Mycobacterium tuberculosis Proteome by ... - ACS Publications

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Characterization of the Mycobacterium tuberculosis Proteome by Liquid Chromatography Mass Spectrometry-based Proteomics Techniques: A Comprehensive Resource for Tuberculosis Research Christina Bell,†,‡ Geoffrey T. Smith,† Michael J. Sweredoski,† and Sonja Hess*,† † ‡

Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, California 91125, United States Institute for Research in Immunology and Cancer, Universite de Montreal, Montreal, Quebec, Canada

bS Supporting Information ABSTRACT: Approximately, one-third of the world’s population is infected with Mycobacterium tuberculosis, the causative agent of Tuberculosis. Secreted and membrane proteins that interact with the host play important roles for the pathogenicity of the bacteria and are potential drug targets or components of vaccines. In this present study, subcellular fractionation in combination with membrane enrichment was used to comprehensively analyze the M. tuberculosis proteome. The proteome of the M. tuberculosis cell wall, membrane, cytosol, lysate, and culture filtrate was defined with a high coverage. Exceptional enrichment for membrane proteins was achieved using wheat germ agglutinin (WGA)-affinity two-phase partitioning, a technique that has to date not yet been exploited for the enrichment of mycobacterial membranes. Overall, 1051 M. tuberculosis protein groups including 183 transmembrane proteins have been identified by LCMS/MS analysis using stringent database search criteria with a minimum of two peptides and an estimated FDR of less than 1%. With many mycobacterial antigens and lipoglycoproteins identified, the results from this study suggest that many of the newly discovered proteins could represent potential candidates mediating hostpathogen interactions. In addition, this data set provides experimental information about protein localization and thus serves as a valuable resource for M. tuberculosis proteome research. KEYWORDS: Mycobacterium tuberculosis, membrane proteins, WGA, enrichment, protein localization, mass spectrometry

’ INTRODUCTION Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the most widespread infectious diseases and thus a significant global health problem (WHO http://www.who.int/mediacentre/factsheets/fs104/en). M. tuberculosis is characterized by a particularly waxy coat that makes it resistant to clearance from the host cells, where it can survive for decades. Membrane and membrane-associated proteins embedded in this waxy coat as well as secreted proteins are known to play pivotal roles in hostpathogen interactions and, therefore, represent potential drug targets and components of vaccines. Membrane proteins are generally known to be notoriously difficult to analyze because of their low abundance, hydrophobicity and poor tryptic digestibility due to the infrequency of lysine and arginine residues. Proteins anchored in or spanning through the membrane of M. tuberculosis are orientated to the outer surface and thus can also be associated with the cell wall. In fact, cell wall and membrane fractions are often cross-contaminated when cells are mechanically broken.1 Furthermore, cross-contamination is not limited to proteins but may include lipoglycans (lipoarabinomannans (LAMs), lipomannans (LMs) and phosphatidylinositol mannosides (PIMs)) r 2011 American Chemical Society

and glycolipids. In previous studies focusing on antigens of Mycobacterium leprae and cell wall components of M. tuberculosis, these contaminants have been successfully removed using partitioning with the nonionic detergent TritonX-114.2,3 Another partitioning system that has been successfully used to enrich glycosylated membrane proteins in mammalian cells but has not been applied to prokaryotes is the wheat-germ agglutinin (WGA) affinity partitioning using polyethylene glycol and WGA activated dextran.4,5 To investigate the M. tuberculosis proteome, a number of proteomics studies were initiated and parts of the proteome have been identified as summarized in Table 1.3,620 For membrane proteins mostly gel-based mass spectrometry techniques were used.6,810,13,1521 These studies were aided by a major breakthrough in tuberculosis research when the M. tuberculosis genome was sequenced in 1998.22 In 2004, Schmidt et al. performed a comprehensive proteome analysis of the M. tuberculosis H37Rv strain.16 Using two-dimensional electrophoresis coupled to mass Special Issue: Microbial and Plant Proteomics Received: August 17, 2011 Published: November 07, 2011 119

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Journal of Proteome Research

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Table 1. Major Proteomics Studies on M. tuberculosis year 1997

title Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional

subcellular

number of proteins

fraction(s)a

identified (FDR)

reference

CF

32 (n.d.)

18

L, CF

107 (n.d.)

10

polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry 1999

Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG

2000

Toward the proteome of Mycobacterium tuberculosis

L, CF

167 (n.d.)

15

2001 2003

The application of proteomics in defining the T cell antigens of Mycobacterium tuberculosis Comprehensive proteomic profiling of the membrane constituents of a Mycobacterium

CF, C M

30 (n.d.) 739 (n.d.)

6

2004

Complementary analysis of the Mycobacterium tuberculosis proteome by two-dimensional

L

361 (n.d.)

16

2005

Mycobacterium tuberculosis functional network analysis by global subcellular protein profiling

CW, M, C

1044 (n.d.)

13

2005

Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by

M

105 (n.d.)

17

M

349 (n.d.)

20

strains: toward functional genomics of microbial pathogens

9

tuberculosis strain electrophoresis and isotope-coded affinity tag technology

proteomics 2005

Identification of Mycobacterium tuberculosis H37Rv integral membrane proteins by one-dimensional gel electrophoresis and liquid chromatography electrospray ionization

2007

Comprehensive analysis of exported proteins from Mycobacterium tuberculosis H37Rv

CF

144 (n.d.)

11

2009

Mycobacterium tuberculosis glycoproteomics based on ConA-lectin affinity capture of

CF

41 (5%, 1 peptide)

8

M

1417 (n.d.b, 1 peptide)

3

CW L

234 (3.5%, 2 peptides) 1668 (n.d.b, 1 peptide)

19

CF, C

101; 137 (n.d.)

14

tandem mass spectrometry

mannosylated proteins 2010

Definition of novel cell envelope associated proteins in Triton X-114 extracts of Mycobacterium tuberculosis H37Rv

2010 2010

Proteomic definition of the cell wall of Mycobacterium tuberculosis Using a label-free proteomics method to identify differentially abundant proteins in closely

2010

Descriptive proteomic analysis shows protein variability between closely related clinical

2011

Comparison of membrane proteins of Mycobacterium tuberculosis H37Rv and H37Ra strains

M

1578 (n.d.b, 1 peptide)

12

2011

Characterization of the Mycobacterium tuberculosis proteome by liquid chromatography mass

CW. C, M, L, CF

1051 (1%; 2 peptides)

This study

7

related hypo- and hypervirulent clinical Mycobacterium tuberculosis Beijing isolates isolates of Mycobacterium tuberculosis

spectrometry-based proteomics techniques: a comprehensive resource for Tuberculosis research

CW = cell wall, C = Cytosol, M = membrane, L = lysate, CF = culture filtrate, FDR = false discovery rate, n.d. = not determined. b False positive rate, false positive probability = false positives/(false positives + false negatives) reported at 0.25%. a

spectrometry they were able to identify 108 proteins. The isotope-coded affinity tag (ICAT) reagent tag method and tandem mass spectrometry yielded 280 M. tuberculosis proteins.16 In the same year, Mawuenyega et al. reported the identification of 1044 proteins from the cell wall, cytosol and membrane fraction of M. tuberculosis.13 In this early study, a low resolution ion trap was used and no information about the false discovery rate (FDR) was provided. Because of its importance in hostpathogen protein interaction, several proteomic studies investigated the membrane component of M. tuberculosis.9,17,20 In 2003, Gu et al. reported the identification of 739 proteins in the membrane fraction of M. tuberculosis H37Rv strain, but the 79 identified membrane proteins were underrepresented.9 A higher enrichment for M. tuberculosis membrane proteins was later achieved using highly concentrated urea and a high pH carbonate solution wash with GeLCMS/MS resulting in the identification of 349 M. tuberculosis proteins out of which 100 were integral membrane proteins.20 Recently, de Souza et al. performed a differential GeLCMS/MS analysis of hypo- and hypervirulent clinical M. tuberculosis Beijing isolates and identified 101 differentially

expressed proteins out of a total of 1668 proteins, to our knowledge the highest number of identified proteins in a proteomic study of M. tuberculosis H37Rv so far.7 A study comparing the protein variability of different M. tuberculosis strains by Mehaffy et al. identified and quantified 101 and 137 secreted and cytosolic proteins, respectively.14 Recently, a study from Wolfe et al.19 as well as three studies from Malen et al.3,11,12 have helped to deepen our knowledge about the M. tuberculosis cell envelope and exported proteins. In an attempt to maximize the number of identifications, proteins detected with just one peptide were accepted in the studies from Malen et al.3,11,12 However, despite these advances, our understanding of the M. tuberculosis proteome, its subcellular and membrane composition, is still incomplete. In this study, we used a variety of methods to study M. tuberculosis proteins by liquid chromatographymass spectrometry. Subcellular fractionation in combination with WGAaffinity based membrane enrichment yielded comprehensive coverage of the M. tuberculosis proteome. M. tuberculosis preparations fractionated into cell wall, membrane, cytosol, lysate and culture filtrate were digested with trypsin or trypsin and chymotrypsin. Overall, 1051 unique M. tuberculosis protein groups 120

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including 183 transmembrane proteins have been identified from 100 to 400 μg M. tuberculosis preparations using stringent database search criteria and a minimum of two peptides per protein group with an estimated FDR of less than 1%. This data set also provides experimental information about protein localization and thus serves as a valuable resource for M. tuberculosis research. A comparison with previously published data sets also revealed that 26 proteins have been identified for the first time and have not been described previously in any M. tuberculosis proteomics publication. Although most previous studies on M. tuberculosis relied on trypsin digestions only, we found a combination of trypsin and chymotrypsin digestion to be complementary to specifically increase the identification of M. tuberculosis membrane proteins.

’ EXPERIMENTAL PROCEDURES

alkylated by addition of 10 mM iodoacetamide and incubated for 15 min at RT in the dark. Proteolysis was initiated with 0.1 μg endoproteinase Lys-C and allowed to proceed overnight at 37 °C in the dark. The sample was diluted to a final concentration of 2 M urea by the addition of 100 mM TRIS 3 HCl, pH 8.5 and adjusted to 1 mM CaCl2. Next, 0.5 μg of sequencing grade trypsin or 0.25 μg of sequencing grade trypsin and 0.25 μg of chymotrypsin were added to the mixture. The mixture was then incubated overnight (ca. 18 h) at 37 °C in the dark. This step was repeated to complete the digestion. The digestion was quenched by the addition of formic acid to a final concentration of 5%. The digested peptides were desalted with a C8 peptide macrotrap (Michrom Bioresources, Auburn, CA) on an Alliance 2795 (Waters, Milford, MA). The collected material was then lyophilized and resuspended in 0.2% formic acid (Sigma, St. Louis, MO).

Chemicals and Reagents

Mass Spectrometry Analysis

Water, methanol, acetonitrile (Chromasolv LCMS quality), trifluoroacetic acid (99+%), formic acid (99%), 2-aminoethanethiol, Dextran-T500, dithiothreitol (DTT), iodoacetamide, lithium sulfate, Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), urea and tresyl chloride were supplied by Sigma-Aldrich, St. Louis, MO. Ammonium acetate, ammonium bicarbonate, ammonium hydroxide, calcium chloride and sodium acetate were from Mallinckrodt, Hazelwood, MO. PEG3350 was supplied by Hampton Research, Aliso Viejo, CA. Sodium periodate was supplied by Thermo Scientific, San Jose, CA. Endoproteinase Lys-C and chymotrypsin were from Roche, Indianapolis, IN. Trypsin (modified sequencing grade) was supplied by Promega, Madison, WI. All other chemicals and reagents were of the highest purity available. M. tuberculosis H37Rv fractions were provided by Colorado State University, Denver, CO (http// www.cvmbs.colostate.edu/mip/tb/) and CFP (05.CS.93.1.12.5. CFP) was obtained from the Biodefense and Emerging Infections Research Resource Repository (Manassas, VA).

For the M. tuberculosis fractions the peptide mixtures were separated using a triphasic microcapillary column modified from the original Multidimensional Protein Identification Technology (MudPIT) method described by Wolters et al.24 and Washburn et al.25 A fused silica capillary with an inner diameter of 100 μm (PolyMicro Technology, Phoenix, AZ) and a 5 μm diameter tip, pulled with a P-2000 capillary puller (Sutter Instruments, Novato, CA) was packed with 6.57 cm 5 μm LUNA C18 reversed phase material (Phenomenex, Torrance, CA) at 500 psi, 3.54 cm 5 μm PolySULFOETHYL A strong cation exchange (SCX; The Nest Group, Southborough, MA) and another 2.53 cm 5 μm LUNA C18 (in this order from the tip) at 750 psi. The peptide solution (10 μL, about 1 mg/mL peptide concentration) was pressure loaded onto the column at 750 psi. The sample loaded column was placed in line between a CapLC (Waters, Milford, MA) and an LTQ-FT electrospray hybrid mass spectrometer (ThermoElectron, Palo Alto, CA). Sample separation was achieved with a twelve step (12 h) chromatography program. Solutions used were 0.2% formic acid in 2% acetonitrile and 98% water (buffer A) and 0.2% formic acid in 2% water and 98% acetonitrile (buffer B). Step one consisted of a 40 min gradient to 95% B within 35 min and then 95% A for the last five min. The following chromatography steps were one hour each. After 10 min of 95% A, the gradient was stepped up to 70% B at 40 min, raised to 95% B after 50 min and then changed back to 95% A for the last 10 min of each step. From step two to twelve the salt concentration was increased gradually using 50, 100, 200, 300, 450, 600, 750, 1000, 2000, 5000, 10000 mM ammonium acetate. Eluting peptides were electrosprayed into the mass spectrometer with a distally applied spray voltage of 2.4 kV. The column eluate was continuously analyzed during the entire twelve step chromatography program. One full range mass scan (4001600 m/z) was run at a resolution of 50000. CID was performed in the LTQ at 35% collision energy and an Activation Q of 0.25. The ion trap-LTQ was run in centroided mode with wideband activation. Data dependent MS/MS scans for the 5 most intense ions at a minimal threshold of 5000 were performed. An exclusion list of 500 was enabled with an exclusion duration of 60s. Charge state screening as well as monoisotopic precursor selection was enabled. Unassigned charge states and charge states greater than four were rejected. For the CFP sample, approximately 2.5 μg of the dissolved digest was loaded and run on a Proxeon EASY-nLC

Preparation of WGA-Dextran

For activation of dextran, all solvents were dried using molecular sieves; all glass materials were dried in an oven (∼120 °C). Dextran-T500 was dissolved in water and then freeze-dried. Prior to use, dextran was dried again in vacuum, activated with tresyl chloride and coupled to WGA as described previously.4,23 Affinity Two-phase Partitioning

Glycosylated proteins together with other membrane proteins were enriched based on affinity purification in an aqueous twophase system consisting of the polymers polyethylene glycol (PEG) and dextran as recently described by Schindler et al.5 Protein pellets obtained through affinity two-phase partitioning were resolubilized in 8 M urea and digested as described in Proteolytic digestion. Because of the appearance of an interphase during the two-phase partitioning steps, the interphases were either enriched by an additional affinity two-phase partitioning step or directly digested and analyzed by mass spectrometry. Proteolytic Digestion

Protein samples were proteolytically digested in solution as follows: lyophilized protein samples (1020 μg) were resolubilized in 40 μL of 8 M urea, 100 mM TRIS 3 HCl, pH 8.5 and reduced by incubation with a final concentration of 3 mM TCEP in LCMS water for 20 min at RT. Reduced cysteines were 121



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(ThermoFisher, Waltham MA) with a 75 μm ID silica capillary column packed in house with 15 cm of C18 Aqua, 3 μm, 120 Å (Dr. Maisch, Ammerbuch-Entringen, Germany) coupled online to an LTQ-Orbitrap Classic. Runs were 160 min each with a gradient of 040% buffer B (80% acetonitrile in 0.2% formic acid). The LTQ Orbitrap was operated in a data dependent mode with a full scan MS performed in the Orbitrap and a dependent scan MS/MS in the LTQ. Ions from m/z = 400 to 1600 were surveyed. For the full scan the Automated Gain Control (AGC) injection target was set at 500000 with a maximum injection time of 700 ms. Charge state screening and rejection were enabled so that charge states of the precursor ion g+2 were accepted and FT master scan preview mode was enabled. For the dependent scan the AGC target was 5,000 ions with a maximum injection time of 150 ms. Injection waveforms were enabled for both. For scan event 1, the 10 most intense peaks were selected for fragmentation in the subsequent scan events. Dynamic exclusion was also enabled for the maximum list size of 500 for a duration of 90 s. Three technical replicates were run for each sample to ensure reproducibility. Data Analysis

Tandem mass spectra were converted to mgf files using ReAdW4Mascot2 (http://peptide.nist.gov/metrics/). All MS/ MS samples were analyzed using Mascot (MatrixScience, London, U.K.; version 2.2.06). A target sequence database was constructed consisting of the M. tuberculosis proteome (3952 entries) and common contaminants (262 entries). In addition, a target/decoy database was created for estimating the false discovery rate (FDR) by reversing each sequence and appending it to the sequence database. Either trypsin ([KR]∧P) alone or trypsin/chymotrypsin ([KRFLYW]∧P) was set as the digestion enzyme(s), respectively. Up to two missed cleavages were allowed. Mass tolerances were set to 10 ppm for parent ions and 0.50 Da for fragment ions. Carbamidomethylation of cysteine was specified in Mascot as a fixed modification. Oxidation of methionine and carbamylation of peptide N-terminus were specified as variable modifications. Mascot search results were further processed by ProteoIQ (NuSep, Bogart, GA, version 2.2) to curate a set of peptides and proteins at a 1% FDR. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Transmembrane domains (TMDs) were predicted with the TMHMM2.0 algorithm (http://www.cbs.dtu.dk/ services/TMHMM/).26,27 Cytoscape (www.cytoscape.org) was used to visualize the overlap between the different WGA samples.28

Figure 1. Experimental overview. M. tuberculosis fractions (whole cell lysate, cell wall, cytosol, culture filtrate and membrane fraction) were reduced alkylated and either double (LysC/trypsin) or triple digested (LysC/trypsin/chymotrypsin). A portion of the membrane fraction was additionally subjected to WGA-affinity two-phase partitioning. All samples were then analyzed by LCMS/MS.

WGA-affinity two-phase partitioning, was applied to the membrane fraction to enrich for membrane proteins. Overall, 1051 protein groups were identified with a minimum of two unique peptides at an FDR of less than 1% on the peptide and protein level. Table S1 (Supporting Information) lists the protein groups, their probabilities, their localization, predicted TMDs, the used digestion and enrichment techniques and whether a protein has previously been identified in any of the major proteomics studies focusing on M. tuberculosis. All original raw files can be downloaded from Tranche and all peptide identifications are available at http://pel.caltech.edu/data/Mtb. The 26 proteins that have been identified for the first time and have not been described previously in any M. tuberculosis proteomics publication are listed in Table S2 (Supporting Information). TMD Proteins and Digestion Methods

Tryptic digestions are commonly used in most proteomics studies. Our own experience with transmembrane proteins30,31 as well as a recently published proteomic analysis suggested that an additional chymotryptic digestion may achieve higher proteome and sequence coverage when compared to trypsin digestion alone.32 To test this on the mycobacterial proteome, LysC and trypsin double digestion were compared to LysC, trypsin and chymotrypsin triple digestion (Table 2). Venn diagrams in Figure 2 illustrate that chymotrypsin indeed increased the protein coverage. When analyzed for all proteins containing either zero, one or two and more TMDs, it is apparent that the highest contribution is achieved for the proteins with two and more TMDs. This is easily explainable by the fact that proteins with TMDs have hydrophobic sequences to pass through the hydrophobic membrane, making them often inaccessible to LysC/tryptic digestion.31,32 In contrast, chymotrypsin, which

’ RESULTS AND DISCUSSION In this study the M. tuberculosis proteome was globally analyzed by complementary mass spectrometry-based proteomics techniques. Figure 1 shows an overview of the experiments conducted in this study. Initially, the entire M. tuberculosis proteome was surveyed by analyzing the following fractions: whole cell lysate, cell wall, cytosol, culture filtrate and membrane using a combination of LysC/trypsin double digestion and LysC/trypsin/chymotrypsin triple digestion. We then focused on an in-depth characterization of proteins of the cell envelope. It was recently noted that the cell envelope of M. tuberculosis is understudied. 29 As membrane proteins are often extensively glycosylated, a glycosylation enrichment technique, 122



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Table 2. Overview of Identified Proteinsa M. tuberculosis subcellular fraction and digestion method CW Try

C Chy

Try b

M Chy

Try b

M-WGA Chy

Chy b

L Try

CF Chy

Try b

Protein ID

533

404 (350)

400

371 (312)

381

207 (190)

299

516

395 (346)

TMD

85

56

14

12

62

33

56

51

37

77

%TMD

16

14

4

3

16

16

19

10

9

17

422

CW = cell wall, C = cytosol, M = membrane, M-WGA = membrane WGA interphase, L = lysate, CF = culture filtrate, Try = trypsin double digestion, Chy = chymotrypsin triple digestion. b Number in brackets shows those proteins that were common in both trypsin and chymotrypsin digestion. a

preferentially cleaves at hydrophobic amino acids (tryptophan, tyrosine, phenylalanine, leucine and methionine), is able to digest these hydrophobic sequences, allowing the identification of substantially more proteins with two and more TMDs. As shown here, this is of particular advantage for proteins containing longer hydrophobic sequence portions (i.e., membrane proteins containing 2 and more TMDs), while proteins with more common distributed lysines and arginines yield shorter tryptic peptides that might be overdigested upon addition of an additional enzyme. Out of the 183 identified TMD containing membrane proteins, 97 were identified in the cell wall fraction with up to 15 TMDs indicating a previously under-appreciated abundance of tightly associated M. tuberculosis proteins in the cell wall. Overall, 23% of the TMHMM 2.0 predicted membrane proteome (801 proteins) has been identified in the present study. As expected, a large number of TMD containing proteins were also found in culture filtrate and membrane fractions and the cytosol contained the fewest TMD containing proteins (Table 2, Figure 3).

abundance membrane proteins an affinity enrichment step was added. For the affinity two-phase system WGA was coupled to dextran. Glycosylated membrane proteins bind to WGA and are thus pulled into the WGA-dextran phase and then released by an excess of N-acteyl-D-glucosamine. Using this procedure, potentially glycosylated proteins were enriched at the protein level before being digested into peptides using LysC, trypsin and chymotrypsin. Effectively, 304 proteins were identified after WGA-affinity capture including 56 TMD proteins (Table S3, Supporting Information). To ensure comprehensiveness, top phases and stable interphases were analyzed separately. Furthermore, the interphase was analyzed with and without an additional WGA affinity step. Figure 4 visualizes the protein identifications in the top phase versus those in the interphases, both with and without the additional affinity step. In the top phase, 107 proteins were identified, out of which 21 were predicted to contain at least one TMD. In the interphase without the additional affinity step, 270 proteins were identified with 42 proteins predicted to contain at least one TMD. The interphase that was subjected to an additional WGA-affinity step yielded 218 protein identifications, out of which 45 proteins were predicted to contain at least one TMD. There was a considerable overlap of proteins that have been identified in the top and the interphases. It is also apparent from Figure 4 that more proteins were identified in the interphases than in the top phase. Due to their amphiphilic nature, many membrane proteins were highly enriched in the interphase, between the more hydrophilic bottom and the hydrophobic top phase. Interestingly, the proteins identified in the top phase were almost entirely a subset of the proteins identified in the interphases, with 94% of the proteins identified in the top phase also being identified in both interphases, while only a third of the proteins identified in the interphases were also identified in the top phase. Common to top phase and both interphases were 100 proteins. The likelihood of protein identification increased due to the partitioning regardless whether an additional WGA affinity step was performed or not. In this study the lectin affinity matrix WGA was used to capture putative glycoproteins with the ultimate goal of identifying cell surface proteins in M. tuberculosis. It is known that C-type lectins exist as immune receptors and Pattern Recognition Receptors (PRRs) on the surface of dendritic cells, macrophages and soluble components of the innate immune system of the host. Mycobacterial cell envelope components associate with these lectins to initiate the phagocytosis event and mediate host cell responses. Mannose receptor (MR), dendritic cell-specific ICAM3 grabbing nonintegrin (DC-SIGN), and surfactant proteinA and D (SP-A and SP-D; surfactant proteins of the lung) play an important role in mycobacterial infection.36 One strategy

WGA-affinity Two-phase Partitioning

Since low abundance membrane proteins are often extensively glycosylated, an enrichment technique based on the properties of the glycoforms has been used. As illustrated in Figure 1, WGAaffinity two-phase partitioning has been conducted using the membrane fraction. Previous proteomic studies focusing on the analysis of glycosylated proteins mostly used Concanavalin A (ConA) affinity enrichment based on the known abundance of mannose residues on the M. tuberculosis surface exposed proteins.8,33 In our preliminary studies, we found WGA to be particularly effective using mycobacterial membrane fractions.34 In contrast to glycosylated proteins, enzymatically deglycosylated membrane proteins were not binding to WGA, indicating high selectivity of the WGA procedure. In contrast to ConA, which preferentially binds to mannose residues, WGA preferentially binds to N-acetylglucosamines and sialic acids, which are known to be present in the cell envelope.35 The enrichment of mycobacterial membrane proteins based on this property has not been exploited to date. Using WGA-affinity two-phase partitioning, membrane proteins were enriched by conventional twophase partitioning prior to the affinity enrichment step. We established an aqueous two-phase system formed by the two polymers polyethylene glycol (PEG) and dextran. According to their charge and hydrophobicity, membrane proteins of M. tuberculosis partitioned into the PEG phase. This partitioning resulted in exceptional enrichment for membrane proteins, presumably due to the elimination of cross-contaminations including LAMs, LMs and PIMs. To further enrich for the low 123



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Figure 3. Contributions of trypsin double and chymotrypsin triple digestion to (A) all protein and (B) transmembrane protein identifications per cellular subfraction.

Figure 4. Graphical representation of proteins identified after WGAtwo phase partitioning with and without additional affinity step. Each node represents an identified protein. Proteins containing no TMD are shown as dots, proteins containing one or more TMDs are shown as squares.

Figure 2. Venn diagrams of (A) all proteins, proteins containing (B) 0, (C) 1 and (D) 2 or more TMDs detected after trypsin double (dark gray) and chymotrypsin triple (light gray) digestion.

only identified in one of the three replicates (Table S3A, Supporting Information). While some of these proteins may be copurified, others are potentially glycosylated. Regardless of their potential glycosylation status, they may play an important role in M. tuberculosis virulence. When the spectral counts of the proteins identified in the membrane fraction were compared with those in the WGA affinity fraction, proteins listed in Table 3 were significantly enriched in the WGA affinity fraction. Notably, one of the two characterized mycobacterial glycoproteins, the probable

that we pursued for the global analysis of membrane proteins was thus to enrich for proteins that may interact with lectins. This generated a database of 218 candidate proteins (Table S3A, Supporting Information) that were identified using WGA-affinity capture including the highest percentage of TMD proteins (45 proteins). The vast majority (166) of the WGA-affinity captured proteins were identified in all three replicates and only 6 were 124



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Table 3. Spectral Count of Peptides from the Membrane Fraction vs. Membrane WGA-Affinity Fractiona GI number

a

protein name

SC Mem

SC M-WGA

P-value

% M-WGA SC

gi|15610041

50S ribosomal protein L19

7

35

0.833

0.001415

gi|15607861

30S ribosomal protein S5

38

65

0.631

2.29 106

gi|15607844

50S ribosomal protein L2

40

87

0.685

2.32 106

gi|15610937

acyl-CoA synthetase

24

43

0.642

0.005506

gi|15610025

amidase

0

8

1

0.002802

gi|15608964

Conserved hypothetical protein CFP17

0

7

1

0.005842

gi|15607807

DNA-directed RNA polymerase subunit beta

7

26

0.788

0.000289

gi|15607808 gi|15608444

DNA-directed RNA polymerase subunit beta’ F0F1 ATP synthase subunit A

27 4

50 18

0.649 0.818

0.002001 0.001185

gi|15608448

F0F1 ATP synthase subunit alpha

27

46

0.63

0.006899

gi|15608334

hypothetical protein Rv1194c

1

11

0.917

0.002079

gi|15609435

hypothetical protein Rv2298

10

28

0.737

0.001147

gi|15609586


1

10

0.909

0.004

gi|15610266


1

13

0.929

0.000552

gi|15608415

Possible Lipoprotein LPRC

0

7

1

0.005842

gi|15607431 gi|15610180

Probable conserved transmembrane protein Probable cytochrome C oxidase polypeptide I CTAD

18 12

48 41

0.727 0.774

3.85 105 1.17 105

gi|15608109

Probable metal cation transporter P-type ATPase CTPV

72

108

0.6

0.000786

gi|15609719

Probable peptidyl prolyl cistrans isomerase B PPIB

1

10

0.909

0.004

gi|15607573

Probable periplasmic superoxide dismutase [CuZn] SODC

9

35

0.795

1.73 105

gi|15609337

Probable transmembrane cytochrome C oxidase (subunit II) CTAC

21

40

0.656

0.004198

gi|15609333

Probable Ubiquinol-cytochrome C reductase QcrB

14

32

0.696

0.002506

gi|15607572

Putative tuberculin related peptide

3

16

0.842

0.001265

gi|15608531

S-Adenosylmethionine synthetase

3

14

0.824

0.003946

SC = spectral count, M = membrane, M-WGA = membrane WGA-affinity.

periplasmic superoxide dismutase SODC,37 was highly significantly enriched in the WGA affinity fraction. In earlier studies, SODC was shown to be membrane-associated38 and be present in the cell envelope39 as well as in the CFP, where it was found to act as an antigen.29 Our results are in accordance with these findings including a more detailed subcellular localization study showing SODC to be dominant in the membrane and cell wall fractions and at lower concentrations in the CFP (Table S1, Supporting Information).37 The probable periplasmic superoxide dismutase SODC has recently been shown to be a glycosylated lipoprotein with B-cell antigen activity.37,40 Also highly significantly enriched were 50S ribosomal proteins L2 and L19. Ribosomal proteins in S. cerevisiae have previously been reported to be glycosylated using ConA and WGA affinity purification.41 In addition, ribosome-UDP-N-acetyl-glucosamine complexes have been shown to be involved in protein glycosylation.42 No attempts were made to determine whether the ribosomal proteins identified after WGA affinity were glycosylated or part of a ribosome-UDP-N-acetylglucosamine complex. Either way, they are likely involved in some glycosylation events. Currently, glycosylation sites are known for only two mycobacterial proteins (SODC and alanine and proline rich secreted protein Apa). Table 3 provides significantly WGA-affinity enriched glycoprotein candidates that warrant further studies. Even beyond the highly significantly enriched proteins, there are additional proteins of interest. A closer investigation of the proteins that have primarily been identified in WGA affinity purification reveals that some probable/possible conserved integral membranes are exclusively (Rv2120C) or highly enriched (Rv2199c) in the WGA affinity fraction. Protein Rv2199c

is predicted to have 3 TMD and has not been identified in any previous proteomics studies. Similarly, 8 TMD containing phosphate-transport integral membrane ABC transporter PSTC1 was only detected after two-phase partitioning, with and without additional WGA affinity step. Probable Lipase LipE has been identified in the cell wall and after two-phase partitioning of the membranes, but not when membranes were analyzed directly, nor in any other fraction (Supplementary Table S1, Supporting Information). The fatty acid synthetase FAS is apparently more prominent in the cell wall, where it has been identified with 40 chymotryptic peptides, than in the membrane, where it has been identified with 4 chymotryptic peptides. When this is compared to the 3 chymotryptic peptides that have been identified after the WGA affinity step, FAS is de-enriched in the WGA affinity step indicating that FAS is more likely copurified and unlikely specifically interacting with WGA. Nevertheless, FAS is an important mycobacterial drug target. Two of the currently used mycobacterial drugs are inhibiting fatty acid synthesis either directly (pyrazinamide) or indirectly (isoniazide).43,44 Similar to previous findings by Malen et al., a number of lipid metabolizing and catabolizing enzymes were found in the membrane.11 Lipids in the cell envelope fulfill two important functions for M. tuberculosis. First, they mediate immune suppression and second, M. tuberculosis actively acquires lipids from the host as nutrients.45 To efficiently make use of the host-derived lipids and resist host defense, lipid catabolizing and metabolizing enzymes are needed in the cell envelope. They may be further exploited as effective mycobacterial drug targets. Identification of M. tuberculosis Protein Antigens

Besides the candidate proteins identified through WGAaffinity, Mycobacteria generally have an extraordinarily large 125



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amount of antigens when compared to other pathogens and minor protein constituents can be presented to host cells under circumstances that favor antigenicity. The combined data sets of all approaches performed in this study confirmed the presence of several previously described M. tuberculosis antigens that bear “antigen” in their name by proteomics analyses (Table S4, Supporting Information). For example, the alanine and proline rich secreted protein Apa (45 kDa antigen) was preferentially detected in the CFP, in line with previous reports.46 This protein was initially suggested to be glycosylated based on a ConA assay33 and characterized in 1995 to be O-glycosylated by mannose residues.47 Apa was identified as a potential adhesion protein to colonize target cells.48 Interestingly, Apa antigen showed a T-cell dependent immune response, most likely mediated through macrophage mannose receptors and/or dendritic cell receptors.49,50 Deglycosylation of Apa reduced this immune response indicating the importance of the glycosylation for the M. tuberculosis-host interactions.49,50 More recently, glycosylated Apa was found to bind to the mannose-specific C-lectin DC-SIGN.51 Furthermore, it also directly interacts with the pulmonary surfactant protein SP-A and has been proposed to interact with the pathogen recognition receptors of the host.48 In addition, the antigens of the Antigen 85 Complex (Ag85-A, Ag85-B (also called α-antigen) and Ag85-C) and the related MPT51 protein have been detected: While Ag85A-C are mycolyl transferases essential for the biosynthesis of mycobacterial cell wall component α,α0 -trehalose mycolate,52 MPT51 was characterized as a noncatalytic α/ß hydrolase.53 The mycolyl transferases Ag85AC and the noncatalytic hydrolase MPT51 can bind the extracellular matrix protein fibronectin, which has been associated with mycobacterial recognition and tissue colonization.54,55 Several other immunogenic proteins such as secreted L-alanine dehydrogenase ALD, secreted proline rich protein MTC28, secreted protein antigen (RV3312), MPT63, MPT64, soluble secreted MPT53 precursor, CFP2 and CFP29, low molecular weight antigen TB8.4, lipoprotein LpqH have also been detected in this study. LpqH is also known to bind to DC-SIGN.51

SECRETORY ANTIGENIC TARGET, ESAT-6) and ESXB (10 KDA CULTURE FILTRATE ANTIGEN, LHP, CFP10) (Table S5, Supporting Information). Like Ag85A, ESAT-6 and CFP10 stimulate human T cells.5860 ESXH (low molecular weight protein antigen 7, CFP-7), and a number of ESAT-6 like proteins (ESXG, ESXJ, ESXL, ESXN, ESXO) were also found. EsxG and EsxH belong to the ESX-3 gene cluster and are believed to be involved in metal ion homeostasis61,62 and cell growth.63 PE-/PPE-Family Proteins

Closely related to the ESX secretion system are the PE and PPE proteins, named after their N-terminal Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs. PE-/PPE-family proteins are acidic proteins with a high glycine and alanine content and are characterized by repetitive sequences. Consequently, there is a relative paucity of tryptic cleavage sites in PE-/PPE family proteins, making them particularly difficult to detect in global proteomics approaches. Thus, it is not surprising that only a few PE and PPE proteins have been determined by global mass spectrometry approaches so far. Many PE and PPE genes are upstream of ESX loci. Recently, it has been shown that PPE proteins are secreted through the ESX secretion system.64 Later reports showed that ESX-5 suppresses the cytokine secretion via toll-like receptor (TLR) signaling cascades.65 Three PPE-family proteins and three PE-family proteins were identified in this study. Table 4 shows clearly that all PPE family proteins were best identified in the cell wall fractions after trypsin double digestion, followed by chymotrypsin triple digestion. Although one would generally expect difficulties in identifying cell wall proteins because of the waxy characteristics of the cell wall, the denaturation of the proteins with urea facilitated their analysis. It is, in fact, quite conceivable that it is easier to extract membrane and membrane-associated proteins from the waxy components of the cell wall than to extract them from the amphiphilic membranes, where they may be more tightly associated. In accordance with this consideration only a few single peptides were identified in the membrane either directly using trypsin digestion or in the interphase of a WGA affinity two-phase partitioning enrichment using chymotrypsin prior to mass spectrometry analysis. In contrast, all PE/PPEfamily proteins found in the cell wall were identified without any enrichment. This is in accordance with previous reports where PE-/PPE-proteins have been described to be localized in the cell wall and cell membrane of M. tuberculosis.66 For comprehensiveness, we also investigated the lysate and culture filtrate and

ESAT-6 Secretion System

A secretion system that is essential for full virulence of M. tuberculosis is the early secreted antigen 6 kDa (ESAT-6) secretion system1 (ESX-1).56 In the M. tuberculosis proteome four additional ESX paralogs comprise 23 proteins.22,57 In the present study, 8 proteins of the 23 could be identified, including the components of ESX-1, namely ESXA (6 KDA EARLY Table 4. PE and PPE Family Proteinsa

M. tuberculosis subcellular fraction and digestion method CW sequence name

GI number

M

Try

Chy

PE

57116857

5

1

PE

57116840

2

4

PE

57117111

2

PPE

57116841

5

1

PPE

57116714

2

4

PPE

57116858

2

1

M-WGA

Try

Chy

L Try

CF Chy

1

Try 2

3 1

1 1 1

1

1

2

a Shown are the numbers of peptides identified in the different preparations. CW = cell wall, M = membrane, M-WGA = membrane WGA interphase, L = lysate, CF = culture filtrate, Try = trypsin double digestion, Chy = chymotrypsin triple digestion.

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filtrate and 13 peptides in the membrane, including 9 peptides in the WGA interphase and 7 peptides in the WGA interphase affinity fractions, indicating a specific enrichment of this lipoglycoprotein in the membrane fraction. Additional lipoproteins that were enriched in the membrane fraction include the SODC, lipoproteins LprA, LprG, LppX, and the periplasmic phosphate binding lipoprotein PSTS1 and PSTS2. Interestingly, Lipoproteins LprA, LprG, LpqH and PSTS1 are toll-like receptor 2 (TLR2) agonists.74,75 In general, signaling through TLR2 leads to host immunity, but M. tuberculosis effectively hijacks this immune response through inhibition of the expression of host macrophage MHC class II molecules after prolonged exposure.76 LprG and LprA have previously been determined to be cell wall-associated.69,77 In this study, we detected both LprG and LprA mostly in the membrane, culture filtrate and cell wall. Lipoprotein LppX, enriched in CFP and membranes, is involved in transporting the mycobacterial lipids phthiocerol dimycocerosates to the outer membrane.78,79 Mutation of the LppX gene leads to an attenuated strain stressing the importance of LppX for virulence.78 Similarly, other lipoproteins such as lpqT and lprG are essential for optimal growth.71

some PE/PPE family proteins could also be identified in these preparations (Table 4). One of the PPE family proteins (57116852) and one of the PE family proteins (57116840) have been identified for the first time in a mass spectrometry-based proteomics study. Heat Shock Proteins

In M. tuberculosis heat shock proteins have been found to have antigenic potential.67 Heat shock proteins are expressed in increased amounts under stressful culture conditions such as high temperature. In this study, six heat shock proteins have been identified. Under these, the highly conserved chaperonins GroEL and GroES, DnaK, probable DnaJ1 and DnaJ2, and the heat shock protein 90 have been found (Table S6, Supporting Information). In addition, heat shock protein F84.1, Hsp and HspX (also known as α-Crystallin homologue) have been detected. Of these, GroEL, GroES, DnaK, HspX have been previously shown to have antigenic properties.67,68 In addition, like other antigens, DnaK has been characterized as a novel ligand of DC-SIGN.69 M. tuberculosis Lipoproteins

Lipoproteins of M. tuberculosis are an important class of cell envelope proteins that are anchored in the membrane via posttranslational modification of a cysteine in the “lipobox” following a type II signal peptide.70,71 This signal peptide directs the proteins to the so-called “pseudoperiplasmic” compartment, an equivalent of the periplasmic compartment in Gram-negative bacteria. This pseudoperiplasmic compartment is characterized by the peptidoglycan-arabinogalactan-mycolic acid wall skeleton, in which the mycolic acids form a lipid permeability barrier. Concanavalin binding assays using an M. smegmatis expression system support the bioinformatic analysis that many lipoproteins are also glycosylated.40 This was further corroborated by individual studies focusing on single proteins. For instance, the 19 kDa LpqH lipoprotein was also characterized as glycoprotein by ConA binding and glycosylation has been shown to be responsible for humoral and cellular immune responses.72,73 Given its location at the mycobacterial cell surface, lipoproteins can be expected to be virulence factors, either by directly being involved in cell adhesion and invasion or indirectly using enzymatic and signaling processes that enable growth of M. tuberculosis in the host. In a comprehensive bioinformatic analysis, Sutcliffe et al. identified 98 lipoprotein of the R37Rv strain, categorized into proven and probable, possible, and anomalous groups.70 When combining the results of this study, 42 lipoproteins of diverse functionalities were detected (Table S7, Supporting Information). As expected from bioinformatics analyses,40,70 of the 42 lipoproteins identified in this study, most (33 lipoproteins) were detected in the culture filtrate indicating that they are secreted either by shedding (release of acylated lipoproteins) or shaving (proteolytic cleavage). Twenty-four of the proteins identified in the culture filtrate were also found in other fractions, predominantly cell wall and membrane (Table S7, Supporting Information). Our data set thus provides a detailed map of protein localization. For instance, lipoproteins LpqL, LppH, LppK, LppO, LprQ, ProX, Subl, thioredoxin and the conserved hypothetical threonine rich protein have been identified exclusively in the CFP. In contrast, lipoprotein GlnH, LppD, LpqD, LpqG, LprB, LprC, Mce1E and OppA have not been identified in the CFP. Other proteins, like the 19 kDa lipoprotein LpqH, a known glycoprotein, was identified with 2 peptides in the culture

Newly Identified M. tuberculosis Proteins

Most of the newly identified proteins are hypothetical, possible, probable or putative and have not been assigned to a specific function (Table S2, Supporting Information). A number of proteins are either membrane or secreted proteins. Interestingly, the previously mentioned virulence factors EsxH, PE, PPE and LprQ proteins were among the newly identified proteins. In addition, PapA5, a phthiocerol dimycocerosyl (DIM) transferase, was found for the first time in a global proteomics approach.80 Together with LppX, PapA5 is involved in translocating DIMs to the outer membrane.78,80 Disruption of the papA5 or lppX gene leads to attenuated strains, stressing the importance of DIMs in the outer membrane for M. tuberculosis virulence.

’ CONCLUSION The initial M. tuberculosis-host interactions play an important role for the microbe’s virulence and ability to evade the host’s immune response. While our understanding of some of these interactions has increased during the past decade, our knowledge is far from complete. Proteomics offers the unique possibility to enhance this knowledge, initially by cataloging the proteins present. From this knowledge, new hypotheses can be formed and tested in subsequent studies. Toward this larger goal, this study provides a comprehensive resource of M. tuberculosis proteome including the subcellular localization of over 1000 proteins. The results from this study suggest that many proteins could represent potential candidates mediating virulence. Furthermore, the results achieved in this work could aid in the design of new antigens for vaccines. This can ultimately result in the development of improved strategies for mycobacterial disease control and prevention through vaccination and immuno-diagnosis. ’ ASSOCIATED CONTENT

bS

Supporting Information Supplementary File 1 contains Table S1 and lists the protein groups, their probabilities, their localization, predicted TMDs, the used digestion and enrichment techniques and whether a protein has previously been identified in any of the major

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proteomics studies focusing on M. tuberculosis. Supplementary File 2 contains Table S2 (Newly identified proteins), S3 (WGA all proteins; i.e. proteins identified from all WGA approaches), S3A (Glycoprotein candidates; i.e. proteins identified from WGA-affinity approach), S4 (Antigens), S5 (ESAT-6 secretion system), S6 (Heat shock proteins), S7 (Lipoproteins).The raw data associated with this manuscript is available at the ProteomeCommons.org Web site (https://proteomecommons.org/ tranche/) using the following hash: IgePczukEULAEB7dCds78PV2hxToWbGeIROwt+NbbGoDHjoadQ4SaL8yjAJ2DNm+q Lbgq9iPRFJKL6kI12ixQV8MEikAAAAAAAE8+g== (passcode: SoMcCl9 cmL757hkGFdEz). This material is available free of charge via the Internet at http://pubs.acs.org.

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’ AUTHOR INFORMATION Corresponding Author

*Sonja Hess, 1200 E California Blvd, Pasadena, CA 91125, Tel. (1)626-395-2339, Fax. (1) 626-449-4159, e-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by the Betty and Gordon Moore Foundation and the Beckman Institute. We thank Colorado State University, Denver, CO (http//www.cvmbs.colostate.edu/ mip/tb/) and the Biodefense and Emerging Infections Research Resource Repository (Manassas, VA) for providing M. tuberculosis H37Rv fractions. ’ ABBREVIATIONS #, number; 2-DE, 2-dimensional electrophoresis; AGC, automatic gain control; Apa, alanine and proline rich secreted protein; CFP, culture filtrate protein; ConA, concanavalin A; DCSIGN, dendritic cell-specific ICAM3 grabbing nonintegrin; FAS, fatty acid synthase; FDR, false discovery rate; ICAT, isotopecoded affinity tag; LAMs, lipoarabinomannans; LCMS, liquid chromatographymass spectrometry; LMs, lipomannans; MS, mass spectrometry; MR, mannose receptor; M. tuberculosis, Mycobacterium tuberculosis; MudPIT, Multidimensional Protein Identification Technology; PEG, polyethylene glycol; PSTC1, phosphate-transport integral membrane ABC transporter; PIMs, phosphatidylinositol mannosides; PRRs, pattern recognition receptors; SODC, probable periplasmic superoxide dismutase; SP-A, surfactant protein-A; SP-D, surfactant protein-D; TB, tuberculosis; TLR, toll-like receptor; TMD, transmembrane domain; WGA, wheat germ agglutinin ’ REFERENCES (1) Barnes, P. F.; Mehra, V.; Hirschfield, G. R.; Fong, S. J.; Abou-Zeid, C.; Rook, G. A.; Hunter, S. W.; Brennan, P. J.; Modlin, R. L. Characterization of T cell antigens associated with the cell wall protein-peptidoglycan complex of Mycobacterium tuberculosis. J. Immunol. 1989, 143 (8), 2656–62. (2) Hunter, S. W.; Rivoire, B.; Mehra, V.; Bloom, B. R.; Brennan, P. J. The major native proteins of the leprosy bacillus. J. Biol. Chem. 1990, 265 (24), 14065–8. (3) Malen, H.; Pathak, S.; Softeland, T.; de Souza, G. A.; Wiker, H. G. Definition of novel cell envelope associated proteins in Triton X-114 extracts of Mycobacterium tuberculosis H37Rv. BMC Microbiol. 2010, 10, 132. 128



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