Global Analysis of Lysine 2-Hydroxyisobutyrylome upon SAHA

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Global analysis of lysine 2-hydroxyisobutyrylome upon SAHA treatment and its relationship with acetylation and crotonylation Quan Wu, Li Ke, Chi Wang, Pingsheng Fan, Zhiwei Wu, and Xiaoling Xu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00289 • Publication Date (Web): 15 Aug 2018 Downloaded from http://pubs.acs.org on August 16, 2018

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Journal of Proteome Research

Global analysis of lysine 2-hydroxyisobutyrylome upon SAHA treatment and its relationship with acetylation and crotonylation *

Quan Wu1, 2, , Li Ke3, Chi Wang1, Pingsheng Fan4, Zhiwei Wu1, Xiaoling Xu5, 1

*

Central Laboratory of Medical Research Centre, The First Affiliated Hospital of USTC,

Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230001, China. 2

Department of Microbiology and Immunobiology, Harvard Medical School, Boston,

Massachusetts, 02115, USA. 3

Department of Thoracic Surgery, The First Affiliated Hospital of USTC, Division of Life

Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230001, China. 4

Department of Oncology, The First Affiliated Hospital of USTC, Division of Life Sciences and

Medicine, University of Science and Technology of China, Hefei, Anhui, 230001, China. 5

Department of Respiration, The First Affiliated Hospital of USTC, Division of Life Sciences

and Medicine, University of Science and Technology of China, Hefei, Anhui, 230001, China. *

Correspondence:

E-mail: [email protected], Phone: +86 18963789002 (Xiaoling Xu) and E-mail: [email protected], Phone: +86 13866729902 (Quan Wu)

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ABSTRACT Lysine 2-hydroxyisobutyrylation is a newly discovered protein acylation and was reported to share acyltransferases and deacylases with the widely studied lysine acetylation. The well-known acetyltransferase Tip60 and histone deacetylases HDAC 2 and HDAC 3 were discovered to be “writer” and “eraser” of this new PTM on histones. However, the acyltransferases and deacylases for non-histone proteins are still unclear. In this work, lysine 2-hydroxyisobutyrylome on both histones and non-histone proteins upon SAHA treatment were intensively studied and 8,765 lysine 2-hydroxyisobutyrylation sites on 2,484 proteins were identified in A549 cells. This is the largest dataset of lysine 2-hydroxyisobutyrylome in mammalian cells by now. It was found that lysine 2-hydroxyisobutyrylation participate in varieties of biological functions and processes including ribosome, glycolysis/gluconeogenesis and transcription. More importantly, it was found that most quantified sites on core histones were up-regulated upon SAHA treatment for all of 2-hydroxyisobutyrylation, crotonylation

and

acetylation

and

the

fold

changes

upon

SAHA

of

2-hydroxyisobutyrylation and crotonylation on non-histone proteins were highly correlated, while their fold changes have little correlations with acetylation on non-histone proteins. KEYWORDS: 2-hydroxyisobutyrylation, crotonylation, acetylation, SAHA

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INTRODUCTION Lysine acylation is one of the new post-translational modification (PTM) types, which influence protein properties including cellular localization, stability, interaction, enzymatic activity and so on.1-3 Over the past decade, eight new PTMs of this type have

been

succinylation,

identified,

such

as

crotonylation,

propionylation, glutarylation,

malonylation,

butyrylation,

β-hydroxybutyrylation

and

2-hydroxyisobutyrylation.4-10 And further studies of the new acylations were successively published in recent years by identification of thousands of modification sites on histones and non-histone proteins, showing the various functions and biological roles of these modifications. Emerging evidence suggests that these new modifications are structurally and functionally different from the widely studied lysine acetylation and play important roles in metabolic regulation of gene expression.11 Lysine 2-hydroxyisobutyrylation (Khib) was initially identified on histones in HeLa cells and mouse embryonic fibroblast cells by Dai and co-workers.5 It was first reported that histone Khib show distinct genomic distributions from histone Kac or histone Kcr during male germ cell differentiation. They identified 60 histone Khib sites in mouse testis cells and 22 histone Khib sites in HeLa cells and reported that histone Khib H4K8 is associated with active gene transcription in meiotic and post-meiotic cells. In a recent study, Huang et al showed that the amount of H4K8hib fluctuates in response to the availability of carbon source in Saccharomyces cerevisiae and the low-glucose

conditions

lead

to

diminished

Khib.12

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The

removal

of

the


2-hydroxyisobutyryl group from H4K8 is mediated by the histone lysine deacetylase Rpd3p and Hos3p in vivo. Furthermore, proteomic analysis revealed that a large set of proteins involved in glycolysis/gluconeogenesis are modified by Khib. They identified 1,458 Khib sites on 369 proteins in Saccharomyces cerevisiae in that study. In a latest research paper, Huang et al reported a global profiling of Khib proteome in HEK293T cell, by identifying 6,548 Khib sites on 1,725 substrate proteins13 and more importantly, both “writers” and “erasers” for histone Khib was discovered. It was showed that Esa1p in budding yeast and its homologue Tip60 in human could add Khib to substrate proteins both in vitro and in vivo. In addition, they identified HDAC2 and HDAC3 as the major enzymes to remove Khib. To investigate its unrevealed biological functions, Khib has drawn increasing attentions recently and this new modification was also investigated in other species. Zhang et al. systematically identified Khib in bacteria of Proteus mirabilis.14 They identified 4735 Khib sites in 1,051 proteins and the 2-hydroxyisobytyrylated proteins were found to be highly involved in different metabolic pathways. Moreover, they demonstrated that Khib on K343 of Enolase was a negative regulatory to its activity. In other studies, Peng and Wu’s research groups studied Khib in plant of Rice and Physcomitrella patens15, 16. They all identified thousands of Khib sites and showed that Khib was widely distributed across different species and involved in a wide range of molecular functions and cellular processes including glycolysis/gluconeogenesis, TCA cycle and so on. These studies also extensively expanded our understanding of Khib about their biological functions and are valuable for further functional

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investigations. Although lysine 2-hydroxyisobutyrylation was studied by a number of research groups and some of the catalyzing enzymes were identified, the regulation and biological roles of this new PTM still need much more investigations. Suberoylanilide hydroxamic acid (SAHA) is a common HDACi known to suppress HDAC class I, IIa and IIb. In previous publications, the impacts of SAHA on lysine acetylome and crotonylome were investigated,17, 18 showing that SAHA treatment enhanced lysine acetylation and crotonylation in both histone and non-histone scale. Furthermore, it was

found

in

recent

studies

that

acetylation,

2-hydroxyisobutyrylation share some deacylases12,

13, 19, 20

crotonylation

and

while the revealed

acyltransferases and some of the deacylases are different for crotonylation and 2-hydroxyisobutyrylation. In this work, we comprehensively studied lysine 2-hydroxyisobutyrylation to investigate the landscape of 2-hydroxyisobutyrylome and SAHA treatment was used to study its cross-talk with other lysine acylations. We performed lysine 2-hydroxyisobutyrylation analysis on proteome level by an integrated system combining pan-antibody enrichment, SILAC technique, high resolution mass spectrometry and bioinformatics. As a result, we dramatically identified 8,765 unique Khib sites across 2,484 proteins in A549 cells. To our knowledge, this is the first quantitative analysis and the largest dataset in cells of lysine 2-hydroxyisobutyrylome by now. Further bioinformatics of the dataset and SILAC-based quantification upon SAHA treatment reveal diverse biological functions of this new post-translational modification and its relationship with acetylation and

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crotonylation.

EXPERIMENTAL SECTION SILAC labeling and SAHA treatment Stable isotope labeling with amino acids in cell culture (SILAC) technique was used for Khib quantification. Briefly, A549 cells were maintained in SILAC DMEM medium (Invitrogen, Carlsbad, CA) with 10% FBS at 37°C in humidified atmosphere with 5% CO2. The SILAC experiment was done as previously reported.17, 18 The cells were isotope labeled for 6-7 generations until the labeling efficiency over 97%. After labeling, the “light” labeled cells (marked as L) were treated with SAHA for 18 hours at a final concentration of 3 µM, while the “heavy” labeled cells (marked as H) were treated with the same volume of DMSO as negative control. After that, the cells were harvested and washed twice with ice-cold PBS. Extraction of proteins was performed according to previous methods17, 18. Protein digestion and high pH RP-HPLC separation Firstly, dithothreitol (DTT) was added to 10 mM (final concentration) and incubated at 37 °C for 60 min. After that, iodoacetamine (IAA) was added to 15 mM and incubated at room temperature in dark for 40 min. The alkylation reaction was quenched by 30 mM cysteine at room temperature for another 30 min. Finally, trypsin was added with a enzyme to protein ratio of 1:25 (w/w) for digestion at 37 °C overnight. The tryptic peptides was then separated by high-pH reverse-phase (RP) HPLC using Agilent 300Extend C18 column (5 µm, 4.6 mm ID, 250 mm length) into 6 / 23


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sub-fractions. Briefly, peptides were separated with a 80 min-gradient of 2% to 60% acetonitrile in 10 mM ammonium formate at pH 9 in into 54 fractions and then the 54 fractions were combined into 18 fractions for global proteome analysis and 6 fractions for lysine 2-hydroxyisobutyrylation analysis. Affinity enrichment of lysine 2-hydroxyisobutyrylated peptides Before enrichment, anti-Khib antibody beads (PTM Biolabs, Hangzhou, China) were washed twice with ice-cold PBS. After that, 5 mg of tryptic peptides were dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) and then incubated with pre-washed antibody beads in a ratio of 15 µL beads/mg protein at 4 °C overnight. The beads were washed four times with NETN buffer and twice with ddH2O after the incubation. Finally, the bound peptides were eluted from the beads with 0.1% TFA for LC-MS/MS analysis. LC-MS/MS analysis Peptides was firstly redissolved in solvent A which contains 0.1% FA, 2% ACN and 98% H2O. After that, peptide solution was loaded onto a reversed-phase analytical column (homemade, 100 µm ID × 15 mm long, 2 µm particle, 100 Å). Then the peptides were separated with a gradient of 6% to 22% solvent B (0.1% FA in 98% ACN) for 24 min, 22% to 36% for 10 min and climbing to 80% in 3 min then holding at 80% for the last 3 min. The flow rate was set at 400 nL/min on an EASY-nLC 1000 UPLC system. The resolution of full MS was set at 70,000, while the resolution of MS/MS were set as 17,500. Peptides were selected for MS/MS using NCE setting as 28. A data-dependent procedure that alternated between one

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MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions. The threshold ion count was set at 1.5E4 and the dynamic exclusion was set at 15 s. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was was set at 5E4 ions for generation of MS/MS spectra. The m/z scan range for MS scans was 350 to 1600. Database search The data mining was performed by using MaxQuant software (version 1.5.2.8). Briefly, the MS/MS data were searched against SwissProt human database (release-2017-08). Trypsin/P was set as the enzyme and up to 4 missing cleavages were allowed. Mass error was set as 5 ppm for precursor and 0.02 Da for fragments. Carbamidomethylation was set as fixed modification on Cys and oxidation on Met, 2-hydroxyisobutyrylation on Lys and acetylation on protein N-terminal were set as variable modifications. False discovery rate (FDR) thresholds for protein, peptide and modification site were all set at 0.01. The site identifications with localization probability less than 0.75 were removed. Bioinformatics Gene Ontology (GO) based analysis were performed by using DAVID software (the Database

for

Annotation,

Visualization

and

Integrated

Discovery).

Kyoto

Encyclopedia of Genes and Genomes (KEGG) database was used to identify pathways. InterPro database was done by using DAVID against the background. The corrected p-value < 0.05 was considered as significance for all of the bioinformatics analysis.

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Western blotting Proteins were firstly separated in a 12% SDS-PAGE gel, and then transferred to a PVDF membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was incubated in blocking buffer for 2 h. After that, the membrane was subsequently incubated with pan anti-2-hydroxyisobutyryllysine antibodies (PTM Biolabs, Hangzhou, China) 4 h at 4 ° C, followed with the incubation of horseradish peroxidase-conjugated goat-anti-rabbit secondary antibody for an additional 2 h at room temperature. Parallel reaction monitoring (PRM) The PRM experiment was followed by our previous method.17 Briefly, 1 mg of proteins of light and heavy labeled were mixed together for digestion and then Khib enrichment. After enrichment, the Khib peptides were analyzed by an EASY-nLC 1000 UPLC system coupled to tandem mass spectrometry (MS/MS) in Q Exactive

Plus

(Thermo). A list of selected Khib peptides were import to the MS method and a data-independent procedure were performed that alternated between one MS scan followed by 20 MS/MS scans of the selected peptides. The data processing was performed by using Skyline (v.3.6) software. The parameters was as follows: enzyme was set as Trypsin, Max missed cleavage less than 2, the peptide length 8-25, variable modification set as Carbamidomethyl on Cys and oxidation on Met, and the max variable modifications set as 3. The precursor charges were set as 2, 3, ion charges were set as 1, 2, ion types were set as b, y, p. The product ions were from ion 3 to last ion, the ion match tolerance was 0.02 Da.

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RESULTS Global analysis of lysine 2-hydroxyisobutyrylome We combined SILAC technique, High-pH HPLC separation, pan-antibody enrichment, high resolution LC-MS/MS and bioinformatics for extensive study of lysine 2-hydroxyisobutyrylome in A549 cells. The entire procedures includes: (1) SILAC labeling of A549 cells; (2) whole protein extraction and tryptic digestion; (3) High-pH HPLC separation of peptides; (4) enrichment of lysine 2-hydroxyisobutyrylated peptides using pan antibody; (5) LC-MS/MS analysis; (6) database searching; (7) bioinformatics for data mining (Fig. 1A). Altogether, in three biological replicates, we identified 8,765 Khib sites on 2,484 proteins from A549 cells upon SAHA treatment, among which 7,315 sites from 2,039 proteins were quantifiable. All the data was listed in Supplementary Information Table S1. This is the first quantitative analysis and also the largest dataset of lysine 2-hydroxyisobutyrylation in mammalian cell lines up to now. To obtain high confident data, all the Khib sites were filtered with strict criteria including less than 1% FDR and over 0.75 localization probability score. The deep profiling of 2-hydroxyisobutyrylome in this work mainly lies on the efficient affinity enrichment at proteome-wide based on an anti-2-hydroxyisobutyryllysin pan-antibody (PTM Biolabs, Hangzhou, China). Of the 8,765 sites, 81% (7,094) were identified in at least two biological replicates (Fig. 1B), showing the high reproducibility of our procedures. Furthermore, the quantitative reproducibility was also evaluated as show in Figure 1C. The linear

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correlation between every two replications with R2 over 0.75 shows good reproducibility of the quantification results. As there were already of some published data for lysine 2-hydroxyisobutyrylome in human, we compared our data with previously published data in which 6549 Khib peptides13, the result was shown in Table S5. It was found that 3036 out of 6548 Khib peptides (46.4%) were also identified in our study, although the two studies were with different conditions and materials, which showed the depth of this study. Functional classification of lysine 2-hydroxyisobutyrylome The identified Khib proteins have varied numbers of modification sites. We found that only 1,040 proteins (41.8%) were modified at only one Khib site, while 58.1% proteins have two or more sits. Especially, we found that there are 142 proteins that have more than 10 Khib sites, including Plectin (PLEC) with 101 sites and Myosin-9 (MYH9) with 71 sites (Fig. 2A). It was found that quite lots of enzymes were highly modified, such as DNA-dependent protein kinase catalytic subunit (PRKDC) with 32 sites, Protein disulfide-isomerase (P4HB) with 27 sites, Retinal dehydrogenase 1 (ALDH1A1) with 25 sites and so on (Fig. 2B and Table S2). These enzymes participate in a number of important metabolic pathways. For example, 10 key enzymes required for glycolysis were all modified with Khib, and 7 of which were heavily modified with more than 10 Khib sites (Table S2). Moreover, 5 out of the 8 enzymes participate in citric acid cycle were identified to be 2-hydroxyisobutyrylated, including Citrate synthase, Isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase and so on (Table S2). These results suggest that lysine 2-hydroxyisobutyrylation may

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paly important biological roles in metabolism of cells. Then we did the subcellular localization analysis by using the data of identified lysine 2-hydroxyisobutyrylated proteins (Fig. 2C). It was showed that most of the Khib were happened in the proteins in cytosol (34.9%), nuclear (28.3%) and mitochondria (13.8%), and there are another 3.7% proteins localized both in cytosol and nuclear. The same analysis of the global proteome data was also performed for comparison. According to the data, the protein localization of Khib proteins and global proteome has no remarkable difference, which suggest that lysine 2-hydroxyisobutyrylation may have a wide localization distribution in A549 cells. SAHA treatment increased lysine 2-hydroxyisobutyrylation level in A549 cells It

was

reported

by

previous

studies

that

lysine

crotonylation

and

2-hydroxyisobutyrylation were both catalyzed by well-known acetyltransferases and deacetylases such as HDAC 2 and HDAC 3.12, 13, 19, 20 Moreover, lysine crotonylation was also proved to be removed by the sirtuin family such as Sirt 1, Sirt 2 and Sirt 3, which has not been reported in 2-hydroxyisobutyrylation studies yet.21 As SAHA is a common HDACi which inhibit class I, IIa and IIb HDAC, our previous results showed that lysine acetylation and crotonylation in both core histones and non-histone proteins were markedly increased by SAHA treatment. Therefore, we compared the Khib changes before and after SAHA treatment in this study, to verify previous reports and to reveal new information for “writers” and “erasers” of Khib. In this work, 7,315 sites from 2,039 proteins were quantifiable. The Khib sites with fold changes over 1.5 and P value less than 0.05 were considered as significant

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difference. Using this criteria, 549 sites on 292 proteins were considered as up-regulated while 55 sites on 36 proteins were down-regulated. A volcano plot was used to show the quantification pattern (Fig. 3A). There are much more proteins up-regulated than down-regulated, showing that lysine 2-hydroxyisobutyrylation was heavily elevated after SAHA treatment. Furthermore, the Khib changes on histones upon SAHA were also investigated. Altogether, 24 Khib sites on core histones were found in this work, 6 of which were novel (Fig. 3B). According to the quantification results, it is found that most of the Khib sites, especially those on histone H2A and H2B were up-regulated after SAHA treatment, while there were only two sites (H3K23 and H4K12) down-regulated. All the data was presented in Table S1 and Fig. 3B. According to above data, Khib level in histones and non-histone proteins were shown to be highly elevated after SAHA treatment. These results are consistent with previous reports that HDAC 2 and HDAC 3, which could be selectively suppressed by SAHA, catalyzed the remove of 2-hydroxyisobutyrylation. Functional

enrichment

of

the

significantly

changed

lysine

2-hydroxyisobutyrylome upon SAHA The subcellular localization and functional enrichment of the differentially expressed Khib proteins were performed. As shown in Fig. 4A, the subcellular localization result was presented. It was found that majority of differentially expressed proteins were identified in cytoplasm and nuclear. The GO-based functional enrichment was shown in Fig. 4B. The up-regulated proteins were enriched in cytosolic part, ribosome and

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some biological processes including translational elongation, transcription and gene expression, while the down-regulated Khib proteins were mainly participate in the cellular components of vacuolar lumen, lysosome, nucleosome and the functions of hydrolase activity, iron binding. KEGG-based functional enrichment was also carried out (Fig. 4C). It was found that the up-regulated Khib proteins were enriched in processes of ribosome and glycolysis/gluconeogenesis and protein export, while the down-regulated Khib proteins were enriched in antigen processing and presentation, lysosome and protein processing in E.R. For detailed analysis of lysine 2-hydroxyisobutyrylome functions, clustering analysis were performed based on GO and KEGG enrichment data. The detailed results were presented in Figure S1. Western blotting and PRM-based validation To confirm the proteomic results, western blotting and PRM validation were performed. Pan anti-2-hydroxyisobutyryllysine antibody was used for western blotting to validate the global Khib level before and after SAHA treatment. Due to no site-specific 2-hydroxyisobutyrylation antibodies were available, we use PRM for the validation of some selected Khib sites. The western blotting result was shown in Figure 6. According to the result, it is obviously that SAHA treatment significantly increased Khib level, which is consisted with the MS data. The PRM result was presented in Table 1 and Supplementary Table S4. I Due to that PRM experiment has no HPLC fractionation step and the number of peptide for monitoring is limited, much fewer Khib sites were quantified in PRM experiment than in SILAC experiments. But the ratios of most quantified sites in PRM were consisted with

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SILAC experiments as shown in Table 1. These results proved that the proteomic results were of high confidence.

DISCUSSION In the recent study, Huang and co-workers revealed that class I HDACs such as HDAC 2 and HDAC 3, are the major enzymes to remove Khib, which suggest its close relationship with Kac.12, 13 Moreover, previous studies also proved that HDAC 2 and 3 are decrotonylases,19, 20 which suggest that Khib and Kcr are very similar modifications and are both closely related with Kac. However, the decrotonylases HDAC1 and HDAC 8 discovered by Wong et al. have no activity for removing Khib according to the data reported by Huang et al. and the known “writer” of the two modifications are also different.13, 19 These fact suggest that the two modifications of Khib and Kcr are different to some extent. As SAHA is a well-known inhibitor for class I, IIa and IIb HDAC, the data we obtained show useful information about Khib and its relationship with Kac and Kcr. Therefore, we compared the data obtained this time with the data in our previous studies on Kac and Kcr.17, 18 Firstly, we compared the modification sites on histones. In total, 16 sites on core histones were undergo all three modifications of acetylation, crotonylation and 2-hydroxyisobutyrylation as shown in Fig. 3B. Among these sites, 10 of which were up-regulated for all three modifications upon SAHA treatment, especially on histone H2A and H2B. This result is consistent with previous reports of the shared deacylases of HDAC 2 and HDAC 3 for these modifications. However, there were still a few Khib sites not changed or even down-regulated upon SAHA treatment. The two Khib sites 15 / 23



down-regulated were H3K23 and H4K12. Strangely, the acetylation level on the same sites after SAHA treatment were also decreased. The mechanism lay behind worth further investigations. The modification level of non-histones after SAHA treatment was also compared. In our previous studies, 1,099 Kac sites and 10,163 Kcr sites were identified, respectively. By comparing the data in this work and previous two studies, the proteins undergo all three modifications on the same site were presented in Table S3. There are 460 sites on 243 proteins modified with all three modifications (Fig. 5A). The relationships of the quantitative fold change upon SAHA treatment of the three modifications on the same site were also analyzed. It was showed that Kac has no linear relationship with Kcr and Khib on non-histone scale (Fig. 5B and 5C), showing that Kcr and Khib are distinct modifications compared with the widely studied Kac. For the comparison of Kcr and Khib, there are 4,969 sites modified with both two modifications and the relationships of the fold changes were also compared (Table S3 and Fig. 5D). It is obviously that the two modifications on non-histone scale were highly related with R2 over 0.5 (Fig. 5D).

CONCLUSIONS In this study, we extensively profiled lysine 2-hydroxyisobutyrylation on both histones and non-histone proteins in A549 cell line. By using an integrated system, we quantitatively compared the Khib sites before and after SAHA treatment. We dramatically identified 8,765 Khib sites on 2,484 proteins, which is the largest dataset of lysine 2-hydroxyisobutyrylation in cell lines by now. Bioinformatics showed that 16 / 23


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lysine 2-hydroxyisobutyrylation participate in a broad range of important biological processes. Furthermore, it was revealed that the 2-hydroxyisobutyrylation, crotonylation and acetylation level of most core histone sites were significantly elevated after SAHA treatment. And the fold changes upon SAHA of 2-hydroxyisobutyrylation and crotonylation were also highly correlated. However, the fold changes of 2-hydroxyisobutyrylation and crotonylation have little correlation with acetylation on non-histone proteins. This study largely broaden our knowledge of Khib and showed its close relationships with Kcr and Kac on proteome level.

SUPPORTING INFORMATION: The following supporting information is available free of charge at ACS website http://pubs.acs.org

Figure S1. Clustering analysis for the quantified 2-hydroxyisobutyrylome. A, molecular function analysis. B, cellular component analysis. C, biological process analysis. D, KEGG pathway analysis. Figure S2. MS/MS spectra of some selected Khib peptides Table S1. The lysine 2-hydroxyisobutyrylome data in this work. Table S2. The Khib sites on enzymes identified in this work. Table S3. The overlapped sites and proteins between Khib and Kcr after SAHA treatment. Table S4. PRM results of the selected Khib sites. Table S5. The comparison of Khib data with previously published data.

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ACKNOWLEDGEMENTS This work is supported by National Natural Science Foundation of China (Grant No. 81572255),

Anhui

provincial

Natural

Science

Foundation

(Grant

No.

1608085MH216). The authors thank Jingjie PTM Biolabs for the help of mass spectrometry analysis and bioinformatics.

ABBREVIATIONS hib:

2-hydroxyisobutyrylation;

cr:

crotonylation;

ac:

acetylation;

SAHA:

suberoylanilide hydroxamic acid; HDAC: histone deacetylase; SILAC: stable isotope labeling with amino acids in cell culture; PTM: post-translational modification; RP: reversed phase; DTT: dithothreitol; IAA: iodoacetamine; ACN: acetonitrile; KEGG: Kyoto Encyclopedia of Genes and Genomes; GO: Gene Ontology.

NOTE The Authors declare no competing financial interest.

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Peng, C.; Lu, Z.; Xie, Z.; Cheng, Z.; Chen, Y.; Tan, M.; Luo, H.; Zhang, Y.; He, W.; Yang, K.;

Zwaans, B. M.; Tishkoff, D.; Ho, L.; Lombard, D.; He, T. C.; Dai, J.; Verdin, E.; Ye, Y.; Zhao, Y., The first identification of lysine malonylation substrates and its regulatory enzyme. Mol. Cell. Proteomics 2011, 10, (12), M111 012658. 7.

Tan, M. J.; Luo, H.; Lee, S.; Jin, F. L.; Yang, J. S.; Montellier, E.; Buchou, T.; Cheng, Z. Y.;

Rousseaux, S.; Rajagopal, N.; Lu, Z. K.; Ye, Z.; Zhu, Q.; Wysocka, J.; Ye, Y.; Khochbin, S.; Ren, B.; Zhao, Y. M., Identification of 67 Histone Marks and Histone Lysine Crotonylation as a New Type of Histone Modification. Cell 2011, 146, (6), 1015-1027. 8.

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14. Dong, H.; Guo, Z.; Feng, W.; Zhang, T.; Zhai, G.; Palusiak, A.; Rozalski, A.; Tian, S.; Bai, X.; Shen, L.; Chen, P.; Wang, Q.; Fan, E.; Cheng, Z.; Zhang, K., Systematic identification of lysine 2-hydroxyisobutyrylated proteins in Proteus mirabilis. Mol. Cell. Proteomics 2018, 17: 482-494. 15. 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. Sci. Rep. 2017, 7, (1), 15553. 16. Meng, X.; Xing, S.; Perez, L. M.; Peng, X.; Zhao, Q.; Redona, E. D.; Wang, C.; Peng, Z., Proteome-wide Analysis of Lysine 2-hydroxyisobutyrylation in Developing Rice (Oryza sativa) Seeds. Sci. Rep. 2017, 7, (1), 17486. 17. Wu, Q.; Li, W.; Wang, C.; Fan, P.; Cao, L.; Wu, Z.; Wang, F., Ultradeep Lysine Crotonylome Reveals the Crotonylation Enhancement on Both Histones and Nonhistone Proteins by SAHA Treatment. J. Proteome Res. 2017, 16, (10), 3664-3671. 18. Wu, Q.; Cheng, Z.; Zhu, J.; Xu, W.; Peng, X.; Chen, C.; Li, W.; Wang, F.; Cao, L.; Yi, X.; Wu, Z.; Li, J.; Fan, P., Suberoylanilide hydroxamic acid treatment reveals crosstalks among proteome, ubiquitylome and acetylome in non-small cell lung cancer A549 cell line. Sci. Rep. 2015, 5, 9520. 19. Xu, W.; Wan, J.; Zhan, J.; Li, X.; He, H.; Shi, Z.; Zhang, H., Global profiling of crotonylation on non-histone proteins. Cell Res. 2017, 27, (7), 946-949. 20. Wei, W.; Liu, X.; Chen, J.; Gao, S.; Lu, L.; Zhang, H.; Ding, G.; Wang, Z.; Chen, Z.; Shi, T.; Li, J.; Yu, J.; Wong, J., Class I histone deacetylases are major histone decrotonylases: evidence for critical and broad function of histone crotonylation in transcription. Cell Res. 2017, 27, (7), 898-915. 21. Bao, X.; Wang, Y.; Li, X.; Li, X. M.; Liu, Z.; Yang, T.; Wong, C. F.; Zhang, J.; Hao, Q.; Li, X. D., Identification of 'erasers' for lysine crotonylated histone marks using a chemical proteomics approach. eLife 2014, 3:e02999. DOI：10.7554/eLife.02999

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Table 1. The comparison between SILAC and PRM results Protein accession

Gene name

Position

SAHA/Control (Khib PRM ratio)

SAHA/Control (Khib SILAC ratio)

Q15293

RCN1

266

1.651

1.685

P04179

SOD2

130

0.651

0.561

P30838

ALDH3A1

269

1.705

1.654

P06733

ENO1

126

1.731

1.502

P04075

ALDOA

13

0.605

0.732

P23528

CFL1

30

1.465

1.654

P31947

SFN

140

1.956

2.317

O60218

AKR1B10

66

0.473

0.634

P0DMV9

HSPA1A

246

1.727

1.786

P62158

CALM1

22

1.587

1.891

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Page 22 of 30

FIGURE LEGENDS Figure

1.

(A)

The

workflow

for

global

quantification

of

lysine

2-hydroxyisobutyrylome in A549 cells upon SAHA treatment. (B) Pie chart shows the experimental reproducibility of three replicates. (C) Comparison of the quantification ratios of Khib sites to show the reproducibility of three biological replicates. Figure 2. (A) Distribution of the number of Khib sites per proteins. (B) The enzymes with more than 20 Khib sites. (C) The subcellular localization of lysine 2-hydroxyisobutyrylome compared to global proteome. Figure 3. (A) Volcano plot to show the changes of Khib upon SAHA treatment. (B) A diagram showing sites of histone Khib, Kcr and Kac identified in this study and our previous data.17, 18 Figure 4. (A) The subcellular localization of significantly changed lysine 2-hydroxyisobutyrylated proteins upon SAHA treatment. (B) GO-based functional enrichment analysis for the quantitative 2-hydroxyisobutyrylome. (C), KEGG-based functional enrichment analysis for the quantitative 2-hydroxyisobutyrylome. Figure 5. (A) The overlap of sites between Khib, Kcr and Kac upon SAHA treatment. (B) Scatter diagram to show the correlation between Khib and Kac upon SAHA treatment. (C) Scatter diagram to show the correlation between Kcr and Kac upon SAHA treatment. (D) Scatter diagram to show the correlation between Khib and Kcr upon SAHA treatment. Figure 6. SAHA treatment significantly increased lysine 2-hydroxyisobutyrylation in A549 cells. A549 cells were treated with or without SAHA for 24 h. Cells were then

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harvested, and the crude proteins from whole cell lysates were extracted. Thirty micrograms of crude proteins was subject to SDS-PAGE followed by Western blot analysis with anti-2-hydroxyisobutyryllysine antibody.

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Figure 1. (A) The workflow for global quantification of lysine 2-hydroxyisobutyrylome in A549 cells upon SAHA treatment. (B) Pie chart shows the experimental reproducibility of three replicates. (C) Comparison of the quantification ratios of Khib sites to show the reproducibility of three biological replicates. 128x98mm (300 x 300 DPI)


Page 24 of 30

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Figure 2. (A) Distribution of the number of Khib sites per proteins. (B) The enzymes with more than 20 Khib sites. (C) The subcellular localization of lysine 2-hydroxyisobutyrylome compared to global proteome. 123x85mm (300 x 300 DPI)



Figure 3. (A) Volcano plot to show the changes of Khib upon SAHA treatment. (B) A diagram showing sites of histone Khib, Kcr and Kac identified in this study and our previous data. 78x114mm (300 x 300 DPI)


Page 26 of 30

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Figure 4. (A) The subcellular localization of significantly changed lysine 2-hydroxyisobutyrylated proteins upon SAHA treatment. (B) GO-based functional enrichment analysis for the quantitative 2hydroxyisobutyrylome. (C), KEGG-based functional enrichment analysis for the quantitative 2hydroxyisobutyrylome. 150x114mm (300 x 300 DPI)



Figure 5. (A) The overlap of sites between Khib, Kcr and Kac upon SAHA treatment. (B) Scatter diagram to show the correlation between Khib and Kac upon SAHA treatment. (C) Scatter diagram to show the correlation between Kcr and Kac upon SAHA treatment. (D) Scatter diagram to show the correlation between Khib and Kcr upon SAHA treatment. 91x70mm (300 x 300 DPI)


Page 28 of 30

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Figure 6. SAHA treatment significantly increased lysine 2-hydroxyisobutyrylation in A549 cells. A549 cells were treated with or without SAHA for 24 h. Cells were then harvested, and the crude proteins from whole cell lysates were extracted. Thirty micrograms of crude proteins was subject to SDS-PAGE followed by Western blot analysis with anti-2-hydroxyisobutyryllysine antibody. 25x35mm (300 x 300 DPI)



for TOC only 66x45mm (300 x 300 DPI)


Page 30 of 30

Global Analysis of Lysine 2-Hydroxyisobutyrylome upon SAHA

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