Abstract Background Protein lysine malonylation, a newly discovered post-translational modification (PTM), plays an important role in diverse metabolic processes in both eukaryotes and prokaryotes. Common wheat is a major global cereal crop. However, the functions of lysine malonylation are relatively unknown in this crop. Here, a global analysis of lysine malonylation was performed in wheat. Results In total, 342 lysine malonylated sites were identified in 233 proteins. Bioinformatics analysis showed that the frequency of arginine (R) in position + 1 was highest, and a modification motif, K^maR, was identified. The malonylated proteins were located in multiple subcellular compartments, especially in the cytosol (45%) and chloroplast (30%). The identified proteins were found to be involved in diverse pathways, such as carbon metabolism, the Calvin cycle, and the biosynthesis of amino acids, suggesting an important role for lysine malonylation in these processes. Protein interaction network analysis revealed eight highly interconnected clusters of malonylated proteins, and 137 malonylated proteins were mapped to the protein network database. Moreover, five proteins were simultaneously modified by lysine malonylation, acetylation and succinylation, suggesting that these three PTMs may coordinately regulate the function of many proteins in common wheat. Conclusions Our results suggest that lysine malonylation is involved in a variety of biological processes, especially carbon fixation in photosynthetic organisms. These data represent the first report of the lysine malonylome in common wheat and provide an important dataset for further exploring the physiological role of lysine malonylation in wheat and likely all plants. Electronic supplementary material The online version of this article (10.1186/s12864-018-4535-y) contains supplementary material, which is available to authorized users. Keywords: Malonylome, Lysine malonylation, Triticum aestivum L., Calvin cycle, Post-translational modification Background Post-translational modifications (PTMs) are dynamic and reversible modifications of proteins and have wide effects in broadening the range of functionality of these proteins [[39]1–[40]3]. Many functional groups, such as phospho, ubiquityl, acetyl, methyl, and malonyl groups, are able to be introduced by PTMs [[41]4]. Among PTMs, protein malonylation is a newly discovered modification, playing an important role in modulating various cellular processes [[42]4–[43]6]. Through reversible addition of a malonyl group to lysine residues, protein malonylation regulates protein localization, enzymatic activity, protein stability and many other biochemical processes. Although Malonyl-CoA is considered to be one of the most common donors of malonyl group, the enzymes controlling the malonylation status of proteins are largely unknown [[44]7, [45]8]. Sirt5, a member of the lysine deacetylases (KDACs), was found to be able to catalyze lysine demalonylation reaction in mammalian cells [[46]7, [47]8]. Therefore, it is speculated that both protein acetylation and malonylation are reversibly regulated by lysine acetyltransferases (KATs) and KDACs, which are located to diverse cell compartments [[48]7–[49]9]. Similar to lysine acetylation, many malonylated proteins localized in the nucleus, cytoplasm, mitochondria and chloroplast have been identified [[50]5, [51]8, [52]10–[53]13], indicating that a wide variety of biological processes are potentially regulated by lysine malonylation. In recent years, as a result of advancements in liquid chromatography-tandem mass spectrometry (LC-MS/MS), a large number of lysine-acetylated proteins have been identified [[54]2]. However, compared to these acetylation profiles, only a few organisms have been studied with respect to lysine malonylation, including Bacillus amyloliquefaciens [[55]4], Escherichia coli [[56]5], Saccharopolyspora erythraea [[57]10], cyanobacteria (Synechocystis) [[58]11], rice [[59]12], HeLa cells [[60]9], and mice [[61]7, [62]13]. Common wheat (Triticum aestivum L.) is one of the most important cereal crops in the world. Systematic analysis of the lysine acetylated [[63]2] and succinylated proteins [[64]3] in common wheat revealed that they were involved in a variety of signaling pathways in development and metabolism. As one PTM that occurs on a lysine residue and competes with succinylation and acetylation, protein malonylation was expected to play a very important role in multiple processes in common wheat. To test this hypothesis, we performed the first proteomics study of lysine malonylation in common wheat. In total, we identified 342 lysine malonylation sites in 233 proteins. The malonylated proteins were associated with diversified biological processes and were distributed in multiple compartments, including the cytosol, chloroplast, nucleus, mitochondria, plasma membrane, cytoskeleton, extracellular space and peroxisome. Importantly, we found that 30% of the malonylated proteins were located to the chloroplast, and further studies showed that these proteins play an important role in photosynthetic carbon fixation in common wheat. Comparative analyses of proteomic profiles among the malonylome, acetylome and succinylome suggest that these three PTMs can occur on the same lysine residues and may coordinately regulate the function of many proteins in common wheat. This systematic analysis provides a rich dataset for further exploring the physiological role of lysine malonylation in this cereal crop and likely all plants. Methods Protein extraction from common wheat Qing Mai 6, a common wheat variety (T. aestivum L.) tolerant to salt stress, was used for lysine malonylome analysis in this research. The seedlings of Qing Mai 6 were grown in a greenhouse for 3 weeks [[65]12] and the excised leaves were subjected to protein extraction as previously described [[66]14]. In brief, the leaves were ground in liquid nitrogen followed by sonicating for three times on ice in lysis buffer containing 8 M urea (Sigma-Aldrich, Saint Louis, USA), 1% TritonX-100 (Sangon Biotech, Shanghai, China), 10 mM dithiothreitol (DTT) (Sigma-Aldrich, Saint Louis, USA), and 1% Protease inhibitor cocktail (Merck Millipore, Billerica, USA) [[67]14]. After centrifugation at 20,000×g 4 °C for 20 min, proteins in the supernatant were precipitated with tricarboxylic acid (TCA) (Sigma-Aldrich, Saint Louis, USA) at − 20 °C for 2 h [[68]3]. The precipitates were washed three times by cold acetone (Hannuo, Lanxi, China) and were then redissolved in buffer (8 M urea, 100 mM NH[4]CO[3], pH 8.0) [[69]3]. Protein concentration was determined using the standard Bradford method. Affinity enrichment of lysine malonylated peptides For affinity enrichment, the wheat proteins were cleaved into peptides through tryptic digestion. The extracted proteins were firstly reduced with 10 mM DTT at 37 °C for 1 h and alkylated with 20 mM iodoacetamide (IAA) (Sigma-Aldrich, Saint Louis, USA) for 45 min, and were then digested by trypsin (Promega, Madison, USA) as described [[70]2]. Trypsin was added at 1:50 and 1:100 trypsin-to-protein mass ratio for the first and the second digestion, respectively [[71]15]. A high pH reverse-phase HPLC with Agilent 300Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length) (Agilent, Santa Clara, USA) was used to fractionate the resulting peptides into 80 fractions [[72]15]. Thereafter, the separated peptides were combined into 6 fractions [[73]15]. To enrich peptides with malonylation sites, the combined peptides dissolved in NETN buffer (100 mM NaCl (Sigma-Aldrich, Saint Louis, USA), 1 mM EDTA (Sigma-Aldrich, Saint Louis, USA), 50 mM Tris-HCl (Sigma-Aldrich, Saint Louis, USA), 0.5% NP-40 (Sigma-Aldrich, Saint Louis, USA), pH 8.0) were incubated with pan anti-malonyllysine antibody conjugated agarose beads (PTM Biolabs, Hangzhou, China) at 4 °C overnight with gentle shaking [[74]2]. The bound peptides were eluted from the beads with 0.1% trifluoroacetic acid (TFA) (Sigma-Aldrich, Saint Louis, USA) after 4 times washing with NETN buffer and were then cleaned using C18 ZipTips (Merck Millipore, Billerica, USA) [[75]13]. LC-MS/MS analysis LC-MS/MS analysis of the malonylation peptides was carried out as described [[76]15–[77]18]. Briefly, the peptides cleaned with C18 ZipTips were separated with a reversed-phase analytical column (ThermoFisher Scientific, Waltham, USA). The gradient was comprised of an increase from 6 to 22% solvent B (0.1% formic acid (Sigma-Aldrich, Saint Louis, USA) in 98% acetonitrile (Sigma-Aldrich, Saint Louis, USA)) for 24 min, 22 to 40% for 8 min and climbing to 80% in 5 min then holding at 80% for the last 3 min, all at a constant flow rate of 300 nl/min on an EASY-nLC 1000 UPLC system. The resulting peptides were analyzed by Q Exactive™ Plus hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific, Waltham, USA) [[78]16]. Intact peptides and ion fragments were detected in the Orbitrap at a resolution of 70,000 and 17,500, respectively, with the NCE setting at 30 [[79]16]. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 5E3 in the MS survey scan with 15.0 s dynamic exclusion. [[80]16]. An electrospray voltage of 2.0 kV was employed to the LC-MS/MS analysis [[81]16]. To generate MS/MS spectra, 5E4 ions were accumulated and automatic gain control (AGC) was used to prevent overfilling of the orbitrap [[82]16]. For MS scans, the m/z scan range was 350 to 1800 [[83]16]. Fixed first mass was set as 100 m/z [[84]15–[85]18]. Database search MaxQuant with integrated Andromeda search engine (v.1.5.1.8) was used to process the obtained MS/MS data [[86]19, [87]20]. The collected tandem mass spectra were searched against 4565_PR_wheat database concatenated with reverse decoy database [[88]3]. Trypsin/P was specified as cleavage enzyme allowing up to 4 missing cleavages, 5 modifications per peptide and 5 charges; minimum peptide length was set at 7 [[89]3]. Mass error was set to 10 ppm and 0.02 Da for precursor ions and fragment ions, respectively [[90]3]. Carbamidomethylation on Cys was set as fixed modification; malonylation on Lys was set as variable modification [[91]2]. False discovery rate (FDR) threshold of 1% was specified for protein, peptide and modification site [[92]2]. All the other parameters in MaxQuant were set as default values [[93]2]. The probability for site localization was set as > 0.75 [[94]2, [95]15–[96]18]. Bioinformatics analysis The malonylated proteins were analyzed by Gene Ontology (GO) annotation derived from the UniProt-GOA database ([97]http://www.ebi.ac.uk/GOA/) based on biological process, cellular component and molecular function [[98]13]. InterPro ([99]http://www.ebi.ac.uk/interpro/) was used to annotate functional domains of all identified proteins [[100]14]. Protein pathways were annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) database [[101]21]. A two-tailed Fisher’s exact test was employed to test the enrichment of the malonylated proteins and a corrected p-value < 0.05 was considered significant [[102]15]. Subcellular localization of the modified proteins was predicated with WoLF PSORT (version PSORT/PSORT II) [[103]22]. The current dataset used to train WoLF PSORT contains over 12,000 animal sequences and more than 2000 plant and fungi sequences respectively. For each protein, WoLF PSORT reported a number to roughly indicate the number of nearest neighbors to the protein which localize to each site. Then, localization sites with the maximum number were selected as the subcellular localization of protein. The model of sequences constituted with amino acids in specific positions of modify-21-mers was analyzed using Motif-x software and the secondary structures of proteins were predicted using NetSurfP software [[104]23]. Only predictions with a minimum probability of 0.5 for one of the different secondary structures (coil, α-helix, β-strand) were considered for analysis. The mean secondary structure probabilities of the malonylated lysine residues were compared with the mean secondary structure probabilities of a control dataset containing all the lysine residues of all the malonylated proteins identified in this study. Cytoscape software was employed to analyze the protein-protein interactions for the malonylated proteins and the protein-protein interaction network was obtained from the STRING database [[105]24, [106]25]. BLASTP was carried out to determine the conservation of lysine malonylated proteins between common wheat and other organisms. Immunoprecipitation of dehydroascorbate reductase The leaves of common wheat were ground in liquid nitrogen and the soluble proteins were extracted as previously described [[107]3]. After overnight incubation of 1 mg of soluble proteins with or without 1 μg of dehydroascorbate reductase specific antibody (Agrisera, Vännäs, Sweden), 50 μl of protein A agarose beads (GE Healthcare, Uppsala, Sweden) were added and the solution was incubated for another 1 h. The bound proteins were eluted from the beads with boiled SDS-PAGE sample buffer following 5 times wash with lysis buffer [[108]3]. Western blot analysis Eluted proteins were separated by SDS-PAGE on a 12% gel and probed with dehydroascorbate reductase antibody (1:5000 dilution) and anti-malonyllysine antibody (1:2000 dilution, PTM Biolabs, Hangzhou, China), respectively [[109]3]. Proteins were visualized using Pierce Fast Western Blot Kit ECL Substrate (ThermoFisher Scientific, Waltham, USA) according to the manufacturer’s instructions [[110]3]. Results and discussion Identification of lysine malonylated proteins in common wheat To elucidate the regulatory functions of lysine malonylation in wheat, a proteome-scale analysis of malonylated proteins was performed (Additional file [111]1: Figure S1a). To validate the MS data, the mass error of all the identified peptides was checked, and the total distribution of the mass error was less than 5 ppm, indicating that the mass accuracy of the MS data fit the requirement (Additional file [112]1: Figure S1b). We further checked the distribution of all the peptides, and the results showed that the length of most of the peptides was between 8 and 20 (Additional file [113]1: Figure S1c), which demonstrated that the sample preparation met the standard [[114]15, [115]17]. After a large-scale lysine malonylome analysis using LC-MS/MS, a total of 342 lysine malonylation sites in 233 protein groups were identified (Additional file [116]2: Table S1). Three representative MS/MS spectra of the malonylated peptides were showed in Additional file [117]1: Figure S2. The number of malonylated proteins in common wheat was higher than that in S. erythraea [[118]4], but less than the number of identified proteins in B. amyloliquefaciens [[119]4], E. coli [[120]5] and mammals [[121]7, [122]13]. Dehydroascorbate reductase (DHAR), a protein involved in redox homeostasis under biotic and abiotic stresses and a key component of the ascorbate recycling system, was found to be malonylated on the lysine residue, K157 (Additional file [123]2: Table S1). Immunoprecipitation coupled with Western blot analysis confirmed malonylation of DHAR in wheat leaves (Additional file [124]1: Figure S3). Proteins can be modified on either one or several amino acid residues. Thus, the number of malonylated sites in the identified proteins was calculated in common wheat. As shown in Additional file [125]1: Figure S4, 75% (175) of malonylated proteins had only one malonylated lysine site, whereas 25% (58) were modified on multiple lysine residues. A similar distribution pattern was also found in S. erythraea [[126]10]. To our knowledge, these findings represent the first report of lysine malonylome in common wheat. Pattern analysis of malonylated sites Previous studies have demonstrated the modified site preferences for