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Global analysis of protein lysine 2-hydroxyisobutyrylation (Khib) profiles in Chinese herb rhubarb (Dahuang)



Lysine 2-hydroxyisobutyrylation (Khib) is a newly discovered protein posttranslational modification (PTM) and is involved in the broad-spectrum regulation of cellular processes that are found in both prokaryotic and eukaryotic cells, including in plants. The Chinese herb rhubarb (Dahuang) is one of the most widely used traditional Chinese medicines in clinical applications. To better understand the physiological activities and mechanism of treating diseases with the herb, it is necessary to conduct intensive research on rhubarb. However, Khib modification has not been reported thus far in rhubarb.


In this study, we performed the first global analysis of Khib-modified proteins in rhubarb by using sensitive affinity enrichment combined with high-accuracy HPLC-MS/MS tandem spectrometry. A total of 4333 overlapping Khib modification peptides matched on 1525 Khib-containing proteins were identified in three independent tests. Bioinformatics analysis showed that these Khib-containing proteins are involved in a wide range of cellular processes, particularly in protein biosynthesis and central carbon metabolism and are distributed mainly in chloroplasts, cytoplasm, nucleus and mitochondria. In addition, the amino acid sequence motif analysis showed that a negatively charged side chain residue (E), a positively charged residue (K), and an uncharged residue with the smallest side chain (G) were strongly preferred around the Khib site, and a total of 13 Khib modification motifs were identified. These identified motifs can be classified into three motif patterns, and some motif patterns are unique to rhubarb and have not been identified in other plants to date.


A total of 4333 Khib-modified peptides on 1525 proteins were identified. The Khib-modified proteins are mainly distributed in the chloroplast, cytoplasm, nucleus and mitochondria, and involved in a wide range of cellular processes. Moreover, three types of amino acid sequence motif patterns, including EKhib/KhibE, GKhib and k.kkk….Khib….kkkkk, were extracted from a total of 13 Khib-modified peptides. This study provides comprehensive Khib-proteome resource of rhubarb. The findings from the study contribute to a better understanding of the physiological roles of Khib modification, and the Khib proteome data will facilitate further investigations of the roles and mechanisms of Khib modification in rhubarb.

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Protein posttranslational modifications (PTMs) are reversible and dynamic modifications of proteins that are crucial to protein maturation, protein structure and function. Currently, PTMs are known to be the most efficient strategies for expanding the functional diversity of proteins and play key roles in diverse biological processes [1, 2]. Among a wide array of types of PTMs, lysine residues of proteins have been found to be the most common modification sites, such as ubiquitination, SUMOylation, methylation, acetylation, butyrylation, succinylation, crotonylation, malonylation, and propionylation at lysine [3]. Recently, lysine 2-hydroxyisobutyrylation (Khib), a new type of PTM at lysine, was discovered to be widespread on human and mouse histone proteins, and it is considered to be a new histone mark in eukaryotic cells [4]. Dozens of Khib sites have been identified on histones, and histone Khib has been shown to regulate gene transcription, which is associated with cell differentiationand cell proliferation [4,5,6,7].

Khib signals have been detected in HeLa cells, mouse embryonic fibroblasts (MEFs), Drosophila S2 cells and yeast Saccharomyces cerevisiae cells on both histone and nonhistone proteins [4]. These findings suggest that Khib should be present in nonhistone proteins and should have functions independent of nucleosomes. To date, Khib has been detected in eukaryotes, including human and animal cells [4, 8] and tissues [9], yeast (S. cerevisiae) [6], plants (Physcomitrella patens [10], Oryza sativa [11, 12]), and bacteria, including Proteus mirabilis [13,14,15] and Toxoplasma gondii [16]. These findings indicate that Khib exists on histone proteins in the nucleus but also widely exists on nonhistone proteins in various parts of the cells. For example, proteome-wide profiling of Khib identified 6,548 Khib sites on 1,725 substrate proteins in mammalian cells [6, 17].

LC-MS/MS-based qualitative proteome analysis in rice (O. sativa) showed that most Khib proteins are located in the cell (38 %), 28 % in organelles, and 15 % in the membrane [12]. Meanwhile, these identified Khib proteins show a variety of functions, with 44 % of the proteins involved in binding, 40 % of proteins involved in catalytic functions, etc. [12]. In summary, based on accumulating proteomic datasets, Khib occurs extensively in proteins and is an evolutionarily conserved PTM observed in various organisms. Therefore, comprehensive detection of Khib-modified proteins and modification sites is essential for understanding the physiological functions and regulatory mechanisms of cells.

However, Khib modification has not been reported thus far in the Chinese herb rhubarb. Rhubarb, also named Rhei or Dahuang, belongs to the Rheum L. genus from the Polygonaceae family, which is one of the most ancient and important herbs in traditional Chinese medicine and has been widely used in clinical applications for over a thousand years [18, 19]. Currently, rhubarb has been suggested to possess various forms of activity such as antibacterial, anti-inflammatory, antifibrotic, and anticancer activity, and the regulation of gastrointestinal function. Due to its wide range of pharmacological activities, rhubarb has been used in the treatment of several diseases, including severe acute pancreatitis (SAP), sepsis, and chronic renal failure (CRF) [18]. To better understand the physiological activities and mechanism of treating diseases with the herb, it is necessary to conduct intensive research on rhubarb. Studies have suggested that Khib is broadly obtainable in plant cells, and this modification is highly conserved and enriched in various biological processes [11]. Therefore, to investigate the Khib modification in rhubarb and the potential regulatory functions of Khib for its physiological activity, we present the first systematic proteome-wide analysis of Khib modification in rhubarb. Our findings provide a promising platform for the further exploration of Khib modification in physiological processes in rhubarb.

Materials and methods

Plant material and cultivation

Plants of rhubarb (Rheum palmatum L.) were used in this study. The specimen of the plant has been deposited in Chinese Virtual herbarium with herbarium ID of WUK 520,024. The rhubarb used in our experiments were identified by Zhu Renbin. Rhubarb plants approximately 2 years old were cultivated in nutrient soil for 20 days in a greenhouse at a relative humidity of 50 % and at 22 °C during the day and 18 °C at night. The leaves of the cultivated rhubarb were collected and used for extraction of protein samples.

Protein extraction, Western blotting and digestion

Whole proteins of rhubarb leaves were extracted by using a modified phenol isolation protocol [20]. In brief, rhubarb tissues were prefrozen in liquid nitrogen and then ground into fine powder with a mortar and pestle in liquid nitrogen and 10 % polyvinylpolypyrrolidone (PVPP). Ground tissues were suspended in 1.6 mL lysis buffer (1 M sucrose, 0.5 M Tris 8.0, 0.1 M KCl, 50 mM ascorbic acid, 1 % NP40, 1 % NaDOC, 10 mM EDTA, 10 mM DTT, and 1 % protease inhibitor cocktail; the final pH was 8.0) and then sonicated on ice for 30 min to promote the dissolution of the sample proteins. The resulting solution was mixed with an equal volume of tris-saturated phenol (pH 8.0) and then homogenized for 10 min. The homogenate was centrifuged for 10 min at 16,000 g, and the upper phenol phase was collected. The collection of phenol was mixed with four volumes of precipitation buffer (methanol with 0.1 M ammonium acetate, precooled at -20 ℃ before use), and protein precipitation was performed at -20 ℃ overnight. The precipitate was collected by centrifugation at 16,000 g for 10 min, and the pellet was rinsed with cold methanol, repeated three times. The final protein pellet was dissolved in 0.4 mL lysis buffer. The protein concentration was determined by a 2D Quant kit (GE Healthcare).

For Western blotting, a total of 30 µg of protein samples was electrophoresed in a 12 % slab SDS-PAGE gel, and Khib-modified proteins were detected with rabbit-derived anti-2-hydroxyisobutyryllysine pan antibody (Micrometer Biotech, China) in a standard procedure. For tryptic digestion, proteins were first reduced by using 5 mM DTT and subsequently alkylated by using 30 mM iodoacetamide (IAM) in a standard procedure. The resulting proteins were suspended in 0.1 M and digested overnight with sequencing grade trypsin (Promega) at a trypsin:substrate ratio of 1:50 (w/w). The tryptic peptides were purified by using a Strata X C18 SPE column (Phenomenex) and vacuum-dried before affinity enrichment.

Immunoaffinity enrichment of Khib-modified peptides

The immunoaffinity enrichment of Khib-modified peptides were performed as described previously [11, 12]. Briefly, the dried peptides were dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM tris-HCl, 0.5 % NP-40, pH 8.0) and incubated with anti-2-hydroxyisobutyryllysine pan antibody conjugated to agarose beads (Micrometer Biotech, China) overnight with gentle oscillation at 4 °C. Then, the agarose beads were washed four times with NETN buffer and once with ice-cold deionized water. Khib-modified peptides were eluted by using 0.1 % trifluoroacetic acid (TFA) and desalted with a C18 ZipTips column (EMD Millipore).

HPLC-MS/MS analysis

HPLC-MS/MS analysis was carried out using a nano HPLC-MS/MS (UltiMate RSLCnano 3000, Thermo Scientific) coupled to a Q Exactive HF-X mass spectrometer (Thermo Scientific). The enriched Khib peptides were dissolved in HPLC buffer A (0.1 % (v/v) formic acid in water) and then separated by a C18 capillary column (Acclaim PepMap RSLC, 75 μm × 50 cm, 2 μm particles, Thermo Fisher Scientific) at a flow rate of 250 nL/min with a 62 min HPLC gradient (2–10 % buffer B (0.1 % formic acid and 80 % acetonitrile in water) in 6 min, 10–20 % buffer B in 45 min, 20–80 % buffer B in 7 min and 80 % buffer B for 4 min). The eluted peptides were then ionized and electrosprayed (2.0 kV voltage) into the mass spectrometer. The mass spectrometric analysis was performed in data-dependent mode with an automatic switch between a full MS scan and an MS/MS scan in the Orbitrap. Peptides were detected in MS at a resolution of 60,000 with a scan range of 350–1800 m/z and with automatic gain control (AGC) of 1e6. The top 15 peaks were selected for MS/MS by higher-energy collision dissociation (HCD) with a normalized collision energy (NCE) of 26 %. The MS/MS spectra were acquired at a resolution of 30,000. The dynamic exclusion for the data-dependent scan was 6 s, and AGC was set at 1E5. HPLC-MS/MS analysis was performed by Micrometer Biotech Company (Hangzhou, China).

Database search

The resulting MS/MS raw data were searched against the Rheum palmatum, L. transcriptome data (NCBI BiopPoject ID: PRJNA714631). The transcriptome data were concatenated using the decoy database using the MaxQuant search engine (v1.5.2.8) with false discovery rates (FDRs) for proteins, peptides and modifications of less than 1 %. Trypsin/P was specified as a cleavage enzyme and allowed a maximum of 4 missed cleavages, 5 modifications per peptide and 5 charges. The mass tolerances for precursor ions and fragment ions were set to 10 ppm and 0.02 Da, respectively. Carbamidomethylation on Cys was specified as a fixed modification, and oxidation on Met, 2-hydroxyisobutyrylation on both Lys and the protein N-terminus were specified as variable modifications. The minimal peptide length was set to 7. Khib sites identified with a localization probability of < 0.75 were removed. The cut-off score for modified peptides was set to 40.

Bioinformatics analysis of Khib-modified peptides and proteins

The bioinformatics analysis of the identified Khib-modified peptides and proteins was performed as described in detail previously [11]. Briefly, amino acid sequence motifs (10 amino acids upstream or downstream of Khib sites) were analysed by using motif-X software ( Motif-based clustering analysis was also performed, and cluster membership was visualized by using a heat map via the “heatmap.2” function in the “gplots” R package.

The Gene Ontology (GO) annotation of disentitled proteins was derived from the UniProt-GOA database ( Proteins were classified into three categories (biological process, cellular compartment, and molecular function) based on GO annotation. The InterPro database was searched for proteins that were not annotated by the UniProt-GOA database. The Kyoto Encyclopedia of Genes and Genomes (KEGG) [21] Automatic Annotation Server (KAAS) tool was used to identify enriched pathways, and the resulting results were mapped to the KEGG pathway database by using the online service tool KEGG mapper that provided by Kanehisa Laboratories ( The functional annotation tool of DAVID bioinformatics resources 6.7 was used for GO and KEGG pathway enrichment analysis. Fisher’s exact test (two-tailed test) was applied to test for GO/KEGG pathway enrichment analysis. Correction for multiple hypothesis testing was performed by using standard FDR control methods, and annotation terms with a corrected p-value < 0.05 were considered significant. Additionally, the subcellular localization of identified proteins was predicted by the WoLF PSORT platform ( The domain functional descriptions of the identified proteins were annotated using the sequence analysis application InterProScan ( based on the protein sequence alignment method, and the InterPro domain database (a resource that provides functional analysis of protein sequences by classifying them into families and predicting the presence of domains and important sites) was used.

Results and discussion

Proteome-wide identification of Khib modification in rhubarb

Lysine Khib modification is a novel protein PTM initially identified on histones and involved in transcriptional activity regulation in mammals [4]. In recent years, this modification has been identified in diverse organisms, including eukaryotes and prokaryotes [4, 10, 14, 22]. However, the Khib modification has not been revealed in rhubarb, a type of widely used traditional Chinese medicine in clinical application in China. In this study, to detect the Khib modification status in rhubarb, total protein samples were isolated from rhubarb leaves and submitted to proteome-wide identification conducted by combining affinity enrichment by the Khib antibody with nano-HPLC-MS/MS.

To obtain more accurate data, we performed three independent tests, and 6062, 7594 and 7660 Khib-modified peptides were separately identified with peptide score > 40, localization probability > 0.75 and most peptide mass error < 2 ppm (the detailed identification information is shown in Supplemental Table S1 and Fig. S1). Overall, a total of 4333 peptides that overlapped in the three tests were identified to be Khib-modified. The lengths of the identified peptides varied from 7 to 43 amide acid residues, but most peptide lengths were between 7 and 20 residues (Fig. 1 A). These Khib-modified peptides matched 1525 proteins with Khib sites from 1 to 31 (Fig. 1 B). Although most proteins contain only one Khib site, 4 proteins were found to have more than 20 Khib sites, including elongation factor 2-like (22 sites), hypothetical protein CCACVL1_04477 (22 sites), ribosomal protein (23 sites), and heat shock cognate protein 80 (Supplemental Table S1), indicating that Khib is a type of multiple PTM. The distribution of modified peptide lengths and modified sites within individual proteins was similar to previous reports [23, 24]. Based on the results described above, Khib modification is an abundant and complex PTM and may have a very important regulatory effect on protein function in plants, which is consistent with previous reports of the Khib modification proteome identified in rice [11].

Fig. 1
figure 1

Distribution of identified lysine 2-hydroxyisobutyrylation (Khib) peptides and proteins. (A) Distribution of Khib-modified peptides based on their length; (B) Distribution of Khib-modified proteins based on the number of Khib peptides identified

Characterization of Khib modification proteins identified in rhubarb

Gene ontology (GO) classifications are often used to help analyse the potential functions of proteins [25]. Therefore, to better understand the potential roles of Khib-modification in rhubarb, all identified Khib-modified proteins were submitted to GO functional classification analysis based on their biological process, cellular component, and molecular function. As shown in Fig. 2 and Supplemental Table S2, the classification results showed that the Khib-modified proteins were involved in a variety of biological processes, cellular components and molecular functions. In the classification of the GO term “biological process”, two major classes of Khib-modified proteins were classified into metabolic process and cellular process, accounting for 37 and 29 % of the total Khib-modified proteins, respectively (Fig. 2, indicated in green). In the classification of the GO term “molecular function”, most of the Khib-modified proteins were classified into two categories, catalytic activity (45 %) and binding (44 %) (Fig. 2, indicated in purple). This result was in good agreement with the classification result of the biological process, and these two results suggest that the enzymatic proteins associated with metabolism and regulatory proteins related to cellular processes are more likely to be Khib-modified in rhubarb. In the “cellular components” category of GO classification, 40 % of all identified Khib proteins were in the cell, 24 % in macromolecular complex, 22 % in the organelle and 13 % in the membrane (Fig. 2, indicated in orange).

Fig. 2
figure 2

GO functional classification of the Khib-modified proteins based on biological process, molecular function, and cellular component. The number of proteins and the percentages of each category are indicated

The subcellular localizations of Khib-modified proteins were also analysed, and the results showed that most of the proteins were distributed in the chloroplast (45 %), cytoplasm (35 %), nucleus (11 %) and mitochondria (6 %) (Fig. 3 and Supplemental Table S3).

Fig. 3
figure 3

Subcellular localization distribution of Khib-modified proteins

The overall trend of GO classification (including biological process, molecular function and cellular components) and subcellular localization of Khib proteins identified in rhubarb were similar to previous findings obtained in other plants such as P. patens [10] and rice [12], although the percentages of each category were slightly different.

GO, KEGG Pathway and Domain Enrichment Analysis of Khib-modified Proteins

To investigate which categories of proteins are preferred targets for Khib modification, the GO, KEGG pathway and protein domain enrichments of the Khib modification proteome obtained in rhubarb were analysed, and the significantly enriched categories for biological process, molecular function and cellular component of Khib-modified proteins are shown in Fig. 4 and Supplemental Table S4. In the GO enrichment of biological process, the terms glycolytic process, single-organism carbohydrate catabolic process, peptide biosynthetic process, translation, amide biosynthetic process and peptide metabolic process were found to be significantly enriched (Fig. 4, indicated in green bars). The enrichment of these biological processes suggests that those proteins associated with carbohydrate metabolism and protein biosynthesis may be preferred to be Khib-modified in rhubarb. Similar observations were also found in the GO enrichment analysis for the molecular function, in which proteins possessing aminoacyl-tRNA ligase activity, ligase activity, translation factor activity and structural constituent of ribosome were highly enriched (Fig. 4, indicated in red bars). These enriched proteins are mainly related to protein biosynthesis. Regarding the GO enrichment analysis for the cellular component, the Khib-modified proteins were significantly enriched in ribosomes, ribonucleoprotein complexes and intracellular ribonucleoprotein complexes (Fig. 4, indicated in blue bars). Overall, the results of GO enrichment analysis strongly suggest that those proteins related to protein biosynthesis and central metabolism may be strictly regulated by Khib modification.

Fig. 4
figure 4

GO enrichment analysis of the Khib-modified proteins. The y-axis denotes the categories of GO terms. The x-axis denotes the fold enrichment

Consistent with these findings, the enrichment analysis of KEGG pathway and protein domains also showed that proteins related to protein synthesis and central metabolism tended to be Khib-modified (Fig. 5, Supplemental Table S5 and Table S6). As shown in Fig. 5 A and Supplemental Table S5, enrichment analysis of KEGG pathways demonstrated that Khib-modified proteins were enriched in the pathways of protein synthesis, including valine, leucine and isoleucine biosynthesis, aminoacyl-tRNA biosynthesis, and ribosome (Khib-modified proteins associated with the ribosome are shown in a schematic diagram, as shown in Supplemental Fig. S5). In addition to the enrichment of Khib proteins associated with protein biosynthesis, proteins related to central metabolic processes, including the citrate cycle (TCA cycle), glycolysis/gluconeogenesis, pentose phosphate pathway, photosynthesis, carbon fixation in photosynthetic organisms, glyoxylate and dicarboxylate metabolism, and pyruvate metabolism, were also found to be highly enriched (Fig. 5 A and Supplemental Table S5). Most of the enzymatic proteins involved in glycolysis/gluconeogenesis, the pentose phosphate pathway, the TCA cycle and oxidative phosphorylation were identified to be Khib-modified (Fig. 6 shows the Khib-modified enzymes in the TCA cycle, and Supplemental Fig. S6 shows the modified enzymes in glycolysis/gluconeogenesis, the pentose phosphate pathway and oxidative phosphorylation).

Fig. 5
figure 5

KEGG pathway and protein domain enrichment analysis of the Khib-modified proteins. The y-axis denotes the categories of KEGG pathways (A) and protein domains (B). The x-axis denotes the fold enrichment.

Fig. 6
figure 6

Khib-modified enzymes enriched in the TCA cycle in rhubarb. The identified Khib enzymes are indicated with a red background

The results of protein domain enrichment analysis showed that proteins possessing the domains of TCP-1-like chaperonin intermediate domain, aminoacyl-tRNA synthetase (class II), GroEL-like equatorial domain, lactate/malate dehydrogenase (both N-terminal and C-terminal), pyruvate kinase (beta-barrel insert domain), pyruvate kinase (barrel), etc., were found to be highly enriched (Fig. 5 B and Supplemental Table S6). It is not difficult to find that these enriched protein domains are involved in two main physiological functions: protein biosynthesis and central metabolism. In combination, the enrichment analysis of GO annotation, KEGG pathway and protein domain demonstrates that proteins with Khib modification in relation to protein biosynthesis and central metabolism are extremely important for the physiological activities of rhubarb.

Notably, the finding that the Khib modification proteins mainly involved in central metabolism and protein biosynthesis are significantly enriched has also been identified in rice [11, 12]. In addition to the Khib modification in plants, other types of PTMs on lysine in other organisms have also been found to tend to occur on proteins closely related to protein biosynthesis and central metabolism such as lysine acetylation and succinylation in Vibrio parahaemolyticus [3, 26] and Mycobacterium tuberculosis [27, 28] and lysine malonylation in Escherichia coli [29]. These findings indicate the fundamental role of lysine modification in different organisms, including eukaryotes and prokaryotes.

Analysis of Khib-modified peptides reveals conserved motifs

Previous studies on both eukaryotic and prokaryotic cells have found a preference for specific amino acid residues around the Khib site [10,11,12, 14]. Therefore, we utilized the Motif-X tool, software for extracting overrepresented patterns [30], to further analyse the conserved sequence motifs from all of the Khib-modified peptides in rhubarb. Figure 7 and Supplemental Table S7 show that a total of 13 conserved motifs, with amino acid residues from − 10 to + 10 surrounding the Khib site, were extracted from 3234 Khib-modified peptides. Survey of these motifs showed that only three amino acid residues are preferred flanking the Khib site: a negatively charged side chain residue of glutamic acid (E), a positively charged residue of lysine (K), and an uncharged residue with the smallest side chain of glycine (G). According to the amino acid residues and their position around the Khib site, these 13 motifs may be classified into three patterns: the + 1 or -1 position is a negatively charged side chain residue (EKhib or KhibE), the − 1 position is an uncharged residue with the smallest side chain (GKhib), the position of + 5 to + 9 or -5 to -9 (except − 8) is a positively charged residue (k.kkk….Khib….kkkkk) (“.” indicates a random amino acid residue, and “k” indicates that a residue at this position in the third pattern of motifs is K). Although a similar motif of EKhib has also been identified in rice [11], the other two motif patterns are unique and are not currently identified in other plants, such as P. patens and O. sativa (Supplemental Fig. S7). Thus, the preference for K and G flanking Khib-modified sites should be a unique feature of catalytic enzymes for Khib modification in rhubarb.

Fig. 7
figure 7

Properties of the identified Khib peptides. (A) The Khib sequence motifs and conservation of Khib sites. The size of the letter indicates the frequency of the amino acid residing at that position. (B) Frequency of each motif occurring in peptides with Khib modification. Motif score was also indicated in the histogram. (C) Heat map showing the frequency of the amino acid residues around the Khib sites. The enrichment [11] and dispersion (green) of amino acids from −10 to +10 flanking the Khib sites.

Furthermore, based on the frequency analysis of amino acid residues flanking the Khib-modified site, as shown in the heat map (Fig. 7 C), aspartic acid (D) at -3, E at + 1 or -1, G at + 2 or + 3, isoleucine (I) at + 3 or + 10, arginine (R) at -8, valine (V) at -4 or + 6, and alanine (A) at several positions were overpresented around the Khib site. However, cysteine (C), histidine (H), methionine (M) asparagine (N), arginine (Q) serine (S), proline (P) and tryptophan (W) were all underpresented flanking the Khib site. Overall, motif analysis provides a better method to understand potential catalytic enzymes in cells.


In this study, we presented a proteome-wide identification of Khib modification in rhubarb, an important model of traditional Chinese medical plants, by using a highly sensitive proteomic method. A total of 4333 overlapping Khib-modified peptides on 1525 proteins were identified in three independent tests. The Khib-modified proteins were evaluated to be distributed mainly in chloroplasts, cytoplasm, nucleus and mitochondria and involved in a wide range of cellular processes through extensive characterization of the Khib proteome. However, the Khib-modified proteins in relation to protein biosynthesis and central metabolism were highly enriched, suggesting that they play particularly important physiological roles in rhubarb. Moreover, the analysis of amino acid sequence motifs revealed that glutamic acid (E), glycine (G), and lysine (K) have a strong bias around the Khib site, and three types of motif patterns were extracted from a total of 13 Khib-modified peptides. Importantly, some motif patterns unique to rhubarb have been identified, and these motif patterns have not been identified in other plants to date. Although the regulatory effects and mechanisms of Khib modification remain to be clarified, our systematic analysis of Khib modification provides a better understanding of the potential physiological activities of Khib-modified proteins in rhubarb.

Availability of data and materials

The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier of PXD026307 ( The transcriptome data were deposited in n the National Center for Biotechnology Information (NCBI) database ( (BioProject ID: PRJNA714631).



Isocitrate dehydrogenase


Isocitrate dehydrogenase (NAD+)


Aconitate hydratase


Pyruvate dehydrogenase E2 component (dihydrolipoamide acetyltransferase)


Pyruvate dehydrogenase E1 component


Dihydrolipoamide dehydrogenase


Citrate synthase


ATP citrate (pro-S)-lyase


Malate dehydrogenase


Fumarate hydratase, class I


Succinate dehydrogenase (ubiquinone) flavoprotein subunit


Succinyl-CoA synthetase alpha subunit


2-oxoglutarate dehydrogenase E2 component (dihydrolipoamide succinyltransferase)


2-oxoglutarate dehydrogenase E1 component


  1. Walsh CT, Garneau-Tsodikova S, Gatto GJ, Jr.: Protein posttranslational modifications: the chemistry of proteome diversifications. Angewandte Chemie (International ed in English) 2005, 44(45):7342–7372.

    Article  CAS  Google Scholar 

  2. Jensen ON: Interpreting the protein language using proteomics. Nature reviews Molecular cell biology 2006, 7(6):391–403.

    Article  CAS  Google Scholar 

  3. Pan J, Chen R, Li C, Li W, Ye Z: Global Analysis of Protein Lysine Succinylation Profiles and Their Overlap with Lysine Acetylation in the Marine Bacterium Vibrio parahemolyticus. Journal of proteome research 2015, 14(10):4309–4318.

    Article  CAS  Google Scholar 

  4. Dai L, Peng C, Montellier E, Lu Z, Chen Y, Ishii H, Debernardi A, Buchou T, Rousseaux S, Jin F et al: Lysine 2-hydroxyisobutyrylation is a widely distributed active histone mark. Nature chemical biology 2014, 10(5):365–370.

    Article  CAS  Google Scholar 

  5. Rousseaux S, Khochbin S: Histone Acylation beyond Acetylation: Terra Incognita in Chromatin Biology. Cell journal 2015, 17(1):1–6.

    PubMed  PubMed Central  Google Scholar 

  6. Huang J, Luo Z, Ying W, Cao Q, Huang H, Dong J, Wu Q, Zhao Y, Qian X, Dai J: 2-Hydroxyisobutyrylation on histone H4K8 is regulated by glucose homeostasis in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America 2017, 114(33):8782–8787.

    Article  CAS  Google Scholar 

  7. Zhang L, Zou Y, He N, Chen Y, Chen Z, Li L: DeepKhib: A Deep-Learning Framework for Lysine 2-Hydroxyisobutyrylation Sites Prediction. Frontiers in Cell and Developmental Biology 2020, 8:580217.

    Article  Google Scholar 

  8. Huang H, Tang S, Ji M, Tang Z, Shimada M, Liu X, Qi S, Locasale JW, Roeder RG, Zhao Y et al: p300-Mediated Lysine 2-Hydroxyisobutyrylation Regulates Glycolysis. Molecular cell 2018, 70(4):663–678.e666.

    Article  CAS  Google Scholar 

  9. Ge H, Li B, Chen W, Xu Q, Chen S, Zhang H, Wu J, Zhen Q, Li Y, Yong L et al: Differential occurrence of lysine 2-hydroxyisobutyrylation in psoriasis skin lesions. Journal of proteomics 2019, 205:103420.

    Article  CAS  Google Scholar 

  10. Yu Z, Ni J, Sheng W, Wang Z, Wu Y: Proteome-wide identification of lysine 2-hydroxyisobutyrylation reveals conserved and novel histone modifications in Physcomitrella patens. Scientific reports 2017, 7(1):15553.

    Article  Google Scholar 

  11. Meng X, Xing S, Perez LM, Peng X, Zhao Q, Redoña ED, Wang C, Peng Z: Proteome-wide Analysis of Lysine 2-hydroxyisobutyrylation in Developing Rice (Oryza sativa) Seeds. Scientific reports 2017, 7(1):17486.

    Article  Google Scholar 

  12. Xue C, Qiao Z, Chen X, Cao P, Liu K, Liu S, Ye L, Gong Z: Proteome-Wide Analyses Reveal the Diverse Functions of Lysine 2-Hydroxyisobutyrylation in Oryza sativa. Rice 2020, 13(1):34.

    Article  Google Scholar 

  13. Dong H, Guo Z, Tian S, Zhai G, Zhang K: Analysis of lysine 2-hvdroxvisobutvrvlation proteins in Proteus mirabilis. Chinese Journal of Chromatography 2016, 34(12):1215–1218.

    Article  CAS  Google Scholar 

  14. Dong H, Guo Z, Feng W, Zhang T, Zhai G, Palusiak A, Rozalski A, Tian S, Bai X, Shen L et al: Systematic Identification of Lysine 2-hydroxyisobutyrylated Proteins in Proteus mirabilis. Molecular & cellular proteomics 2018, 17(3):482–494.

    Article  CAS  Google Scholar 

  15. Dong H, Zhai G, Chen C, Bai X, Tian S, Hu D, Fan E, Zhang K: Protein lysine de-2-hydroxyisobutyrylation by CobB in prokaryotes. Science advances 2019, 5(7):eaaw6703.

    Article  CAS  Google Scholar 

  16. Yin D, Jiang N, Zhang Y, Wang D, Sang X, Feng Y, Chen R, Wang X, Yang N, Chen Q: Global Lysine Crotonylation and 2-Hydroxyisobutyrylation in Phenotypically Different Toxoplasma gondii Parasites. Molecular & cellular proteomics 2019, 18(11):2207–2224.

    Article  CAS  Google Scholar 

  17. Huang H, Luo Z, Qi S, Huang J, Xu P, Wang X, Gao L, Li F, Wang J, Zhao W et al: Landscape of the regulatory elements for lysine 2-hydroxyisobutyrylation pathway. Cell research 2018, 28(1):111–125.

    Article  CAS  Google Scholar 

  18. Xiang H, Zuo J, Guo F, Dong D: What we already know about rhubarb: a comprehensive review. Chinese medicine 2020, 15:88.

    Article  Google Scholar 

  19. Zhuang T, Gu X, Zhou N, Ding L, Yang L, Zhou M: Hepatoprotection and hepatotoxicity of Chinese herb Rhubarb (Dahuang): How to properly control the “General (Jiang Jun)” in Chinese medical herb. Biomedicine & pharmacotherapy 2020, 127:110224.

    Article  CAS  Google Scholar 

  20. Chitteti BR, Tan F, Mujahid H, Magee BG, Bridges SM, Peng Z: Comparative analysis of proteome differential regulation during cell dedifferentiation in Arabidopsis. Proteomics 2008, 8(20):4303–4316.

    Article  CAS  Google Scholar 

  21. Kanehisa M, Goto S: KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 2000, 28(1):27–30.

    Article  CAS  Google Scholar 

  22. Tan M, Luo H, Lee S, Jin F, Yang JS, Montellier E, Buchou T, Cheng Z, Rousseaux S, Rajagopal N et al: Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification. Cell 2011, 146(6):1016–1028.

    Article  CAS  Google Scholar 

  23. Jin W, Wu F: Proteome-Wide Identification of Lysine Succinylation in the Proteins of Tomato (Solanum lycopersicum). PloS one 2016, 11(2):e0147586.

    Article  Google Scholar 

  24. Shen C, Xue J, Sun T, Guo H, Zhang L, Meng Y, Wang H: Succinyl-proteome profiling of a high taxol containing hybrid Taxus species (Taxus × media) revealed involvement of succinylation in multiple metabolic pathways. Scientific reports 2016, 6:21764.

    Article  CAS  Google Scholar 

  25. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT et al: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nature genetics 2000, 25(1):25–29.

    Article  CAS  Google Scholar 

  26. Pan J, Ye Z, Cheng Z, Peng X, Wen L, Zhao F. Systematic analysis of the lysine acetylome in Vibrio parahemolyticus. J Proteome Res. 2014;13(7):3294–302.

    Article  CAS  Google Scholar 

  27. Xie L, Liu W, Li Q, Chen S, Xu M, Huang Q, Zeng J, Zhou M, Xie J. First succinyl-proteome profiling of extensively drug-resistant Mycobacterium tuberculosis revealed involvement of succinylation in cellular physiology. J Proteome Research. 2015;14(1):107–19.

    Article  CAS  Google Scholar 

  28. Xie L, Wang X, Zeng J, Zhou M, Duan X, Li Q, Zhang Z, Luo H, Pang L, Li W, et al. Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis. Int J Biochemistry Cell Biol. 2015;59:193–202.

    Article  CAS  Google Scholar 

  29. Qian L, Nie L, Chen M, Liu P, Zhu J, Zhai L, Tao SC, Cheng Z, Zhao Y, Tan M. Global Profiling of Protein Lysine Malonylation in Escherichia coli Reveals Its Role in Energy Metabolism. J Proteome Res. 2016;15(6):2060–71.

    Article  CAS  Google Scholar 

  30. Chou MF, Schwartz D: Biological sequence motif discovery using motif-x. Current protocols in bioinformatics 2011, Chap. 13:Unit 13.15-24.

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We are grateful to Micrometer Biotech Company (Hangzhou, China) for helping to analyze the proteomics data.


This work was supported by Qingdao People’s Livelihood Science and Technology Project (19-9-1-254-nsh).

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Authors and Affiliations



Q.T. and L.J.P. carried out plant cultivation and experimental material collection, and wrote the manuscript. W.H. performed the protein preparation and peptide enrichment. W.H., H.X. and L.J.R. participated in the experimental design, analysed all data. D.J. supervised the overall study and assisted in manuscript preparation and revision. The author(s) read and approved the final manuscript.

Corresponding author

Correspondence to Jinzhe Du.

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All methods involving rhubarb samples were carried out in accordance with relevant guidelines of “List of National Key Protected Wild Plants of China”.

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The authors declare that they have no competing interests.

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Supplementary Information

Figure S1

, Mass accuracy of MS; Figure S2, The bubble chart shows the fold enrichment for GO analyses of Khib-modified proteins in terms of biological process, cellular component and molecular function; Figure S3, The bubble chart shows the fold enrichment of KEGG pathways of Khib-modified proteins; Figure S4, The bubble chart shows the fold enrichment of the protein domains of Khib-modified proteins; Figure S5, Pathway map of the Khib-modified enzymes in central carbon metabolism in rhubarb. Central carbon metabolism includes glycolysis/gluconeogenesis, the pentose phosphate pathway, the TCA cycle and oxidative phosphorylation. The identified Khib-modified enzymes are indicated in red boxes. The Khib-modified enzymes in the TCA cycle are shown in Fig. 6; Figure S6, Schematic diagram showing the association of Khib-modified proteins with the ribosome in rhubarb. The identified Khib-modified proteins are indicated in red boxes.

Table S1

, All identified Khib-modified peptides and proteins in rhubarb.

Table S2

, GO functional classification of Khib-modified proteins.

Table S3

, Classification of subcellular localizations of Khib-modified proteins.

Table S4

, GO enrichment analysis of Khib-modified proteins.

Table S5

, KEGG pathway enrichment analysis of Khib-modified proteins.

Table S6

, Protein domain enrichment analysis of Khib-modified proteins

Table S7

, Motif analysis of Khib modified peptides.

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Qi, T., Li, J., Wang, H. et al. Global analysis of protein lysine 2-hydroxyisobutyrylation (Khib) profiles in Chinese herb rhubarb (Dahuang). BMC Genomics 22, 542 (2021).

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