Effects of Gentamicin on the Proteomes of Aerobic and Oxygen

Mar 21, 2013 - Queen Mary, University of London, Mile End Road, London, E1 4NS, U.K.. J. Med. Chem. , 2013, 56 (7), pp 2904–2910. DOI: 10.1021/jm301...
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Effects of Gentamicin on the Proteomes of Aerobic and OxygenLimited Escherichia coli Zubida M. Al-Majdoub,†,‡ Abiola Owoseni,†,‡ Simon J. Gaskell,§ and Jill Barber*,†,‡ †

Manchester Institute for Biotechnology, 131 Princess Street, Manchester, M1 7DS, U.K. School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, M13 9PT, U.K. § Queen Mary, University of London, Mile End Road, London, E1 4NS, U.K. ‡

S Supporting Information *

ABSTRACT: The key role of the bacterial ribosome makes it an important target for antibacterial agents. Indeed, a large number of clinically useful antibiotics target this complex translational ribonucleoprotein machinery. Unfortunately, the development of resistant bacterial strains has compromised the effectiveness of most classes of antibacterial agent, including the classes that target the ribosome. Combinations of two or more drugs can be used to help overcome resistance, and in certain circumstances their action may be synergistic. In this study we have used proteomic techniques to establish the effects of gentamicin on the proteomes of aerobic and oxygen-limited Escherichia coli. Ribosomal proteins L1, L9, L10, and S2 were found to be up-regulated in both conditions, and we postulate that these are candidate drug targets for the development of synergistic combinations with gentamicin.

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INTRODUCTION Gentamicin (Figure 1) is a clinically useful antibiotic of the aminoglycoside class. It was discovered in 1963, a product of

for respiratory tract infections, septicaemia, and intra-abdominal infections.5 Gentamicin distributes poorly into the plasma because it is very hydrophilic and unable to cross membranes readily, and oral administration is ineffective for the same reason. Gentamicin is therefore administered by intravenous or intramuscular injection. Gentamicin, like other aminoglycosides, may be inactivated by aminoglycoside-modifying enzymes,7 or it may fail to accumulate because of efflux pumps;8 however, aminoglycoside resistance is actually comparatively rare in clinical isolates. More important in limiting the use of gentamicin and other aminoglycosides is their toxicity to both kidneys and ears. Nephrotoxicity is found in up to 20% of cases after long periods of therapy; drug metabolites accumulate, and these can cause severe side effects.8 Gentamicin also affects the vestibular cells of the ear, causing irreversible hearing loss.6 It is therefore common for the serum levels of gentamicin to be carefully monitored during therapy. Antibiotic combinations are nearly always advantageous in combating bacterial resistance. A bacterial cell is much less likely to develop resistance to two or more antibiotics than to a single compound. However, some antibiotic combinations have the additional advantage of being synergistic, and this is especially advantageous when one of the components is a toxic drug, like gentamicin. Gentamicin is often used in combination with benzyl penicillin in the treatment of Enterococcal, Enterobacterial, and Staphylococcal infections,9 synergy having been demonstrated in vitro.

Figure 1. Gentamicin.

the fungus Micromonospora purpurea.1 In common with other aminoglycoside antibiotics, such as streptomycin and kanamycin, it is chemically stable and very water-soluble.2 Gentamicin binds specifically to the A site of 16S ribosomal RNA, thereby inhibiting bacterial protein synthesis.3 This results in a bactericidal effect on most pathogenic bacteria, whether Gram-positive or Gram-negative. Gentamicin acts similarly to other aminoglycoside antibiotics such as neomycin and kanamycin. They are transported across cell membranes in an oxygen-dependent process. Strict anaerobes are therefore expected to be resistant to gentamicin, and this resistance has been reported.4,5 In human medicine, gentamicin is a firstchoice drug for the treatment of serious aerobic bacterial infections caused by pathogenic Gram negative bacilli such as E.coli and Klebsiella; indeed aminoglycosides are the most commonly used drugs worldwide in the treatment of Gramnegative infections.6 Gentamicin concentrates in the urine, so despite the relatively anaerobic environment of the urinary tract, it is often used for the treatment of urinary tract infections as well as © 2013 American Chemical Society

Received: December 17, 2012 Published: March 21, 2013 2904

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Figure 2. Effect of gentamicin on the growth rate of E. coli K12 in (A) aerobic conditions, exposed to 6 μg mL−1 gentamicin-treated and (B) oxygenlimited conditions, exposed to 0.5 μg mL−1 gentamicin-treated. The experiments were performed in triplicate, and the results are expressed as mean values. Error bars are so small that they do not extend beyond the boundaries of the symbol.

chosen for aerobic E. coli and 0.5 μg mL−1 for oxygen-limited E. coli. These concentrations of antibiotic could be used without significant effects on the growth rates of the bacteria over the range A600 of 0.2−0.8 (aerobic) or 0.15−0.5 (oxygen-limited). The minimum inhibitory concentration (MIC) of gentamicin to aerobic E.coli aerobic cells is normally around 4 μg mL−1,15 which is (as expected) close to but not identical with the concentration required to permit two doublings. Preparation of Control and Drug-Treated E. coli. E. coli triplicate cultures (controls and drug-treated, aerobic and oxygen-limited) were prepared as described in the Experimental Section. The resazurin indicator changed color from blue to pink when the oxygen-limited cultures reached an A600 of approximately 0.3. Although pathogenic E. coli are sometimes described as anaerobic and inhabit parts of the human body where oxygen is limited, the urinary tract (for example) is not a strictly anaerobic environment. This is why we made no efforts to eliminate oxygen completely, only to limit the concentration in the cultures. Isolation of Protein Fraction, Generation of Peptides, and LC−MS/MS. Proteins from gentamicin-treated and control samples were isolated using the FASP (filter aided sample preparation) protocol,17 which was adapted in several minor ways to the use of bacterial cultures (see Experimental Section). Peptides suitable for LC−MS/MS analysis were generated using trypsin in the case of oxygen-limited cultures. For aerobic E. coli, endopeptidase Lys-C digests and tryptic digests were analyzed and the data pooled, increasing the number of protein hits. This was judged unnecessary for the oxygen-limited cultures where (presumably because the proteome is not dominated to the same degree by proteins necessary for rapid growth) tryptic digests gave greater numbers of confident identities. The use of an Amazon iontrap mass spectrometer allowed both CID (collision induced dissociation) and ETD (electron transfer dissociation) data to be collected. The data were analyzed using an in-house Mascot server for identification of peptides and proteins. Peptide samples were not fractionated prior to LC−MS/MS because preliminary experiments had indicated that gentamicin perturbs the proteome quite significantly. Our objective was to identify drug targets for potential synergizers; many candidates could be identified without probing deeply into the proteome.

Bacteria show adaptive responses to antibiotic challenge, upregulating some proteins that help combat antibacterial action.10 Proteomic studies based on two-dimensional gel electrophoresis have been used to identify markers of antibiotic treatment; glyceraldehyde 3-phosphate dehydrogenase was upregulated on erythromycin treatment of Streptococcus pneumonia.11 Limited mass spectrometric studies have also been performed on antibiotic-treated bacteria.12 In this study we exploit the powerful and sensitive approach of mass spectrometry based proteomics to describe the adaptive response of E. coli to gentamicin treatment, with the ultimate aim of developing novel synergistic antibiotic combinations in which gentamicin is one partner.

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RESULTS E. coli was chosen as the test organism. In this instance it is more than a model organism because urinary tract infections in which gentamicin is sometimes indicated are commonly based on E. coli. In the laboratory, E. coli is normally grown under fully aerobic conditions, but in this work, we aimed also to grow the bacterium in oxygen-limited conditions, resembling more closely the environment of the urinary tract. Experiments were designed to monitor the effect of the drug on the proteome of E. coli in both aerobic and oxygen-limited conditions. Bacterial Growth Conditions. The workflow we used was adapted from the work of Mehta and Champney on the effect of anti-ribosomal drugs on bacterial ribosome assembly.13 Antibiotic was added when the bacteria entered the exponential phase (A600 = 0.2 for aerobic cultures; A600 = 0.15 for oxygenlimited cultures), and the bacteria were harvested after two doublings. The amount of antibiotic was chosen to permit two doublings without a significant change in growth rate and had been determined using preliminary experiments in which a range of antibiotic concentrations had been explored. Both aerobic and oxygen-limited E. coli were challenged with gentamicin. Oxygen limitation was achieved by growing the bacteria in sealed tubes containing resazurin indicator. The optical densities of the cultures were estimated by visual comparison with McFarland standards14 made up in the same type of tube. Correlation of measured optical density with estimates was previously shown to be ±5%. Growth curves for aerobic and oxygen-limited E. coli are shown in Figure 2. On the basis of these data, a drug concentration of 6 μg mL−1 was 2905

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information, each of the other experiments contributed additional data. Comparison of the Proteomes of Aerobic and Oxygen-Limited E. coli. Label-free quantification has been achieved in several ways in proteomic studies. These include the emPAI score, the average peptide score of the highest scoring three peptides, and peptide counting.16−18 In this work, we were not concerned with precise quantification of proteins within a sample; we aimed only to identify proteins that were up- or down-regulated. A semiquantitative method is therefore sufficient. We considered all of these three methods of labelfree quantification. (i) The emPAI score was calculated by Mascot19 for the trypsin-CID data. (ii) Rather than consider simply the number of peptides detected for a protein, we calculated the percentage of the total. (iii) We also calculated the average peptide score of the most intense three peptide signals. We considered only proteins for which at least two peptides were detected in all three replicates of one or both data sets (i.e., aerobic or oxygen-limited), and peptide detection was defined as a Mascot score of at least 15 for unique peptides. In this way 85 proteins were selected for comparison. For each of the three quantification parameters, we calculated the mean values and standard deviations of the replicates and the differences between the mean values in terms of the standard deviation (σ). Table 1 in the Supporting Information shows that this value is fairly consistent for two measurements: the average peptide score of the most intense three signals and the percentage of total peptides. The third parameter, the emPAI score, is much less consistent. In order to overcome the “look elsewhere effect”,20 particle physicists require a 5σ difference between the mean values.21 In proteomics experiments, where “elsewhere” is finite, a 3σ effect (a 1 in 1000 chance of a false positive) indicates a very strong likelihood of a

The number of proteins identified together with the corresponding number of peptides is shown in Table 1. Table 1. Number of Proteins and Peptides from Each Sample Identified by LC−MS/MS sample (control) C1a C2a C3a C1ol C2ol C3ol

no. of proteins (peptides) detected

sample (drug treated)

no. of proteins (peptides) detected

Aerobic E. coli 133 (503) G6A 143 (433) G6B 116 (453) G6C Oxygen-Limited E. coli 112 (359) G0.5A 120 (374) G0.5B 117 (376) G0.5C

112 (359) 120 (374) 117 (376) 124 (374) 131 (426) 151 (560)

Table 2. Numbers of Proteins, Peptides, and Residues Detected by Different Enzyme/Fragmentation Method enzyme/ionization method

no. of proteins

no. of peptides

no. of residues

trypsin/CID trypsin/ETD LysC/CID LysC/ETD total

90 55 90 59 133

312 196 270 204 503

4518 2816 4267 3089 7102

Table 2 shows the distribution of peptides detected from each proteolytic enzyme/fragmentation method for a typical (aerobic control) data set. Although the classical tryptic digest with CID as the fragmentation method yielded the most

Table 3. E. coli Proteins Most Clearly Up- or Down-Regulated on Oxygen Depletion mean no. of peptides observed protein ATPB TDCE ATPA SYK2 PTA CLPB ASPA ODP1 KPYK2 GLDA DEOC MBHM OPPA TPX RRF CSPE OSMY CSPC SUCD DGAL SLYD YAHO

function

mass (Da)

Proteins Up-Regulated on Oxygen Depletion ATP synthase β subunit 50351 keto acid formate acetyltransferase 86166 ATP synthase α subunit 55416 Lysyl-tRNA synthetase, heat inducible 57847 phosphate acetyltransferase 77466 chaperone protein clpB 95697 aspartate ammonia lyase 52950 pyruvate dehydrogenase E1 component 99948 pyruvate kinase II 51553 glycerol dehydrogenase 39087 deoxyribose phosphate aldolase 27944 hydrogenase-2 large chain precursor 62908 Proteins Down-Regulated on Oxygen Depletion periplasmic oligopeptide-binding protein precursor 60975 thiol peroxidase 17995 ribosome recycling factor 20683 cold-shock-like protein cspE 7459 osmotically inducible protein Y precursor 21061 cold-shock-like protein cspC 7398 succinyl-CoA ligase [ADP-forming] α subunit 30044 D-galactose-binding periplasmic protein precursor 35690 FKBP-type peptidyl-prolyl cis−trans isomerase slyD 21182 UPF0379 protein yahO precursor 9889 2906

aerobic

oxygen limited

0.3 0 0 0 0 0 2.7 0 1.3 0 0 0

9 8.7 7 6.3 5.7 5.7 8.3 4.6 6 2.3 2 2

15.3 5.7 5.7 4 3.7 4 3.3 3.3 2 2

4.3 0.7 0.7 0 0 0 0 0 0 1

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Table 4. Proteins Most Clearly Up- or Down-Regulated on Gentamicin Treatment of Aerobic E. coli mean no. of peptides observed protein CH60 RS1 RL10 RL1 CLPB RS2 SUCC RL9 OMPX CSPC TREC RAIA FABD GPMI RRF GLPQ GRCA KAD ASPG2 PGK MALE PFLB

function

mass (Da)

Proteins Up-Regulated on Gentamicin Treatment 60 kDa chaperonin 57464 30S ribosomal protein S1 61235 50S ribosomal protein L10 17757 50S ribosomal protein L1 24714 chaperone protein clpB 95697 30S ribosomal protein S2 26784 succinyl-CoA synthetase β chain 41652 50S ribosomal protein L9 15759 outer membrane protein X 18648 Proteins Down-Regulated on Gentamicin Treatment cold-shock-like protein cspC 7398 trehalose 6-phosphate hydrolase 64082 ribosome-associated inhibitor A 12777 malonyl CoA-acyl carrier protein transacylase 32682 2,3-bisphosphoglycerate-independent phosphoglycerate mutase 56272 ribosome recycling factor 20683 glycerophosphoryl diester phosphodiesterase 40874 autonomous glycyl radical cofactor 14332 adenylate kinase 23628 L-asparaginase 36942 phosphoglycerate kinase 41264 maltose-binding periplasmic protein 43360 formate acetyltransferase 1 85588

control

treated

1.7 0 0 0 0 0 2 0 0

11.3 6.3 5.7 4.3 4 3.3 5.3 3.7 2.7

4 2.3 2.7 3.3 4 5.7 6 6.7 8 7 15.7 11.3 13.3

2 0 0 0.3 0.3 2 0.7 1 2 0.3 8 3.3 1

methods have normally been used to derive intraproteomic quantifications;25−27 it becomes progressively more difficult to achieve accuracy as the range of protein size, hydrophobicity, and isoelectric point increases (see, for example, the very low concentration of elongation factor Tu given in ref 28). The proteins of the translation machinery include both small, basic ribosomal proteins and acidic proteins, such as EF-Tu, and are therefore especially vulnerable. Our use of label-free methods is, however, much more conservative. We have compared only like with like. We have made no attempt to quantify, only to ascertain whether a protein is up- or down-regulated, and we have required a high standard of proof over a range of data analysis methods. We were interested in the effect of gentamicin on both aerobic and oxygen-depleted E. coli because the drug is used clinically in varying degrees of oxygenation. Clearly the proteomes of the organism in these two states are different, as shown in Table 3. Oxygen depletion appears to have heatshock-like characteristics, with CH60, CLPB, and SYK2 upregulated and CSPE and CSPC down-regulated. ATP synthase, which is significantly up-regulated on oxygen depletion, is also up-regulated as a part of the alkaline response.29 Oxygenlimited E. coli appears to be an order of magnitude more sensitive to gentamicin than E. coli grown in aerobic conditions. This may because oxygen limitation represents a nonspecific insult to the cells, rendering them more sensitive to a second insult (antibiotic), but the high levels of ATP synthase in the oxygen-limited cells might alternatively lead to the tentative speculation that active transport of gentamicin is facilitated under conditions of oxygen limitation. While our primary interest in this work was the search for gentamicin synergizers, it is also possible that proteins up-regulated on oxygen depletion, such as the ATP synthase components, would be potential drug targets in anaerobic infections.

perturbation in concentration. Table 3 shows all the proteins for which a 5σ effect is seen in at least two of the parameters monitored. A full list (including proteins showing a 3σ effect in at least two parameters) is shown in the Supporting Information. Effect of Gentamicin on Aerobic E. coli. The effect of gentamicin on the aerobic E. coli proteome was now assessed similarly. Sixty-nine proteins were selected for comparison, and Table 4 shows the proteins most clearly up- or down-regulated in the presence of gentamicin. In this case, all proteins showing a 3σ effect in at least two parameters are shown. Effect of Gentamicin on Oxygen-Limited E. coli. The effect of gentamicin on the oxygen-limited E. coli proteome was also assessed. Eighty-three proteins were selected for comparison, and Table 5 shows the proteins most clearly upor down-regulated in the presence of gentamicin. Again, all proteins showing a 3σ effect in at least two parameters are shown.

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DISCUSSION We have used a very simple proteomic strategy to identify potential drug targets for synergizers with gentamicin. The FASP protocol is a simple, but powerful, method of isolating whole proteomes. Trypsin, however, is not always the best enzyme for digestion and analysis of small, basic proteins, and we therefore analyzed both Lys-C and tryptic digests, facilitating detection of ribosomal proteins. It would be possible to use stable-isotope labeling22−24 and extensive chromatography to gain detailed, quantitative insights into the effects of drugs on bacterial proteomes. We set out, however, to understand gross effects because it is these that are most likely to assist the drug discovery process. A label-free mass spectrometric approach allowed complete flexibility in the medium and conditions used for bacterial growth. Label-free 2907

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Table 6. E. coli Ribosomal Proteins and Their Operons30

Table 5. Proteins Most Clearly Up- or Down-Regulated on Gentamicin Treatment of Oxygen-Limited E. coli

name of operon

mean no. of peptides observed protein LDCI RL1 RL9 OMPA RS2 RL16 LACI RS7 IPYR RS10 RL10 RS8 DPS DBHA DCEA HNS DEOC MBHM FRDB GLDA USPG DAPD UDP ODP1 YGEY GLPK WRBA 6PGD ATPA ATPB EFG

function

mass (Da)

SPC S10

control

Proteins Up-Regulated on Gentamicin Treatment lysine decarboxylase, inducible 81607 0 50S ribosomal protein L1 24714 1 50S ribosomal protein L9 15759 0 outer membrane protein A 37292 1.7 30S ribosomal protein S2 26784 0 50S ribosomal protein L16 15271 1 lactose operon repressor 38737 1 30S ribosomal protein S7 20007 0.7 inorganic pyrophosphatase 19805 0 30S ribosomal protein S10 11728 0 50S ribosomal protein L10 17757 0 30S ribosomal protein S8 14175 0 DNA protection during starvation 18684 0 protein DNA-binding protein HU-α 9529 2 glutamate decarboxylase α 53221 1 Proteins Down-Regulated on Gentamicin Treatment DNA-binding protein H-NS 15587 4.7 deoxyribose phosphate aldolase 27944 2 hydrogenase-2 large chain 62908 2 fumarate reductase iron−sulfur 27732 2.7 subunit glycerol dehydrogenase 39087 2.3 universal stress protein G 15925 2.7 2,3,4,5-tetrahydropyridine30044 2.7 2,6-dicarboxylate N-succinyltransferase uridine phosphorylase 27313 3 pyruvate dehydrogenase E1 99948 4.7 component uncharacterized protein ygeY 45288 3.7 glycerol kinase 56480 4.7 flavoprotein wrbA 20832 5 6-phosphoglycerate dehydrogenase, 51563 8 decarboxylating ATP synthase subunit α 55416 7 ATP synthase subunit β 50351 9 elongation factor G 77704 12.3

treated L11 α Str L35 trmD S15 L10 S20

7 7.3 5.3 5 3.3 4.3 4.3 3.7 3 2.7 2.3 2.3 2.3

proteins in operon L14, L24, L5, S14, S8, L6, L18, S5, L30, L15, SecY, and L36 S10, L3, L4, L23, L2, S19, L22, S3, L16, L29, and S17 L11, L1 S13, S11, S4, RpoA, L17 S12, S7, EF-G, EF-Tu IF3, L35, L20 S16, rim, trmD, L19 S15, pnp (polynucleotide phosphorylase) L10, L12, RpoB, RpoC S20

regulatory protein S8 L4 L1 S4 S7 L20 S15 L10, L12 S20

several proteotypic peptides on trypsin treatment, are upregulated at the high standard of proof we have applied. Ribosomal proteins may therefore represent attractive drug targets for use in combination with gentamicin. The cell responds to gentamicin injury by up-regulation of the proteinsynthesis machinery. Inhibition of a component of this machinery is likely to disrupt ribosomal assembly, and our data suggest that ribosomal L1, which functions as a regulator of ribosomal protein synthesis, is a candidate target. Ribosomal S2 L9 and L10 are also significantly up-regulated, and L10 has regulatory function. In addition S1, S7, S8, and S10 are upregulated in either aerobic or oxygen-limited conditions, and more complex experiments may indicate general up-regulation in response to gentamicin. In our study, we analyze the total proteome of the E. coli cell, not just the ribosomal subproteome. We are therefore confident that ribosomal proteins accumulate because they are up-regulated, not just because they fail to assemble into functional ribosomes. The up-regulation is not uniform for all ribosomal proteins, and quantitative proteomic methods will undoubtedly illuminate this aspect further. We propose that the regulatory ribosomal proteins L1, L9, L10, and S2 have been identified as candidate drug targets for synergizers with gentamicin, under both aerobic and oxygen-limited conditions.

4 2.3 3.3 0 0 0.7 0 0.3 0 0 1.3 0 0.7 0.3 3.3

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2 3 5.7

EXPERIMENTAL SECTION

Strain and Materials. E. coli K12 (strain KS1000) from New England Biolabs (U.K.) was used in all experiments. Reagents were obtained from Sigma-Aldrich (Poole, U.K.) unless otherwise stated. Mass spectrometry grade lysyl endpeptidase (Lys-C) was purchased from Wako (Osaka, Japan). Protease inhibitor cocktail was purchased from Roche. Bugbuster and Benzonase were from Novagen. Gentamicin was supplied by Sigma and prepared freshly in sterile water before each experiment. Aerobic Growth of E. coli Cells and Cell Lysis. E. coli K12 cells were grown at 37 °C in 100 mL of Luria broth (LB) medium with aeration until the A600 reached 0.2. At this point, gentamicin was added to a final concentration of 6 μg mL−1; no antibiotic was added to the control cultures. The cultures were then incubated for a further 3 h at 37 °C. The cells were collected at OD600 = 0.8 and completely resuspended in BugBuster protein extraction reagent, using 5 mL of BugBuster per gram of wet cell paste. Then 200 μL of protease inhibitor solution (prepared by dissolving a tablet in 2 mL of sterile water) was added, followed by 1 μL (25 units) of Benzonase per mL of BugBuster reagent added. The cells were incubated with gentle mixing for 20 min at room temperature. Lysates were clarified by centrifugation at 16000g for 20 min at 4 °C. The supernatants were transferred to fresh tubes and frozen at −20 °C until needed. All experiments were carried out in triplicate.

Effects of gentamicin treatment include the up-regulation of outer membrane proteins, OMPX in the aerobic cultures, and OMPA in the oxygen-limited cultures. Changes in membrane structure in response to antibiotic insult are expected.31−35 Down-regulated proteins tend to be house-keeping proteins, with no very consistent patterns emerging. The striking feature in both aerobic and oxygen-limited cultures, is, however, the up-regulation of ribosomal proteins. Ribosomal proteins are contained in a small number of operons in the E. coli genome, as shown in Table 6. Although there is some level of co-regulation, certain individual proteins, such as L1, are able to autoregulate apparently without effects on expression of proteins in the same operon.30 Our results do not suggest operon-specific up-regulation of ribosomal proteins. Rather, total ribosomal protein is up-regulated and certain individual proteins, especially the larger proteins that generate 2908

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Anaerobic Culture of E. coli Cells. An amount of 1 mL of 0.001% resazurin indicator solution was added to 300 mL of sterile LB broth culture medium, and the medium was agitated until a homogeneous blue coloration was obtained. Then 30 mL universal bottles were filled to the brim to displace undissolved oxygen. An amount of 10 μL of an overnight E.coli culture was transferred into the medium, and each universal bottle was immediately covered with a sterile rubber stopper. The culture was then incubated at 37 °C and 60 rpm, and the growth rate of the cells was monitored by visual comparison with McFarland standards. When OD600 of about 0.15 was achieved, the cells were treated by injecting gentamicin to a final concentration of 0.5 μg mL−1. Controls were untreated. At the end of the experiment, when an OD600 of 0.6 was obtained, the absorbance was measured to confirm the estimations made. Cell lysis was carried out as above. MS and MS/MS Analysis on the Amazon Ion Trap. For the analysis of peptide mixtures, liquid chromatography was performed using an Ultimate 3000 nano-HPLC system (Dionex, Surrey, U.K.) comprising a WPS-3000 well-plate microautosampler, a FLM-3000 flow manager and column compartment, a UVD-3000 UV detector for chromatogram acquisition usually set at 214 nm, an LPG-3600 dualgradient micropump, and an SRD-3600 solvent rack controlled by Hystar (Bruker Daltonics) and Trap software. Samples were concentrated on a trapping column (Dionex, 300 μm × 0.1 cm) at a flow rate of 30 μL min−1. For the separation, a C18 Pepmap column (75 μm × 15 cm, Dionex) was used as generated by a cap-flow splitter cartridge (1/1000), the column oven was set to 30 °C to maintain a constant temperature, and a flow rate of 300 nL min−1 was applied. At this flow rate, typical column pressure was around 120 bar, master pressure was 200 bar, and loading pressure was 65 bar. Peptides were eluted by the application of a 90 min linear gradient: solvent A (95% H2O, 5% acetonitrile, 0.1% formic acid), 0%−95% solvent B (95% acetonitrile, 5% water, 0.1% formic acid). The LC was interfaced directly with ion trap mass spectrometer (Amazon, Bruker Daltonics) utilizing fused silica PicoTip emitter (New Objective, Woburn, MA, U.S.) using a capillary voltage of 1700−2200 V and nano-ESI mode. The typical setting on the ion trap was tuned as follows: dry gas temperature was set to 150 °C; dry gas was set to 6 L min−1; the scan mode was set to standard enhanced with a m/z range of 200−3000 with a speed of 8100 Th s−1. Ions were accumulated in the trap until the ion charge count (ICC) reaches 200 000 with a maximum accumulation time of 200 ms. Sample tables were inserted into HyStar (Bruker, Bremen, Germany) which incorporates Esquire control, version 6.2 (for the control of the mass spectrometer), and Chromeleon, version 6.8 (for the control of the LC system). In MS/MS analysis, up to three precursor ions were selected per cycle with active exclusion (1.5 min) for both CID and ETD, excluding singly charged ions. CID fragmentation was achieved using helium gas and a 30−200% collision energy sweep with amplitude 1.20 (ions are ejected from the trap as soon as they fragment). For electron-transfer dissociation experiments, the source temperature was set to 60 °C with ionization energy 80 eV and emission current 3 μA. The ion trap instrument was calibrated monthly using Bruker tunes mix. Data Analysis. Protein identification was performed by searching the raw data using the Mascot search algorithm on an in-house Mascot server (http://msct.bms.umist.ac.uk/mascot/home.html; Matrix Science Ltd., London, U.K.) against the E. coli K12 database. The mascot generic file (mgf file) contain the m/z values of the precursor ions, the m/z values of the correspondent fragment ions, the charge states of these ions, and their respective signal intensities. The MASCOT score refers to −10 log(p), where p is the likelihood that an observed event is random. Scores over 15 indicate identity and confidence levels are p < 0.05 for each peptide. Tandem mass spectra were processed using DataAnalysis 3.4 software (Bruker Daltonics). Two searches were performed for CID and ETD, using the E. coli database with the following parameters: maximum missed cleavages, 2; carbamidomethyl (C) as fixed modification; N-acetyl (protein) and oxidation (M) as variable modifications; peptide tolerance, 0.8 Da; fragment tolerance, 0.6 Da; instrument type, ESI-TRAP (for CID) and ETD-TRAP (for ETD) for the two separate data sets. The peptides

analyzed included all possible charge states of those Lys-C and tryptic peptides, where 300 ≤ m/z ≤ 1800 and the peptides contained at least five amino acids. We also used the reverse database functionality in Mascot 2.2 (Decoy), and these values were less than 3%.

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ASSOCIATED CONTENT

S Supporting Information *

An Excel file consisting of expanded results tables for proteins showing a 3σ effect. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +44 (0)161 275 2369. Fax: +44 (0) 161 275 2396. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Drs. Kathryn Lilley and Ruth Ledder for careful and constructive review of the manuscript. ABBREVIATIONS USED E. coli, Escherichia coli; FASP, filter aided sample preparation; CID, collision induced dissociation; ETD, electron transfer dissociation; LysC, endopeptidase Lys-C; emPAI, exponentially modified protein abundance index; σ, standard deviation; EFTu, elongation factor Tu; CH60, 60 kDa chaperonin; CLPB, chaperone protein clpB; SYK2, lysyl-tRNA synthetase heat inducible; CSPE, cold shocklike protein cspE; CSPC, coldshock-like protein cspC; OMPX, outer membrane protein X; OMPA, outer membrane protein A; LB, Luria broth; Th, thomson; mgf, Mascot generic file

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REFERENCES

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