig. 2d).
Functional annotation and cellular localization of crotonylated proteins in peanut
To obtain further information on the distribution and function of lysine crotonylated proteins identified in peanut, we summed the number of the proteins in each level 2 Gene Ontology (GO) term, namely biological process, cellular component and molecular function (Fig. 3, Additional file 2: Table S2). The GO analysis showed that the crotonylated proteins were involved in diverse biological processes, cellular components and molecular functions. The identified crotonylated proteins with respect to biological process being related to metabolic process, cellular process, single-organism process, localization, biological regulation, response to stimulus, cellular component organization and biogenesis and others, accounting for 1106 (36%), 852 (28%), 671 (22%), 151 (5%), 110 (4%), 88 (3%), 69 (2%) and 28 (1%), respectively (Fig 3a, Additional file 3: Table S3). In the cellular component classification, 443 (38%), 268 (23%), 254 (21%), 197 (17%) and 13 (1%) were associated with cell, macromolecular complex, organelle, membrane and others, respectively (Fig. 3b, Additional file 3: Table S3). In the molecular function classification, proteins related to binding, catalytic activity, structural molecule activity, transporter activity, and others, accounted for 1088 (46%), 1015 (42%), 121 (5%), 78 (3%) and 88 (4%), respectively (Fig. 3c, Additional file 3: Table S3). These data indicated that crotonylated proteins take part in a range of biological processes. According to subcellular location annotations of the crotonylated proteins, we summed the number of proteins in each subcellular location. The crotonylated proteins identified in peanut leaf were located in the chloroplast (1068, 43%), cytosol (618, 25%) and nucleus (428, 17%), with a further 116 (5%) proteins being located in the plasma membrane, with 112 (4%) proteins located in the mitochondria, 53 (2%) proteins located to the extracellular space and 30 (1%) proteins located in the cytoskeleton (Fig. 3d, Additional file 4: Table S4).
Functional enrichment analysis of crotonylated proteins
In order to identify the types of proteins that were involved in lysine crotonylation, we performed GO enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. GO enrichment analysis included biological process, cellular component and molecular function classifications (Fig. 4a, Additional file 5: Table S5). The enrichment analysis of the biological process grouping indicated that crotonylated proteins were enriched with respect to biosynthetic processes and catabolic processes. In the cellular component category, the crotonylated proteins were significantly enriched with respect to the cytoplasmic component. There were also many crotonylated proteins associated with thylakoids, intracellular non-membrane-bounded organelles and the proteasome core complex. With respect to the enrichment of the molecular function category, most crotonylated proteins were enriched with respect to ligase activity, cofactor binding, structural constituents of ribosomes, oxidoreductase activity, and isomerase activity (Fig. 4a, Additional file 5: Table S5).
The KEGG pathway indicated that Kcr was associated with many proteins involved in carbon fixation in photosynthetic organisms, aminoacyl-tRNA biosynthesis, glutathione metabolism, the pentose phosphate pathway, biosynthesis of amino acids, ribosomes, carbon metabolism, proteasomes, photosynthesis, glyoxylate and dicarboxylate metabolism, oxidative phosphorylation, pyruvate metabolism, glycolysis/gluconeogenesis, the citrate cycle (TCA cycle), photosynthesis-antenna proteins, and alanine, aspartate, and glutamate metabolism (Fig. 4b, Additional file 6: Table S6). These results imply that crotonylated proteins play an important regulatory role in a wide range of important processes. In line with these results, the ClpP/crotonase-like domain, Glutathione S-transferase, C-terminal-like and Glutathione S-transferase N-terminal domains were clearly enriched in crotonylated proteins (Fig. 4c, Additional file 7: Table S7).
Crotonylated proteins involved in photosynthesis
Photosynthesis is a vital biological process in all plants, including peanut. A total of 29 crotonylated proteins were involved in photosynthesis. LC–MS/MS analysis showed that many subunits of these proteins were crotonylated (Fig. 5a): nine subunits of photosystem II (PsbC/B/O/P/Q/R/S/27/28 ), six subunits of photosystem I (PsaD/E/F/G/H/N), one subunit of the cytochrome b6/f complex (PetC), two subunits of photosynthetic electron transport (PetE/H) and three subunits of F-type ATPase (gamma, delta, b). The data from the functional enrichment analysis showed that crotonylated proteins may play an important role in photosynthesis. LC–MS/MS analysis also showed that a large number of subunits were crotonylated in ribosomes (Fig. 5b).
Interactive networks among crotonylated proteins in peanut
To further investigate the regulatory role of crotonylation in photosynthesis and ribosomes, we used the algorithm from the Cytoscape software to establish protein interaction networks (Fig. 6a, Additional file 8: Table S8). In the peanut photosynthesis network, 26 crotonylated proteins were identified as nodes in the protein interaction database. In the peanut ribosome network, 145 proteins were mapped to the protein interaction database (Fig. 6b, Additional file 9: Table S9). These maps provide a view of how crotonylated proteins regulate photosynthetic and ribosome-associated biological processes.