Comprehensive Profiling of Protein Lysine Acetylation in Escherichia

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Comprehensive Profiling of Protein Lysine Acetylation in Escherichia coli Kai Zhang,*,1 Shuzhen Zheng,1 Jeong Soo Yang,2 Yue Chen,2 and Zhongyi Cheng2 1 2

State Key Laboratory of Medicinal Chemical Biology and Department of Chemistry, Nankai University, Tianjin 300071, China Ben May Department for Cancer Research, The University of Chicago, Chicago, Illinois 60637, United States S Supporting Information *

ABSTRACT: Protein lysine acetylation plays a key role in regulating chromatin dynamics, gene expression and metabolic pathways in eukaryotes, and, thus, contributes to diverse cellular processes like transcription, cell cycle regulation, and apoptosis. Although recent evidence suggests that acetylated proteins impact broadly cellular functions in prokaryotes, the substrates and localization of this modification remain widely unknown due to the limitations of analytical methods. Comprehensive identification of protein acetylation is a major bottleneck due to its dynamic property and pretty low abundance. A complete atlas of acetylome will significantly advance our understanding of this modification functions in prokaryotes. To achieve this goal, we have developed an intergraded approach to identifying lysine acetylation. Combining immunoaffinity enrichment with high sensitive mass spectrometry, we identified 349 acetylated proteins and addressed 1070 acetylation sites in Escherichia coli. To our knowledge, the acetylated proteins and acetylated sites were increased to 3 times and 8 times, respectively, compared to that in previous report. To further characterize this modification, we classified acetylated proteins into several groups according to cell components, molecular functions and biological process. Additionally, interaction networks and high confident domains architectures of acetylated proteins were investigated with the aid of bioinformatics tools. Finally, the acetylated metabolic enzymes were analyzed on the basis of acetylated proteins identified by proteomic survey in E. coli. Our study has demonstrated that the combined approach is powerful for identification and characterization of protein lysine acetylation on a large scale. These results not only greatly expand the number of acetylated proteins, but also provide a series of important information including localization, networks and characterization of acetylome. KEYWORDS: lysine acetylation, post-translational modifications (PTMs), mass spectrometry (MS), Escherichia coli (E. coli)

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INTRODUCTION Protein lysine acetylation, including histone acetylation and non-nuclear protein acetylation, is a dynamic and reversible post-translational modification (PTM) for cellular regulation. Lysine acetylation was first discovered in histones, and it is now identified to occur in more than 80 transcription factors as well as many other nuclear regulators. The acetylation status of histones plays a critical role in the regulation of gene transcription via the modulation of nucleosomal DNA package.1 The discovery of non-histone acetylated proteins has greatly expanded the understanding of these modification functions.2 Although lysine acetylation impacts many biological processes like transcription, cell cycle regulation and apoptosis, a global analysis of lysine acetylation is still relatively difficult due to its dynamic property and pretty low abundance for traditional analysis technologies.3,4 Advances in mass spectrometry (MS)-based proteomics and high affinity purification of acetylated peptides make it possible for proteomic survey of this modification. Thus, tremendous efforts have been made to develop MS-based proteomic technologies for this purpose. Kim et al. developed an © 2013 American Chemical Society

enrichment method for lysine-acetylated peptides by use of affinity purification strategy, which followed by a nano-HPLC/ MS/MS analysis for lysine acetylation characterization. A total of 388 acetylation sites in 195 acetylated proteins from HeLa cells and mouse liver mitochondria were mapped in the first systematic analysis of lysine-acetylated proteins in 2006.3 Three years later, a large-scale identification of acetylation proteins, more than 3600 lysine acetylation sites on 1750 proteins, was achieved by combining this strategy with isoelectric focusing separation and high-resolution high accuracy MS analysis.5 Acetylome studies have significantly expanded our understanding of this PTM in cellular process. First, protein lysine acetylation involves in almost every aspect of cellular physiology including mRNA splicing, protein synthesis, cell cycle and so on.3,5 Second, lysine acetylation has been identified in a variety of cell organelles from nuclei to mitochondria.3 Third, lysine acetylation regulates diverse protein properties including DNA−protein interactions, subcellular localization, Received: September 27, 2012 Published: January 7, 2013 844

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Figure 1. The analytical strategy and method for global profiling of lysine acetylation in E. coli..

transcriptional activity, and protein stability.6 Fourth, lysine acetylation regulates central metabolic pathways as well.7,8 Finally, lysine acetylation is a novel type of therapeutic target.9 Escherichia coli is one of the best understood of the simple model organisms and its genome has also been fully sequenced.10,11 So it is an ideal experimental cell system for understanding acetylome. Recent evidence suggests that lysine acetylation broadly impacts bacterial physiology.8,12−14 Although 138 lysine acetylation sites in 91 proteins were reported in E. coli, the number of lysine acetylated proteins is far more than anticipated.15 The gap should be filled to determine a complete acetylation status of E. coli, to improve our understanding of protein acetylation function in prokaryotes. Combining high affinity anti-acetyllysine pan antibodies with high sensitive mass spectrometry and bioinformatics tools, we developed an intergraded approach for comprehensive profiling of protein lysine acetylation in E. coli. Our study identified 349 acetylated proteins and addressed 1070 acetylation sites. To characterize acetylation proteins, we classified identified acetylated proteins into groups according to cell components, molecular function and biological process. Identified acetylated proteins are involved in important physiology, and some novel identified acetylation sites were found to locate in protein active intervals. With the aid of bioinformatics tools, protein−protein interaction networks and high confident domains were further analyzed. Finally, the acetylated metabolic enzymes were characterized on the basis of acetylated proteins identified by proteomic survey in E. coli. Our study has demonstrated that the combined approach is powerful for global identification and analysis of protein lysine acetylation, and greatly expanded the catalog of acetylated proteins in E. coli.

immobilized on protein A-conjugated agarose beads, and then affinity purification of lysine acetylated peptides was performed according to a modified procedure. The resulting peptides were cleaned with C18 ZipTips (Millipore Corp.) according to the manufacturer’s instructions, prior to nano-HPLC/mass spectrometric analysis. The tryptic digest was dissolved in 10 μL of HPLC buffer A (0.1% (v/v) formic acid in water), and 2 μL was injected into a Nano-LC system (Eksigent Technologies, Dublin, CA). Peptides were separated on a homemade capillary HPLC column (100-mm length × 75-μm inner diameter) containing Jupiter C12 resin (4-μm particle size, 90-Å pore diameter, Phenomenex, St. Torrance, CA) with a 120 min HPLC-gradient from 5 to 90% HPLC buffer B (0.1% formic acid in acetonitrile) at a flow rate of 200 nL/min. The HPLC elute was electrosprayed directly into an LTQ-Orbitrap Discovery mass spectrometer (Thermo Fisher Scientific, Waltham, MA) using a nanospray source. The LTQ-orbitrap mass spectrometer was operated in a data-dependent mode. The 10 most intense ions were sequentially isolated in the linear ion trap and subjected to collision-activated dissociation (CAD) with a normalized energy of 32%. The resulting MS/ MS data were searched against the E. coli entries of NCBInr protein sequence database using Mascot search engine (v2.2) and Maxquant software (v1.0.13.13) with an overall false discovery rate (FDR) for peptides of less than 1%. Trypsin was specified as the proteolytic enzyme, and up to 6 missed cleavage sites per peptide were allowed. Carbamidomethylation of cysteine was set as a fixed modification and oxidation of methionine, Acetyl (Protein N-term) and acetylation of lysine as variable modifications. The characterization of acetylated proteins was investigated according to cell components, molecular function, biological process, protein−protein interaction, domain and pathways via bioinformatics tools and database including Cytoscape (2.8.3), DAVID and STRING. For further details on the above procedures and analysis of acetylated proteins, see the Supporting Information.

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EXPERIMENTAL SECTION Antiacetyllysine pan antibodies were from PTM Biolabs, Inc. (Chicago, IL). Colloidal Blue Staining Kit and Luria−Bertani (LB) medium were from Invitrogen (Carlsbad, CA). Protein Aconjugated agarose beads were from Amersham Biosciences. C18 ZipTips and iodoacetamide were from Millipore (Bedford, MA). E. coli strain K12 DH10 was grown in LB medium at 37 °C. The cultured cells were harvested during the exponential growth phase by centrifugation. Five milligrams of proteins was extracted and purified according to our protocol and the resulting pellet was digested according to a previously described procedure.16 The anti-acetyllysine pan antibodies were

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RESULTS AND DISCUSSION

Analysis Strategy and Identification of Acetylated Proteins

Lysine acetylation has been thought to be an important modification in the regulation of protein function in diverse ways in eukaryotic cellular. Recently, the modification was indicated to impact prokaryotic physiology including cellular metabolism.13,14 However, the study of global acetylated 845

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Figure 2. Annotation of novel lysine acetylation sites in identified acetylated proteins. Ac, acetylation; P, phosphorylation; Succ, Succinylation; Malon, Malonylation.

acetylated sites of overlapped proteins in Figure 4A, 241 novel acetylated sites (70%) were identified in this study. To our knowledge, the lysine acetylated proteins and acetylation sites were increased to 3 times and 8 times in E. coli, respectively. The expansion of catalog of acetylated proteins may attribute to the improvement of antibody specificity and recognition range as well as mass spectrometry sensitivity. In this study, antiacetyllysine monoclonal antibodies and anti-acetyllysine polyclonal antibodies were mixed to prepare the antibody conjugated agarose for immunoaffinity acetylated peptide enrichment, which significantly improved antibody recognition range and efficiency. To examine the significance of novel acetylation sites, we investigated their localization. Four lines of evidence suggest that the novel acetylation sites may be involved in important biological process. First, novel acetylation modification may share the same lysine sites with other lysine modifications. For example, six novel acetylation sites of fructose-bisphosphate aldolase were found to localize in the same positions with the succinylation, a novel evolutionarily conserved PTM,17 as shown in Figure 2. As well, the acetylation of glyceraldehyde-3phosphate dehydrogenase was identified at K331, where two other novel lysine modifications, succinylation and malonylation, were reported recently (Table S6).17,18 While it is still unknown what roles the lysine acetylations could play in the novel positions, it is potentially possible that the acetylations compete with other PTMs at the same lysine residues. Second, novel lysine acetylation appeared to be near other modifications. For example, acetylation of phosphoglycerate kinase was identified at lysine 197 where three known phosphorylation modifications (S192, T196 and T199) adjoin.19 Similarly, two acetylation sites (K177 and K314) of Glyceraldehyde-3phosphate dehydrogenase A were observed close to phosphorylation sites reported (S174 and S313) as well.19 The spectra of

bacterial proteins was performed only on two bacterial species, E. coli15 and Salmonella enterica.8 Even for the two species, the number of acetylated proteins is far more than that expected due to the limited conditions. Therefore, it is highly expected that catalog of acetylated bacterial proteins will continue to expand greatly.13 Herein, a global view of acetylome of E. coli was achieved using integrated and reasoned proteomic techniques. We combined highly specific enrichment of lysine acetylated peptides with highly sensitive orbitrap mass spectrometry and bioinformatics tools for systematic identification of lysine acetylated proteins in E. coli. The combined approach includes 4 key steps, as shown in Figure 1: (1) purification and digestion of the protein lysate of E. coli; (2) affinity enrichment and purification of lysine acetylated peptides; (3) analysis and separation of lysine acetylated peptides using nano-HPLC/MS/ MS; (4) identification of lysine acetylation sites by sequence aliment and characterization of protein lysine acetylation in protein function, pathway and interaction networks. Using the approach above, we identified 349 acetylated proteins and addressed 1070 acetylated sites in E. coli (see Table S1 and S2 in the Supporting Information). A previous report identified 91 acetylated proteins from E. coli,15 68 (75%) of these were present in our data set (Figure 4A). Our data set not only included one-third of known acetylated proteins like ACS (acetyl-CoA synthetase), a simple on−off switch to control the activity of this stand-alone enzyme via reversible acetylation of a single lysine residue, but also filled in two-thirds of lysine acetylated substrates that were not reported in previous study in E. coli. For example, isocitrate lyase was identified as a novel acetylated protein in the analysis (the spectra of MS/MS fragment of a typical acetylated peptide was shown in the Figure 3A). Additionally, our study discovered a large number of novel acetylation sites in E. coli. Compared to 846

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Figure 3. (A) MS/MS spectra of a tryptic peptide ion HYVEK(Ac)VQQPEFAAA K from Isocitrate lyase; (B) MS/MS spectra of a tryptic peptide ion FESEVILSK(Ac)DEGGR from Elongation factor Tu; (C) MS/MS spectra of a tryptic peptide ion TAAILLDTK(Ac)GPEIR from Pyruvate kinase I.

MS/MS fragment of the related peptide was dissected, as shown in Figure 3B. Given the fact that the acetylation of lysine is involved in intra-protein cross-linking,20 we guess that these novel lysine sites may affect the function of other PTMs nearby. Third, novel lysine acetylations occur in the range of regions. For example, acetylation of elongation factor G at lysine 143 localized at the nucleotide binding regions (142−145). Likewise, acetylation of Glyceraldehyde-3-phosphate dehydrogenase A was observed to be close to nucleotide binding regions too, as shown in Figure 2. It is also unclear whether the acetylation plays a role in these important regions. Finally, novel lysine acetylation sites were identified at or near protein active sites or metal binding sites. For instance, acetylation of pyruvate kinase I was identified at lysine 68, and metal binding sites (Potassium) have been known at D66 and T67. Likewise, acetylation of Isocitrate lyase at lysine 194 is closed to the protein’s active site C195.21 Coincidentally, acetylation of phosphoglycerate kinase happens at the ATP binding site of the protein. Because lysine acetylation can alter charge and hydrophobicity of protein, it is possible that the novel acetylation plays an important role at some important positions.

in cytosol (60%), ribosome (17%) and membrane (9%), as shown in Figure 4A. The functional characterization of acetylated proteins was further assigned according to the gene ontology annotationl. Of 349 lysine acetylated proteins, 170 substrates are metabolic proteins, 58 proteins are translational proteins, 24 proteins are transcriptional proteins (Figure 4B). Compared to the previously reported results,15 the number of acetylated proteins in these three functions was increased by 250%, 190%, and 500%, respectively. (Figure S4) As far as molecular function is concerned, the acetylated proteins are assigned to several grouped including 101 proteins associated with nucleotide binding, 88 proteins involved in metal ion binding and 27 proteins related with structural constituent of ribosome (Figure 4C). The gene ontology annotational analysis of acetylome demonstrated that acetylated proteins are relatively broad in cell components, biological processes and molecular functions in E. coli. Our study expands greatly the catalog of lysine acetylation substrates and sites in prokaryotes.

Characterization of Acetylated Proteins in E. coli

We analyzed protein−protein interaction for identified acetylated proteins using Cytoscape software. The E. coli protein−protein interaction network has 331 acetylated proteins as nodes, connected by 3690 identified direct physical interactions obtained from STRING database. A complete network of acetylated proteins was created. Therefore Figure 5 offers an insight into the probability of interactions of acetylated proteins in E. coli. To determine the characteristics

Interaction Networks of Acetylated Proteins in E. coli

To further investigate the characterization of acetylated proteins, we classified the acetylated protein groups according to cell component, molecular function and biological process. The cell component of acetylated proteins was assigned based on Gene Ontology annotation. Our results showed that the acetylated proteins distribute in a variety of organelles, mainly 847

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Figure 4. Characterization of acetylated proteins. (A) Comparison of overlapped acetylated proteins: this study and ref 15; (B) comparison of acetylated sites for above overlapped proteins: this study and ref 15; (C) the distributions of acetylated proteins in cell components; (D) the classification of acetylated proteins in biological process; (E) the categories of acetylated proteins in molecular function.

of network described, the parameters are listed in the Figure S1 in the Supporting Information. The degree of node is an important parameter to evaluate the correlation of protein in network. The degree of each acetylated protein was calculated in the protein−protein interaction network (see Table S3 in the Supporting Information). Five acetylated proteins showed the highest degree in the network including atpA, eno, asps, tpiA, and rpsB. Extensive analysis of acetylome from eukaryotic cells were performed in previous studies,5 which showed the diverse functions of lysine acetylation in different pathways, while it is still unknown whether lysine acetylation plays a similar role in prokaryotic cells due to the limited data set. In this study, we performed function analysis of the large Kac data set and classified the acetylome against the STRING database of physical and functional interaction. Several highly confident protein networks (P < 10 × 10−20) are shown in the Figures S2−S7 in the Supporting Information. A large number of acetylated proteins were revealed in a variety of cellular process and protein-interaction networks. For example, 27 acetylated proteins were involved in ribosome, and they were connected in a relatively high dense protein−protein interaction network, as shown in Figure S2. Many acetylated protein of ribosome were involved in important biological process. For example, rpsE (30S ribosomal protein S5), which

was identified to be acetylated at lysine 4, 16, and 126 (see Table S2 in the Supporting Information), plays an important role in translational accuracy with S4 and S12. Many suppressors of streptomycin-dependent mutants of protein S12 are found in this protein.22 Additionally, 30S ribosomal protein S1 was identified for seven acetylation sites at lysine 150, 229, 272, 279, 320, 350 and 464, respectively. Four acetylation sites of them were found to localize at S1_RPS1_repeat_ec4, which is thought to be involved in the recognition and binding of mRNAs during translation initiation.23 The high dense protein interaction networks were also involved in translation and nucleotide-binding, as shown in Figures S3 and S4. Other connected protein clusters were also evaluated by means of STRING tool (see Figures S5−S7 in the Supporting Information). The protein−protein interaction information suggests that lysine acetylation is relatively active in multiple biological processes in E. coli. Domain Architecture of Acetylated Proteins in E. coli

Protein interactions involving domains are regulated by posttranslational modification of the smaller protein motif. For examples, bromodomain is a protein domain that recognizes acetylated lysine residues such as those on the N-terminal tails of histones in eukaryotes. In our study, acetylome pointed to several domains including nucleic acid-binding (OB-fold), 848

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Figure 5. Protein−protein interaction network of acetylated proteins in E.coli.

Figure 6. Acetylation of metabolic enzymes identified by MS based proteomics in TCA cycle in E. coli. The novel protein identified as lysineacetylated in the E. coli screen are underlined. The proteins previously identified as lysine acetylated in the E. coli are marked with an asterisk.

aminoacyl tRNA synthetase, thioredoxin fold and so on (see Table S5 in the Supporting Information).

Here, we identified 13 acetylated proteins of OB-fold family including RPSA, NUSA, ASPS, RPLB, CSPE, SSB, PNP, RPSQ, 849

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phosphogluconate dehydrogenase (gnd), ribose-5-phosphate isomerase (rpiA), transaldolaseA (talA), transaldolaseB (talB), fructose-bisphosphate aldolase class I (fbaB), fructose-bisphosphate aldolase,classII (fbaA), fructose-bisphosphatase I (fbp), and fructose-bisphosphatase II (glpX). These acetylated proteins were known to be associated with important biological process. For example, Glucose-6-phosphate dehydrogenase, which was identified to be acetylated at lysine 25 and 139, is the rate-controlling enzyme of this pathway. It has been known that an NADPH-utilizing pathway forms NADP+, which stimulates Glucose-6-phosphate dehydrogenase to produce more NADPH. Although pyruvate has been considered to be one of the major products of glycolysis in E. coli, the pyruvate metabolic pathway was missed in acetylome due to limited data set of lysine acetylated proteins in previous study.15 Our study showed that pyruvate metabolic pathway was highly enriched in acetylome data of E. coli. As shown in Figure S9 in Supporting Information, all enzymes of pyruvate metabolic pathway were identified to be acetylated, which is consistent with the knowledge of acetylation of metabolic enzymes in S. enterica8 This further expands the current catalog of acetylated enzymes in metabolic pathways in prokaryotes.

LYSS, LYSU, RNE, ASNC, RHO. The nucleic acid-binding, OB-fold is a compact structural motif used for recognition of single-stranded and unusually structured nucleic acids, and therefore it has been identified as critical for DNA replication, DNA repair, transcription, translation, and telomere maintenance.24 Of identified acetylated nucleic acid proteins, Rho, a hexameric RNA-DNA helicase, can bind two to three nucleotides of RNA with the standard polarity across the OBfold. It is thought that it utilizes both L23 and the canonical OB-fold ligand-binding site for interactions with the nucleic acid.25 Here, 3 acetylation sites were identified at lysine 115, 224 and 385. Metabolic Pathways of Acetylated Proteins in E. coli

Growing evidence suggests that lysine acetylation can regulate the flux of carbon through central metabolism.8 Thus, lysine acetylation has been considered to play a major role in metabolic regulation.26 Although lots of acetylated proteins were identified in the eukaryotes,3,5,7 current data set of acetylated metabolic enzymes is very limited in prokaryotes.8,15 It impedes the understanding of lysine acetylation regulation for metabolic pathways due to the absence of key acetylated proteins. Pathway analysis showed that 164 acetylated proteins involved in multiple metabolic pathways, including glycolysis, TCA cycle, carbohydrate and pyruvate metabolism, which is consistent with previous knowledge on lysine acetylation.7,8,15 Here, we discussed several metabolic pathways in detail below. We identified 12 acetylated enzymes associated with TCA cycle in this study. Six of them were not reported in the previous analysis,15 including aconitate hydratase 1 (acnA), aconitate hydratase 2 (acnB), malate dehydrogenase (mdh), fumarate hydratase (fumA), succinate-CoA ligase (sucD), and dihydrolipoyllysine-residue succinyltransferase (sucB) (Figure 6). The six novel enzymes have been known to play an important role in the central metabolic pathway. For examples, aconitate hydratase is an enzyme that catalyzes the reversible isomerization of citrate and iso-citrate via cis-aconitate in the tricarboxylic acid cycle. The imbalance of aconitate hydratase may contribute to disrupted metabolism in diabetics and other diseases.27 Additionally, we identified eight acetylation sites in the malate dehydrogenase (MDH) including lysine 54, 82, 99, 107, 134, 273, 279, and 301, respectively. The Malate dehydrogenase acetylation had been thought to mediate glucose-induced activation in mammals.7 A recent study further suggests that acetylation of malate dehydrogenase significantly increases its enzymatic activity and is involved in the cross-talk mechanisms between adipogenesis and the intracellular energy level.28 Another interesting pathway, the pentose phosphorylation pathway, was also highly enriched in acetylome data (see Figure S8 in the Supporting Information). The pentose phosphate pathway is a process that generates NADPH and pentose. And it is an alternative to glycolysis. While it does involve oxidation of glucose, its primary role is anabolic rather than catabolic. Dietary pentose sugars derived from the digestion of nucleic acids may be metabolized through the pentose phosphate pathway, and the carbon skeletons of dietary carbohydrates may be converted into glycolytic/gluconeogenic intermediates.29 As for as we have known, the acetylation of enzymes has not been analyzed carefully in this metabolic pathway in the previous study.15 Nine novel acetylated enzymes were identified in this study including glucose-6-phosphate dehydrogenase (zwf),

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CONCLUSIONS

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ASSOCIATED CONTENT

Combining high affinity enrichment of acetylated peptides with high sensitive mass spectrometry and bioinformatics tools, we developed an integrated approach to comprehensively investigate the lysine acetylation profiling in E. coli. The identification of 349 acetylated proteins and 1070 acetylation sites expanded greatly the catalog of acetylated proteins in E. coli, including two-thirds of novel acetylated proteins and seven-eighths of novel acetylation sites. Therefore, a more complete atlas of the acetylome was constructed in E. coli. In addition, many acetylation sites may be associated with regulation of protein function due to their localization near to protein modifications, active binding sites or regions. Characterization of acetylated proteins further indicates that acetylome may play an important role in biological process, protein interaction and metabolic pathways. Our study demonstrated that the combined approach is powerful for identification and characterization of protein acetylation on a large scale. These results represent a significant expansion of our current understanding of acetylome and suggest that lysine acetylation is highly abundant and deeply involved in multiple cellular processes in bacteria.

S Supporting Information *

Additional experimental details. Tables showing identified acetylated proteins and peptides, domain architecture of acetylated proteins, overlapped protein function, and acetylated proteins in interaction networks. Seven figures showing acetylation modulated functional networks in different cellular biological processes. The parameters of protein−protein interaction network in Figure 5. Acetylation of metabolic enzymes identified by MS based proteomics in pentose phosphate and pyruvate pathways in E. coli. This material is available free of charge via the Internet at http://pubs.acs.org. 850

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(15) Zhang, J.; Sprung, R.; Pei, J.; Tan, X.; Kim, S.; Zhu, H.; Liu, C. F.; Grishin, N. V.; Zhao, Y. Lysine acetylation is a highly abundant and evolutionarily conserved modification in Escherichia coli. Mol. Cell. Proteomics 2009, 8 (2), 215−25. (16) Kim, S. C.; Chen, Y.; Mirza, S.; Xu, Y.; Lee, J.; Liu, P.; Zhao, Y. A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea. J. Proteome Res. 2006, 5 (12), 3446−52. (17) Zhang, Z.; Tan, M.; Xie, Z.; Dai, L.; Chen, Y.; Zhao, Y. Identification of lysine succinylation as a new post-translational modification. Nat. Chem. Biol. 2011, 7 (1), 58−63. (18) 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. (19) Macek, B.; Gnad, F.; Soufi, B.; Kumar, C.; Olsen, J. V.; Mijakovic, I.; Mann, M. Phosphoproteome analysis of E. coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation. Mol. Cell. Proteomics 2008, 7 (2), 299−307. (20) Guo, X.; Bandyopadhyay, P.; Schilling, B.; Young, M. M.; Fujii, N.; Aynechi, T.; Guy, R. K.; Kuntz, I. D.; Gibson, B. W. Partial acetylation of lysine residues improves intraprotein cross-linking. Anal. Chem. 2008, 80 (4), 951−60. (21) Robertson, A. G.; Nimmo, H. G. Site-directed mutagenesis of cysteine-195 in isocitrate lyase from Escherichia coli ML308. Biochem. J. 1995, 305 (Pt 1), 239−44. (22) Takyar, S.; Hickerson, R. P.; Noller, H. F. mRNA helicase activity of the ribosome. Cell 2005, 120 (1), 49−58. (23) Meka, H.; Werner, F.; Cordell, S. C.; Onesti, S.; Brick, P. Crystal structure and RNA binding of the Rpb4/Rpb7 subunits of human RNA polymerase II. Nucleic Acids Res. 2005, 33 (19), 6435−44. (24) Theobald, D. L.; Mitton-Fry, R. M.; Wuttke, D. S. Nucleic acid recognition by OB-fold proteins. Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 115−33. (25) Bogden, C. E.; Fass, D.; Bergman, N.; Nichols, M. D.; Berger, J. M. The structural basis for terminator recognition by the Rho transcription termination factor. Mol. Cell 1999, 3 (4), 487−93. (26) Arif, M.; Selvi, B. R.; Kundu, T. K. Lysine acetylation: the tale of a modification from transcription regulation to metabolism. ChemBioChem 2010, 11 (11), 1501−4. (27) Ye, H.; Rouault, T. A. Human iron-sulfur cluster assembly, cellular iron homeostasis, and disease. Biochemistry 2010, 49 (24), 4945−56. (28) Kim, E. Y.; Kim, W. K.; Kang, H. J.; Kim, J. H.; Chung, S. J.; Seo, Y. S.; Park, S. G.; Lee, S. C.; Bae, K. H. Acetylation of malate dehydrogenase 1 promotes adipogenic differentiation via activating its enzymatic activity. J. Lipid Res. 2012, 53 (9), 1864−76. (29) Kruger, N. J.; von Schaewen, A. The oxidative pentose phosphate pathway: structure and organisation. Curr. Opin. Plant Biol. 2003, 6 (3), 236−46.

AUTHOR INFORMATION

Corresponding Author

*Tel.: 86-22-24387742. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Basic Research Program of China (Grants 2012cb910601 and 2013CB910903) and National Natural Science Foundation of China with Grants (20975053 and 21275077).

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REFERENCES

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dx.doi.org/10.1021/pr300912q | J. Proteome Res. 2013, 12, 844−851