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00046 Ma Hughes 2006 Jpr V5p54


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Profiling proteins in microbial systems/M. bovis

Profiling proteins in microbial systems/M. bovis

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  • 1. Quantitative Proteomic Analysis of Drug-Induced Changes in Mycobacteria Minerva A. Hughes,† Jeffrey C. Silva,‡ Scott J. Geromanos,‡ and Craig A. Townsend*,† Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore Maryland 21218, and Waters Corporation, 34 Maple Street, Milford Massachusetts 01757-3696 Received July 29, 2005 A new approach for qualitative and quantitative proteomic analysis using capillary liquid chromatog- raphy and mass spectrometry to study the protein expression response in mycobacteria following isoniazid treatment is discussed. In keeping with known effects on the fatty acid synthase II pathway, proteins encoded by the kas operon (AcpM, KasA, KasB, Accd6) were significantly overexpressed, as were those involved in iron metabolism and cell division suggesting a complex interplay of metabolic events leading to cell death. Keywords: isoniazid • tuberculosis • protein profiling • liquid chromatography • mass spectrometry Introduction Additional gene expression profiling studies offered further insights into the metabolism of M. tuberculosis defining adap- Tuberculosis (TB) continues to be a major cause of disease tive responses to intracellular phagocytosis,8 heat shock,9 and mortality with an estimated 2 million deaths annually.1 oxidative stress,10 nutrient depletion,11 and responses to various Approximately one-third of the world’s population is thought chemotherapeutics.12 to be infected with Mycobacterium tuberculosis, the etiological agent of TB. Despite the reported success of directly observed Though informative, genomic analysis alone provides only treatments, short course (DOTS), noncompliance is one of the a limited view of the dynamics associated with cellular re- factors leading to the emergence of multi-drug resistant strains sponses to a particular stimulus or at steady state. A systemic (MDR-TB). MDR-TB refers to those strains resistant to two or analysis of the proteomic and metabolic fluctuations is needed more of the five first line anti-TB drugs (isoniazid, rifampin, to complement existing genomic studies. Compared to mRNA pyrazinamide, ethambutol, and streptomycin). Patients infected studies, proteomic analysis provides a more accurate assess- with an MDR-TB strain have a mortality rate of 60-90%, ment of the conditional changes because the measurement equivalent to those without treatment.2 The matter is further focuses on the functionally relevant species. Furthermore, a complicated by a rapid progression of the disease and increased direct correlation between mRNA expression and changes in risk of reactivation of latent TB in individuals co-infected with the protein population at either steady-state or in response to Human Immunodeficiency Virus (HIV).3 The growing global a stimulus does not exist due in part to post-translational burden of TB creates an urgent need to define new classes of control mechanisms.13 Since proteins are the target for most therapeutics effective against MDR strains and with improved drugs, our understanding of drug-related responses at the level sterilizing activity. of the proteome will undoubtedly unravel the important An incomplete understanding of the physiology of myco- dynamics of a drug’s mechanism of action and define new bacteria and the mechanisms associated with drug sensitivity pathways for drug discovery. While our primary interests are has been the greatest impediment toward significant advances in understanding drug related effects in mycobacteria, the in drug-development. Recently the completed genome se- methodology applied in this study is applicable to studying a quences for M. tuberculosis H37Rv and other mycobacterial wide array of adaptive responses. strains have become available.4-6 They have made it possible Traditionally, quantitative proteomics relied on the resolving to apply existing bioinformatic, genomic, and proteomic power of two-dimensional gel electrophoresis coupled with techniques to obtain a better understanding of the pathophysi- mass spectrometry (2DE-MS) for qualitative and quantitative ology of mycobacteria as well as pathways of heightened protein identification. Despite its popularity, the accuracy of sensitivity exploitable for rational drug-discovery efforts. For protein quantitation using this method can be ambiguous due example, comparative genomic studies using DNA microarray to post-translational modifications resulting in multiple spots technology have identified ‘core’ genes of the M. tuberculosis for a single protein or multiple proteins in a single spot, protein complex that could provide highly selective drug targets.7 degradation, presence of protein isoforms and variability in protein recovery from in-gel digests. To compensate for the * To whom correspondence should be addressed. Tel: (410) 516-7444. limitations of 2DE-MS, standardized gel-free methods have Fax: (410) 261-1233. E-mail: † Department of Chemistry, The Johns Hopkins University. been developed. They involve the combination of stable- ‡ Waters Corporation, Milford, MA. isotope labeling during sample preparation coupled with 54 Journal of Proteome Research 2006, 5, 54-63 10.1021/pr050248t CCC: $33.50 © 2006 American Chemical Society Published on Web 12/02/2005
  • 2. Quantitative Proteomic Analysis research articles automated liquid chromatography (LC) and subsequent mass LC-MS data sets were acquired with a Waters CapLC/Waters spectrometry (MS) and/or tandem mass spectrometry (MS/ CapLC autosampler equipped with a Waters NanoEase Atlantis MS). Labels are introduced either by chemical modification, C18, 300 µm × 15 cm reversed-phase column configured onto enzymatic derivatization, or metabolic labeling.14,15 Of these a modified Waters/Micromass Q-TOF Ultima API. Chromatog- techniques, the isotope-coded affinity tag (ICAT) strategy has raphy was performed using an aqueous mobile phase (mobile achieved some popularity.16,17 Although these labeling strategies phase A) containing 1% acetonitrile in water with 0.1% formic have provided an alternative to 2DE-MS, they require several acid and an organic mobile phase (mobile phase B) containing steps for sample preparation. Methods that entail multiple sam- 80% acetonitrile in water with 0.1% formic acid. Peptides were ple preparation steps can lead to an increase in the quantitative loaded onto the column with 6% mobile phase B and eluted variability and decrease the accuracy of the experiment. with a gradient of 6-40% mobile phase B over 100 min at 4.4 In this study, we apply a simple, gel-free, label-free approach µL/min followed by a 10 min rinse with 99% mobile phase B to qualitative and quantitative proteomic analyses using LC- then equilibration with 6% mobile phase B for 20 min. Mass MS.18 In light of well-defined biochemical19-24 and supporting spectrometry was performed using positive mode ESI fitted genomic12,25-29 data, we chose to examine the protein expres- with a NanoLockSpray source. The mass spectrometer was sion profile of M. bovis var. BCG (a member of the M. calibrated with a [Glu-1]-fibrinopeptide (GFP) solution (100 tuberculosis complex) in response to isoniazid (INH) treatment fmol/µL) delivered through the reference sprayer of the as a model system. The choice of INH for this study allows for NanoLockSpray source. The doubly charged ion [(M + 2H)2+] a robust cross-validation of this new LC-MS method to its was used for initial single point calibration (Lteff), and MSE extensive biochemistry of action, and provides a vital compo- fragment ions of GFP were used to obtain the final instrument nent to the comprehensive analysis of the drug’s mechanism calibration. Full scan mass spectra were acquired from 300 to of action. Though widely accepted as a cell wall biosynthesis 2000 m/z at a frequency of 1.8 s with an interscan delay time inhibitor targeting enzymes of the fatty acid synthase (FAS) type of 0.2 s. Accurate mass LC-MS and LC-MSE data were II system, the complete cellular dynamics surrounding INH collected using 10 eV for MS and 28-35 eV for MSE such that toxicity are still unclear and remain a topic of active research. one cycle of MS and MSE data was acquired every 4.0 s.30 Experimental Procedures Peptide Clustering and Data Normalization. Each sample M. bovis BCG Growth Conditions and Protein Expression. was analyzed in triplicate. Data were acquired in a continuous Mycobacterium bovis BCG (Pasteur strain, ATCC 35734) was fashion by alternating low and elevated energies as prescribed grown to mid-log phase OD600 0.4-0.5 in Middlebrook 7H9 me- by the Waters Protein Expression System (PLGS v2.2 build 39, dium supplemented with 10% ADC (0.5% BSA, 0.2% dextrose, Waters Corporation). All ion detections from both the low and 0.085% NaCl), 0.025% Tween 80. One liter cultures were treated elevated energy channels have been de-isotoped and charge- with 0.4 mL diluent (DMSO) or INH (1 µg/mL final) and state reduced. Coincident fragmentation data were assigned incubated at 37 °C. At 6 h post addition, the cells were collected to each detected precursor peptide obtained from the low by centrifugation, washed with PBS (1×), ambic buffer (50 mM energy channel by aligning the ion detections from the elevated NH4HCO3, 5 mM EDTA) (1×) and suspended in lysis buffer [50 energy channel (y-, b-ions, neutral losses, immonium ions) with mM NH4HCO3, 5 mM EDTA, 0.05% RapiGest (Waters Corpora- a retention time tolerance of approximately (0.05 min. Identi- tion, Milford, MA)]. An equal volume of 0.1 mm zirconia/silica cal peptide components from each of the replicate injections beads was added to the bacteria suspension and the cells were for both conditions were clustered together by mass precision disrupted with a Mini Bead-beater 8 (BioSpec Bartlesville, OK). (typically <10 ppm) and a retention time tolerance (typically Protein concentrations were estimated using the One Plus 2D <0.25 min) using the PLGS software. The clustered data set quantitation kit (Amersham Biosciences, Piscataway, NJ). was exported from PLGS and further evaluated with Excel and Pre-fractionation with Ammonium Sulfate. Crude lysates Spotfire. Ion detections with a replication rate of one were of M. bovis BCG containing approximately 50 mg of total considered noise and discarded. The observed intensity mea- protein per condition were fractionated into five portions using surements were adjusted for injection variability within each ammonium sulfate according to established protocols: 0-30%, condition and also for variation in protein load between each 30-40%, 40-45%, 45-50%, and 50-90% cuts. The concentra- condition based on those components which replicated through- tions used to fractionate the proteins were chosen to produce out the entire experiment (6 out of 6 injections). The summed approximately the same size protein pellet. The resulting pellets intensity measurements (within one standard deviation) were were suspended in the minimal amount of buffer for complete used to generate the appropriate scaling factors to adjust for resolubilization (50 mM NH4HCO3, 5 mM EDTA, 1 M urea) and injection variability and protein load, respectively.31 dialyzed overnight against the same buffer. Protein concentra- Protein Identification and Quantitation. Processed ions tions were estimated using the Coomassie Plus Protein Assay were sequenced mapped against the M. bovis database (NCBI: reagent (Pierce, Rockford, Il). NC 002945) using PLGS and MASCOT V1.9 (Matrix Science, Liquid Chromatography and Mass Spectrometry. Aliquots Boston, MA). The PLGS search parameters were defined by the of M. bovis BCG lysates containing approximately 200 µg total software (automatic setting used). Peptides were restricted to protein were reduced in the presence of 5 mM DTT at 60 °C trypsin fragments with up to one missed cleavage and cysteine for 30 min followed by alkylation with 15 mM iodoacetamide carbamidomethylation. The MASCOT search parameters were for 30 min in the dark at room temperature. Proteolytic restricted to tryptic peptides (up to one missed cleavage and digestion was initiated with the addition of modified trypsin cysteine carbamidomethylation) with a mass tolerance of (25 (Sequencing Grade, Promega, Madison, WI) in an equal volume ppm and fragments within a mass tolerance of (0.03 Da. All of ambic buffer to a final concentration of 1:75 (w/w trypsin: protein identifications were manually assessed for fidelity. total protein) and incubated at 37 °C overnight. An equal The relative quantitation was performed using the precursor volume of 50 mM NH4HCO3 was added to reduce the concen- intensity measurements available in the clustered output file. tration of RapiGest to 0.025%. The redundant quantitative measurements provided by the Journal of Proteome Research • Vol. 5, No. 1, 2006 55
  • 3. research articles Hughes et al. Figure 1. LC-MS/MSE analysis of control and INH-treated BCG cultures. (A) LC-MS/MSE analysis of a tryptic digest of soluble proteins from the control (left) and INH-treated (right) taken at a timepoint of 6 h. The base peak intensity chromatograms for the tryptic precursors and associated fragment ions are provided in each of the two acquisition channels [low energy (top, LC-MS) and elevated energy (bottom, LC-MSE)]; (B) An overlaid scatter plot of the monoisotopic mass (MH+) and retention time measurements from the replicates of each condition detected throughout the entire LC-MS/MSE analysis and within a restricted mass and retention time window. multiple tryptic peptides from each protein were used to peptide component lists were assembled into a single matrix determine an average relative fold-change. A 95% confidence such that each peptide component was associated across the interval was determined for each average fold-change from the entire experiment. The ion detections which occurred once out standard deviation of the observed quantitative measurements of the six injections were considered background noise and and the total number of observed tryptic peptides. removed. These discarded components corresponded to 7.8- 13.5% of the total number of detected peptide components, Results but only represent 1.5-3.0% of the total detected intensity for Sample Complexity and Ion Detection. Standard 2-DE was each replicate injection, Table 1. The peptide detection ef- performed on cell-free extracts of control and INH-treated ficiency (defined as those peptide components which replicated cultures to assess the protein population using an MS-friendly at least two out of the three replicate injections for either buffer system and to illustrate how INH treatment affected the sample) was 70%, constituting the majority of detected AMRTs. protein profile of the model bacterium, Figure S1 in Supporting More importantly, these components accounted for 98% of the Information. Tryptic peptides generated for each sample were total detected intensity from each condition. analyzed by LC-MS/MSE in triplicate. Precursor ions and their Data Quality Assessment. Before conducting quantitative associated fragment ions were obtained in a continuous comparisons between the two conditions, the observed inten- fashion, alternating low and elevated energies, and aligned sity measurements were normalized for injection (volume) and based on their chromatographic attributes. The inventory of sample (protein load) variability by multiplying each intensity detected peptide components was converted to a text file con- measurement by the appropriate scaling factor obtained as taining all of the mass spectrometric and retention time data, outlined in the Experimental Procedures. This process was easily importable into external programs for data quality assess- performed for both conditions. The scaling factors used for the ment. Each detected component is referred to as an AMRT replicate injections of the control sample were 1.0243, 1.0005 (accurate-mass, retention time pair). An appreciation for the and 1.0000, respectively. The scaling factors used for the complexity of the sample is readily obtained by visualizing the replicate injections of the INH-treated sample were 1.0083, total number of extracted accurate-mass-retention-time-pairs, 1.0000, and 1.0094, respectively. These results indicate that a (AMRTs) detected across both conditions, Figure 1. maximum deviation of 2.5% was observed during the replicate After obtaining an inventory of the detected peptide com- analysis of these two conditions, which is within the accepted ponents from the replicates of each sample, the individual analytical deviation of the auto injector. The scaling factors 56 Journal of Proteome Research • Vol. 5, No. 1, 2006
  • 4. Quantitative Proteomic Analysis research articles Table 1. Summary of Extracted Peptide Components and Peptide Detection Efficiency AMRT inventorya Control Isoniazid-treatment inj 1 inj 2 inj 3 Cv inj 1 inj 2 inj 3 Cv total AMRTs 7387 7081 7337 2.1% 7192 7101 7145 3.8% s•intensity 4.12 × 107 4.19 × 107 4.25 × 107 1.5% 5.10 × 107 5.06 × 107 5.04 × 107 0.6% Peptide Detection Efficiency rep rate 1 2 3 1 2 3 total AMRTs 3276 1827 5840 3292 1889 5654 f•peptides 29.9% 16.7% 53.4% 30.4% 17.4% 52.2% s•intensity 9.96 × 105 2.53 × 106 4.04× 107 1.12 × 106 2.99 × 106 4.74 × 107 f•intensity 2.3% 5.8% 92.0% 2.2% 5.8% 92.0% a Ion detections filtered for a replication rate of 2-6 across both conditions and intensities have been normalized to account for injection variability within each sample and protein load between both conditions; abbreviations: s•intensity, summed intensity of the peptide components associated with the indicated injection or replication rate; rep rate, number of times an AMRT was detected for each condition; f•peptides, fraction (%) of peptides associated with the indicated replication rate; f•intensity, fraction (%) of intensity associated with the indicated replication rate; Cv, coefficient of variance. used to normalize against the total protein load for the control treated and control samples, Figure 4B. Once the matched and the INH sample were 1.0337 and 1.0000, respectively. peptide components were plotted according to their observed A variety of quality control measures was performed on the relative fold-change, the quantitative comparison of the matched replicates of each condition to determine the reproducibility peptide components provided a useful filter to identify peptide of the analytical method. The mass precision of the extracted components of interest. A set of peptides within a determined peptide components was typically within (5 ppm of the mean fold-change, and within a specified measurement tolerance, mass measurement. These data are illustrated in Figure 2A and as determined by the variability of the analytical method, demonstrate the stability of the mass measurement instru- should originate from a limited subset of the proteins in the mentation. The variability of the quantitative intensity mea- natural proteome. In essence, the filter allows one to perform surements among the replicate injections is summarized in a more restrictive peptide-mass-fingerprint (PMF) search with Figure 2B. These results indicate that the average and median a reduced subset of peptide components. The coupling of the coefficient of variation (Cv) among the replicate injections was relative quantitation along with the PMF search will be referred 11.7% and 8.2%, respectively. Figure 2C illustrates the repro- to as a Q-PMF search (quantitative-peptide-mass-fingerprint) ducibility of the chromatography collected during this study. throughout this manuscript. The average and median Cv was 0.3% and 0.1%, respectively. Three sets of peptide components have been highlighted to These observations are within the typical measurements ob- illustrate the utility of the Q-PMF methodology, Figure 4B. tained from previous reports with the same instrument and There are four red peptide components which exhibit a ln- an earlier version of the software.18 (minimum) and ln(maximum) fold change of 0.80 and 1.04, The intensity variation observed with the analytical method respectively. A total of 204 peptide components lie within this can be appreciated by conducting binary comparisons of the observed fold-change range. When the subset of 204 peptide intensity measurements of the matched peptide components components is submitted for a Q-PMF search against the M. for each replicate injection. Figure 3 illustrates the scatter plot bovis protein database without allowing for any missed cleav- of the resulting three binary comparisons for each condition. ages, the search results indicate that the 4 red peptide Under ideal conditions, the binary comparisons would yield a components match to AcpM (meromycolate extension acyl perfect diagonal line that intersected through zero and would carrier protein), Rv2244, to within a 10 ppm mass tolerance display a minimum degree of deviation throughout the signal and comprise 43% total protein sequence coverage. Each of detection range. The data do illustrate a diagonal line where the four peptide components has been validated with support- the minimum deviation between matched peptide components ing sequence information from the associated fragment ions is seen with peptide components of higher intensity. This plot collected in the alternating elevated energy acquisitions. The also illustrates what one would expect to see if there were no Supporting Information for the IPDEDLAGLR peptide (1098.5795 apparent changes between the two conditions. MH+) can be seen in Figure 5. The four peptide identifications Quantitative Analysis and Protein Identification. Quantita- to AcpM illustrate an average fold-change of 2.41 with a tive analysis was performed using the same set of experiments. standard deviation of 0.27, which is within the established Figure 4A illustrates the binary comparison for the average analytical variability. Eight peptides from a subset of 384 were intensity measurements between control and INH-treated identified with Icl (isocitrate lyase), Rv0467, to within a 10 ppm replicate injections. The average standard deviation associated mass tolerance and comprise 20% total protein sequence with the relative intensity ratio of matched peptide components coverage. Seven peptides from a subset of 563 were identified between the control and INH-treated was approximately 0.53, with Ino1 (myo-inositol-1-phosphate synthase) to within a 10 roughly two-times that observed from internal pairwise com- ppm mass tolerance and comprise 18% total protein sequence parisons of the analytical replicates (Figure 3). The natural coverage. The scatter plot in Figure 4C has converted the logarithm of the ratio of the average intensities from each redundant quantitative peptide information for the 103 char- condition, INH-treated (numerator) versus control (denomina- acterized proteins to summarize the overall effect of the protein tor), was determined and plotted against the average intensity in a single average measurement with the appropriate 95% of the 6699 matched peptide components between the INH- confidence interval as dictated by the number of individual Journal of Proteome Research • Vol. 5, No. 1, 2006 57
  • 5. research articles Hughes et al. relative fold change) g (0.39) was the criteria used for an AMRT to be considered significantly differentially expressed. Validation by Fractionation Enrichment. The results ob- tained from the un-fractionated M. bovis BCG extracts provided a list of protein identifications based on corresponding peptide fragments validated not only by Q-PMF, but also by supporting primary sequence coverage from the data obtained in the elevated energy channel. To further validate those peptide/ protein identifications which contained Q-PMF data without sufficient supporting sequence information, the original protein extracts were fractionated by ammonium sulfate precipitation to produce five protein fractions as outlined in the Experimen- tal Procedures. Each of the five fractions was analyzed by LC- MS/MSE in triplicate and the data processed as previously described. By simplifying the protein mixture and reducing the complexity and dynamic range of the samples, increased peptide coverage was obtained for those protein identifications made with only Q-PMF data. To cross-correlate the processed LC-MS and LC-MSE data from the fractionated samples with the un-fractionated data set, a database containing the mass, retention time, and intensity measurement for each sequence- validated tryptic peptide along with the associated protein identification for the fractionated samples was created (protein ion maps). The ionization efficiencies of the characterized tryptic peptides also allowed us to predict which subset of peptides was expected to ionize at low levels in the un- fractionated fractions. The protein ion maps added additional validation for those peptides/proteins identified in the un- fractionated samples which were not of sufficient abundance to produce adequate sequence information. A subset of peptides is indicated in Table 2 with their corresponding MASCOT scores before and after fractionation to illustrate the utility of the fractionation protocol. The MASCOT search results from the un-fractionated control provided 21proteins with a MASCOT score at or above 50, while the results from the un-fractionated INH-treated sample provided 32 proteins with a MASCOT score at or above 50. After consolidating the search results from the fractioned control sample, a total of 177 nonredundant proteins were identified with a MASCOT score at or above 50; 91 of the 177 protein identifications were obtained with a protein score at or above 100. Similarly, the consolidated MASCOT search results from the fractionated INH-treated samples provided a total of 154 nonredundant protein identifications with a protein score at or above 50; 88 of the 154 protein identifications were obtained with a protein score at or above 100. Figure 2. Assessment of the analytical reproducibility. (A) The Discussion mass precision measurements from the 9903 replicating AMRTs (in at least 2 out of 6 injections) across the entire experiment. In this study, we sought to validate a newly developed LC- The median and average mass errors were (2.9 and (3.4 ppm, MS method capable of simultaneously providing qualitative and respectively; (B) Error distribution associated with the intensity quantitative information to study differential protein expression measurements for the 7543 replicating AMRTs detected in the within the complex mycobacterial proteome following INH INH-treated BCG sample (similar profile observed for the control). treatment. Using an MS-friendly buffer system, the soluble The median and average intensity errors were 11.7% and 8.2%, protein extracts were digested and loaded directly onto an LC respectively. (C) The relative standard deviation of the 9903 column to minimize losses due to sample manipulation, replicating AMRTs detected for the entire experiment. The precipitation and/or fractionation, simplifying the overall median and average retention time error was 0.1% and 0.3%, sample preparation process. Only proteins identified under respectively. conditions of high stringency compiled for at least two out of three parallel analyses and by at least three sequenced peptide peptide measurements. The natural logarithm of the average fragments were considered positive identifications. The low relative fold-change for AcpM, Icl and Ino1 were 0.91, 0.69, and analytical variability associated with the mass and retention 0.38, with a 95% confidence interval of (0.11, (0.07, and (0.05, time measurements of the detected replicating AMRTs sup- respectively. A differential expression of (-0.39) g ln(average ported the efficient clustering of identical peptide components. 58 Journal of Proteome Research • Vol. 5, No. 1, 2006
  • 6. Quantitative Proteomic Analysis research articles Figure 3. Comparison of the log intensity measurements obtained for each matched set of AMRTs from each of the replicate injections for the control: (A) injection 1 vs 2; (B) injection 1 vs 3; (C) injection 2 vs 3. The standard deviations associated with the relative intensity ratios of matched AMRTs were 0.25, 0.27, and 0.26, respectively. Comparison of the log intensity measurements obtained for each matched set of AMRTs from each of the replicated injections for the INH-treated cultures: (D) injection 1 vs 2; (E) injection 1 vs 3; (F) injection 2 vs 3. The standard deviations associated with the relative intensity ratio for the matched AMRTs were 0.27, 0.28, and 0.25, respectively. For those low abundant peptide components incapable of et al.,31 the ion inventory of accurate mass measurements for generating sufficient fragmentation data with the instrumenta- the precursors and associated fragment ions obtained from a tion used in this study, the quantitative comparison allowed single LC-MS/MSE analysis was used to construct a more for a more stringent PMF search for subsequent peptide/ comprehensive list of protein identifications present in the un- protein identifications by restricting the number of AMRTs in fractionated sample. These protein results were used to gener- any given search. The quantitative analysis did not incorporate ate protein ion maps that were compared with the Q-PMF any labeling or enrichment strategy, which is an ideal approach identifications obtained from the un-fractionated experiments to minimize sample variation and allow for multiple (more than in order to validate the identifications with supporting frag- two) conditions to be cross correlated (e.g. time courses, drug mentation data. Though similar to the approach of Smith et concentrations, different antibiotics). al.,31 the method in this study uses a single experiment to Although a PMF protein identification strategy was used for provide the qualitative and quantitative information needed precursor ions of insufficient intensity to produce fragment to assemble the protein ion maps as opposed to performing ions, supporting structural validation was obtained by parti- the analysis on two different instruments. tioning the crude protein mixture into various fractions, thereby A total of 103 proteins were confidently identified from 956 reducing the complexity and dynamic range of the proteins of the total 6699 matched peptide components, 14%, based on within each analytical sample. Using a similar strategy to Smith the stringent search parameters employed. Common modifica- Journal of Proteome Research • Vol. 5, No. 1, 2006 59
  • 7. research articles Hughes et al. Figure 5. LC-MS/MSE identification of AcpM. (A) Illustration of the chromatographic alignment for the precursor and four associated fragment ions to the IPDEDLAGLR peptide to AcpM obtained during the LC-MS/MSE acquisition. Five selected ion chromatograms from the raw continuum data of the precursor and four consecutive y-ions indicate that the apex retention times are within a single scan (0.03 min) of the originating precursor; (B) The time resolved, de-isotoped and charge-state reduced fragmentation data associated with the precursor mass of 1098.5873 (MH+) at 46.00 min. The corresponding y-,b-ions and neutral losses associated with the IPDEDLAGLR peptide of AcpM are highlighted in the MSE spectrum. with Accd6 exhibiting the largest deviation with a ln(I/C) ( 95% CI of 1.41 ( 0.23 (4.1-fold). Previous proteomic analysis using 2DE-MS only identified two of the five proteins involved in Figure 4. Relative quantitation. (A) Binary comparisons of the this operon (AcpM and KasA).21 These enzymes are involved log intensity measurements obtained from the 6699 matched in the synthesis of mycolic acids through a type II FAS system. AMRTs for INH-treatment vs control. (B) The natural log of the Mycolic acids are R-alkyl, β-hydroxy acids comprising anywhere intensities (INH-treated vs control) plotted against the natural log from 30 to 60% dry weight of the cell and can range in length of the replicating ions detected in the INH-treated sample. between C70-C90. Differential mRNA analysis and DNA mi- Colored peptides illustrate the redundancy associated with croarray studies have pointed to this gene cluster as a diag- subsequent protein identifications: red, AcpM (4 peptides, 43% nostic response for drugs exerting a primary affect on fatty acid sequence coverage); blue: Icl (8 peptides, 20% sequence cover- biosynthesis in mycobacteria.12 age); green, Ino1 (7 peptides, 18% sequence coverage). (C) Summarized overall effect for the 103 proteins identified in this Additional proteins involved in fatty acid biosynthesis show- study as a single average measurement with the appropriate 95% ing differential expression include FadD26 and DesA2. FadD26 confidence interval: red, up; green, down; gray, slight to no shows homology to fatty acid CoA ligases and is up-regulated change. 1.5-fold with INH treatment, a property consistent with ge- nomic data.28 DesA2 is a potential desaturase possibly involved tions such as glycosylation, acetylation, methylation, phospho- in the synthesis of mycolic acids. M. tuberculosis encodes for rylation and methionine oxidation were not considered as three potential desaturases DesA1, DesA2, DesA3. Biochemical criteria for the characterized protein identifications. Since a studies have shown that desA3 is involved in the synthesis of total inventory of precursors and fragments was generated, the oleic acid (cis-∆9-C18:1) and is likely not involved in modification remaining AMRTs are available with their corresponding of mycolic acids.33 DesA2 is down-regulated during INH treat- fragmentation data to pursue additional identifications in the ment both at the level of the protein, -1.5 fold, and transcript.28 future. The proteins deemed to show significant differential The known inhibition of mycolic acid synthesis by INH seems expression support the extensively characterized biochemical to suggest a functional role for DesA2 in the biosynthetic effects of mycobacteria following INH exposure, Table 3. pathway. Lipid Metabolism. Four of the five proteins encoded by the Intermediary Metabolism and Respiration. The metabolic kas operon, (FabD-AcpM-KasA-KasB-AccD6), were identified switch isocitrate lyase, Icl, was upregulated following exposure 60 Journal of Proteome Research • Vol. 5, No. 1, 2006
  • 8. Quantitative Proteomic Analysis research articles Table 2. MASCOT Scores for 10 Peptides before and after Ammonium Sulfate Fractionationa INH total Rt peptide protein sample protein peptides Mrb ∆Mrc Rt (Cv) (%) score score expectd sequence F04 GlnA1 9 2101.90 -0.02 56.06 1.0 98 456 6.6 × 10-11 DGAPLMYDETGYAGLSDTAR Crude GlnA1 5 2101.92 -0.01 55.51 10 64 4.5 × 10-2 DGAPLMYDETGYAGLSDTAR F05 FadA 3 1565.74 -0.01 41.51 1.7 35 52 3.4 × 10-4 FCASGLEAVNTAAQK Crude FadA 1 1565.75 0.00 40.79 17 17 2.1 × 10-2 FCASGLEAVNTAAQK F05 Mdh 7 1476.70 -0.02 32.73 -2.1 88 215 1.9 × 10-9 GASSAASAASATIDAAR Crude Mdh 6 1476.71 -0.01 33.44 35 111 5.1 × 10-4 GASSAASAASATIDAAR F01 AtpD 13 2841.42 0.01 82.02 0.7 44 205 3.7 × 10-5 GIFPAVDPLASSSTILDPSVVGDEHYR Crude AtpD 3 2842.42 0.01 81.46 9 44 1.1 × 10-1 GIFPAVDPLASSSTILDPSVVGDEHYR F05 KasA 6 1520.72 -0.01 46.89 1.2 59 167 1.3 × 10-6 IVESYDLMNAGGPR Crude KasA 3 1520.72 -0.01 46.32 19 19 1.6 × 10-2 IVESYDLMNAGGPR F03 DnaK 18 1644.85 -0.02 58.12 1.3 47 443 3.2 × 10-5 IVNEPTAAALAYGLDK Crude DnaK 16 1644.86 -0.01 57.38 11 330 1.3 × 10-1 IVNEPTAAALAYGLDK F03 Tig 8 1799.91 -0.01 64.86 0.3 70 122 1.9 × 10-7 LIAGLDDAVVGLSADESR Crude Tig 4 1799.92 -0.01 64.69 21 37 1.4 × 10-2 LIAGLDDAVVGLSADESR F01 AtpA 9 1884.99 -0.02 68.72 0.4 37 116 3.4 × 10-4 LSDDLGGGSLTGLPIIETK Crude AtpA 2 1884.99 -0.01 68.45 1 19 1.40 LSDDLGGGSLTGLPIIETK F01 MoxR1 14 2243.18 -0.02 84.59 0.3 96 375 3.8 × 10-10 LVLTYDALADEISPEIVINR Crude MoxR1 2 2243.19 -0.02 84.31 38 54 2.4 × 10-4 LVLTYDALADEISPEIVINR F03 RpoA 11 1060.52 -0.02 46.11 0.8 59 438 1.9 × 10-6 TESDLLDIR Crude RpoA 9 1060.53 -0.01 45.72 9 221 2.3 × 10-1 TESDLLDIR a Abbreviations: Rt, retention time b Experimental monoisotopic mass measurement c Difference between experimental and calculated monoisotopic masses d MASCOT expectation value: number of times it is expected to obtain an equal or higher score purely by chance Table 3. Differentially Expressed Proteins with Associated Relative Fold Changea Rv no. protein description pep SC (%) ln(I/C) ( 95% CI fn. cat. Rv0058 DNAB Probable replicative DNA helicase 13 16 0.48 ( 0.02 2 Rv0183 Rv0183 Possible lysophospholipase 7 28 -0.39 ( 0.04 7 Rv0467 ICL Isocitrate lyase 8 20 0.69 ( 0.07 7 Rv0475 HBHA Heparin binding hemagglutinin 4 30 0.53 ( 0.20 3 Rv0788 PURQ Probable phosphoribosylformylglycinamidine 6 35 0.59 ( 0.04 7 synthase I Rv0860 FADB Probable fatty oxidation protein 6 11 0.54 ( 0.06 1 Rv1094 DESA2 Possible acyl-ACP desaturase 4 21 -0.39 ( 0.07 1 Rv1160 MUTT2 Probable mutator protein 3 19 1.90 ( 0.11 2 Rv1437 PGK Probable phosphoglycerate kinase 9 22 0.55 ( 0.07 7 Rv2145c WAG31 Conserved hypothetical protein 4 14 1.31 ( 0.21 3 Rv2244 ACPM Meromycolate acyl carrier protein 4 43 0.91 ( 0.11 1 Rv2245 KASA β-ketoacyl synthase 1 3 23 1.20 ( 0.17 1 Rv2246 KASB β-ketoacyl synthase 2 8 28 0.57 ( 0.14 1 Rv2247 ACCD6 Acetyl CoA carboxylase 4 19 1.41 ( 0.23 1 Rv2405 Rv2405 Conserved hypothetical protein 9 42 0.68 ( 0.02 10 Rv2449c Rv2449c Conserved hypothetical protein 7 22 0.52 ( 0.03 10 Rv2493 Rv2493 Conserved hypothetical protein 3 61 -0.77 ( 0.09 10 Rv2522c Rv2522c Conserved hypothetical protein 3 8 -1.68 ( 0.12 10 Rv2744c Rv2744c Conserved 35 kDa alanine rich protein 14 49 0.43 ( 0.07 10 Rv2930 FADD26 Fatty acid-CoA ligase 9 21 0.39 ( 0.08 1 Rv2981c DDLA Probable D-alanine-D-alanine ligase 3 11 -1.49 ( 0.12 3 Rv3778c Rv3778c Possible aminotransferase 7 20 0.43 ( 0.07 7 Rv3841 BFRB Bacterioferritin 7 37 0.41 ( 0.08 7 Rv3858c GLTD Putative NADH-dependent glutamate synthase 6 20 -0.39 ( 0.06 7 a Abbreviations: Pep, peptides detected; SC, protein sequence coverage; ln(I/C), the natural log of the average relative intensity measurement for associated peptide ions from INH-treatment divided by the average relative intensity measurement for the same ions found in the control; Fn. Cat., functional category (TubercuList): 1, lipid metabolism; 2, information pathways; 3, cell wall and cell processes; 7, intermediary metabolism and respiration; 10, conserved hypotheticals. to INH. Icl is the branch point between the Krebs and glyoxylate M. bovis, a single ORF is encoded with the protein product cycles. Activation of Icl switches on the glyoxylate cycle yielding being 100% identical to AceAb. It is unclear if functional protein succinate and glyoxylate from isocitrate. In M. tuberculosis, icl arises in H37Rv, however in M. tuberculosis CSU93, both is up-regulated in response to phagasomal uptake8 and growth enzymes are active with AceA being the less efficient of the on fatty acids.34 It is required for intracellular survival and two.34 Peptide ions associated with AceA were detected in our persistence in mice.35 Unlike the E. coli system, in which Icl studies (Table S1 in Supporting Information), however, only requires induction based on the available carbon sources, a the isocitrate lyase encoded by icl was differentially expressed. constitutive level of activity exists in mycobacteria.34,36 The This would suggest that the primary isocitrate lyase in M. bovis genome of M. tuberculosis and M. bovis encodes for two BCG, much like CSU93, is Icl with AceA contributing negligible different isocitrate lyases. In M. tuberculosis H37Rv, the gene activity. encoding the other form, termed aceA, has a single-base pair Conflicting reports exist in the literature for the transcrip- overlap resulting in two ORFs (aceAa and aceAb) whereas in tional response of icl following INH treatment. This observation Journal of Proteome Research • Vol. 5, No. 1, 2006 61
  • 9. research articles Hughes et al. addresses a fundamental problem with the vast differences it was previously shown to be up-regulated 2.6-fold by tran- among reported DNA array data sets. These discrepancies are scriptional profiling.25 The effects observed at the protein level in part due to differences in sample preparation, the arrays seems to suggest possible post-translational control mecha- themselves, and statistical analysis methods. The effects ob- nisms and reiterates the need for a more dynamic view of served in this study seem to support the work of Waddle et al. cellular responses to drug-related perturbations. in which the gene icl was found to be affected by INH Since functional roles for unknown proteins are constantly treatment.25 being annotated, an attempt was made to postulate a metabolic Up-regulation of the bacterioferritin protein BfrB was ob- role for the conserved hypothetical proteins identified based served upon INH treatment. This protein is involved in iron on primary sequence homology using the latest NCBI database storage in mycobacteria and the gene has previously been (GenBank, last updated June 15, 2005). No homology with shown to be up-regulated under conditions of excess iron.37 functionally characterized proteins could be determined for Iron is an essential cofactor for the INH activating enzyme Rv2493, Rv2405, or Rv2449c. Rv2522c was homologous to KatG, a catalase-peroxidase. The differential expression of BfrB proteins within the peptidase family (30-57% identity), and seems to suggest a connection between regulation/activity of Rv2744c contained a phage shock protein A (PspA) signature proteins involved in INH activation and iron metabolism. domain. Interestingly, PspA from E. coli is thought to facilitate Although INH has been shown to primarily target the enoyl- the maintenance of the proton motive force and is expressed reductase enzyme, InhA, in the FAS II pathway, the observed in response to a variety of environmental stressors, including IC50 of 7.3 µM20 is much higher than the bactericidal potency inhibitors of lipid biosynthesis.44 (0.2-0.4 µM)38 suggesting additional affects beyond inhibition In conclusion, like transcriptional profiling, a great deal of of InhA.23 A disruption in iron metabolism could, in part, be information is generated by proteome profiling. However, frank mediated through formation of reactive NO species, as sug- targets of drugs are not so obviously identified. The complexity gested by others.39,40 of responses seen by either method is not surprising as the Cell Wall and Cell Processes. Ddla, D-alanine:D-alanine cells cope with imminent death. The connection between the ligase, is an ATP-dependent enzyme involved in peptidoglycan initial insult caused by INH, or any drug, and the sequence of biosynthesis. Peptidoglycan is one of the three major compo- events leading to cell death is an important unanswered nents of the mycobacterial cell envelope. It is covalently linked question where time-dependent or concentration-dependent to arabinogalactan chains, via phosphoryl-N-acetylglucosami- profile effects might afford some insight. For example, Wag31, nosyl-rhamnosyl linkage units, which are in turn esterified to likely involved in cell division, was significantly overexpressed mycolic acids. In our study, this protein was markedly down and presents a possible explanation for the morphological regulated following INH treatment, -4.4-fold. This result is changes associated with the bacterial cell poles following INH consistent with disruptions in the cellular morphology previ- treatment that cannot be explained by inhibition of mycolic ously observed following INH treatment.41 Reduced expression acid synthesis alone. Similarly, D-Ala-D-Ala ligase, a key enzyme levels for Ddla seems to suggest reduced substrate, D-alanine, of bacterial cell wall biosynthesis, declines dramatically on levels which may be the result of inhibiting enzymes associated exposure to INH. This decrease certainly correlates to the lytic with D-alanine synthesis. The drug-induced effect on Ddla events that take place during cell death, but appear remote could also suggest a common regulatory pathway between from the proposed primary action of the drug. Nonetheless, mycolic and peptidoglycan biosynthesis. compelling clues can be seen in the changes in protein Although described as a conserved hypothetical, Wag31 (also expression that correlate to known activities of INH. These data known as antigen 84) is grouped with this functional category take us one crucial step closer to the intimate workings of a because it bears some homology (20-48% identity) to cell living cell carried out by its proteins, where a number of division initiation proteins, DivIVA, from other gram-positive metabolic responses is occurring, but clear among them is bacteria. The gene encoding Wag31, Rv2145c, is essential in elevation of the targeted mycobacterial FAS machinery. It is M. tuberculosis based on Himar1-transposon mutagenesis.42 In noteworthy that this identification was so clearly discernible Bacillus subtillus, overexpression of divIVA is lethal to the cell since only 103 proteins were considered, less than 1% of the and is associated with a filamentous phenotype.43 The protein, theoretical proteome of this organism, but their differential DivIVA, is localized to the bacterial cell poles, the primary site expression separates them from the vast majority of detected for morphological alterations following INH treatment in proteins. mycobacteria.41 The possible involvement of Wag31 in cellular division and its response profile observed following INH- Acknowledgment. M.A.H. and C.A.T. are grateful to the treatment highlights the involvement of non-FAS related National Institutes of Health for partial financial support of this proteins in the mechanisms leading to cell lysis, a known work (U01 AI054842). bactericidal property of INH. Supporting Information Available: 2-DE gel images Conserved Hypotheticals. Conserved hypothetical proteins of the crude soluble protein samples for control and INH- account for more that 25% of the M. tuberculosis proteome. treatment extracted with an MS-friendly buffer system and a These proteins comprise hypothetical proteins of unknown complete list of the 103 proteins identified with the number of functions but are shared between organisms. It is likely that detected peptides and average fold change (95% confidence they may prove to have important functions related to cellular intervals. This material is available free of charge via the homeostasis and require further studies to define their roles Internet at in mycobacteria. Several ORFs in this category respond to INH at the transcript level.12,25,28 In our study, several conserved hypothetical proteins were found to be differentially expressed References as well (Rv2405, Rv2449c, Rv2493, Rv2522c, Rv2744c). 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