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Prospective on Mycobacterium tuberculosis Proteomics M. Carolina Mehaffy, Nicole A. Kruh-Garcia, and Karen M. Dobos* Department of Microbiology, Immunology and Pathology, Colorado State University, 1619 Campus Delivery, Fort Collins, Colorado 80523, United States ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis, remains one of the most prevalent human pathogens in the world. Knowledge regarding the bacilli’s physiology as well as its mechanisms of virulence, immunogenicity, and pathogenesis has increased greatly in the last three decades. However, the function of about one-quarter of the Mtb coding genome and the precise activity and protein networks of most of the Mtb proteins are still unknown. Protein mass spectrometry and a new interest in research toward the field of functional proteomics have given a new light to the study of this bacillus and will be the focus of this review. We will also discuss new perspectives in the proteomics field, in particular targeted mass spectrometry methods and their potential applications in TB research and discovery. KEYWORDS: Mycobacterium tuberculosis, functional proteomics, targeted proteomics, multiple reaction monitoring, MRM
’ INTRODUCTION Mycobacterium tuberculosis (Mtb), the causal agent of tuberculosis (TB), infects one-third of the world population, and its global incidence is increasing approximately 1% per year with a projection of 8 million new cases every year.1 In 2009 alone, the World Health Organization (WHO) estimated 9.4 million new cases of TB, and although mortality rates showed a slight decrease, 1.7 million lives were lost due to TB in 2009.2 While the majority of people infected with Mtb will not develop the disease, about 10% will progress to active tuberculosis after a latent interval that can vary from weeks to decades.3 There is also a whole spectrum of clinical presentations, from subclinical to rapidly fatal. While this variation is only partially understood, it is certain that it depends on a complex interplay between environmental, bacterial, and host characteristics.4 Mycobacterial lipids have been extensively studied in the context of virulence and infection.510 While some individual Mtb proteins have been identified to be important during infection,1115 the Mtb proteome remains largely uncharacterized in terms of virulence and pathogenesis. In the past two decades, there has been an increase in the number of descriptive studies on the Mtb proteome. Gel-based proteomics, where protein fractions from different Mtb strains are analyzed by two-dimensional gel electrophoresis (2D-GE), provided the first insights toward the proteome of the bacilli.1622 r 2011 American Chemical Society
These original studies opened the field of proteomics in TB research and provided useful annotation of proteins and 2D-GE maps. However, it was evident that 2D-GE alone was not able to provide all the answers to the questions investigators were looking for. Shotgun proteomics using liquid chromatography coupled to tandem mass spectrometry (LCMS/MS), both labeled and label-free, provided a more ample application, and the number of proteins identified in a single experiment increased considerably. Shotgun strategies when complemented with 2D-GE provided the most complete analysis of Mtb proteins.23 In addition, shotgun proteomics, as opposed to gel-based methods, can be more easily applied to characterization of membrane and cell wall proteomes that, due to their intrinsic insoluble characteristics, are difficult to resolve using 2DGE.24 This application has resulted in extensive descriptions of cytosolic and cell wall proteomes,20,24,25 whole cell lysates,19,26 and membranes.25,2730 Reviews of these descriptive studies and their relevance are provided elsewhere31,32 and will not be the focus of this article. Special Issue: Microbial and Plant Proteomics Received: September 1, 2011 Published: October 11, 2011 17
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abundance and localization in Mtb. However, hypotheses-driven studies that address specific questions about the patho-physiology of the bacilli in the context of hostpathogen interactions have started to move the Mtb proteomics field a step ahead toward “functional proteomics”. This activity-based approach represents, in our opinion, the future of Mtb proteomics and will be the main focus of this review. Finally, perspectives on Mtb proteomics in the years to come, with particular interest in the quantification of specific Mtb proteins in complex mixtures using state of the art instrumentation and techniques, will be discussed.
In addition to these descriptive studies, some proteomics analyses focused on the identification of proteins expressed under conditions mimicking the host environment. These studies contribute to our current knowledge of the mechanisms of infection and provide important information that can be utilized in the context of vaccine and immunodiagnostic development. Proteins such as the immunogenic proteins Mpt32 (a.k.a Apa, ModD), Mpt64 (Rv1980c), and a protein similar to the trigger factor (Tig) were found to be less abundant during starvation, suggesting that their expression during natural infection might be low. In contrast, two hypothetical proteins, Rv2557 and Rv2558, in addition to the heat shock protein, HspX, were found to be present in higher abundance under nutrient-starvation conditions.33 Other proteomic approaches have also shown HspX to be predominantly present during hypoxic conditions,34,35 in aging cultures,36 in standing versus shaking cultures,37 and during intracellular growth,38 all of which suggest that this protein has an essential role during latency. Similar to HspX, other proteins have been shown to be higher under low oxygen conditions, including L-alanine dehydrogenase (Ald), the molecular chaperone GroEL2, probable fructosebiphosphate aldolase (Fba), bacterioferritin (BfrB) and elongation factor EF-Tu (Tuf).34,39 Interestingly, these proteins have also been identified as differentially present when compared between the virulent strain, Mtb H37Rv and the vaccine strain, M. bovis BCG (Bacillus Calmette-Guerin),17,18,38 emphasizing their potential role in the pathogenesis of Mtb. Several other proteins showing differential levels between different stages of nonreplicating persistent Mtb have also been identified using an isotope-coded affinity tags (ICAT) approach. The great majority of the differential proteins belong to the small molecule metabolism category, especially proteins involved in energy metabolism and degradation.40 Studies of in vivo expression of mycobacterial proteins are considerably scarce; however they are very relevant since they represent a more realistic view of what occurs during natural infection. Metabolic labeling of Mtb and infection of THP-1 cells showed the differential expression of 44 proteins, from which 16 were more abundant and 28 were repressed after infection.41 In another study, proteins induced upon phagocytosis of M. bovis BCG by THP-1 cells included the popular protein HspX, and othere molecular chaperones GroEL1, GroEl2 and the Elongation factor EF-Tu (Tuf).38 Several enzymes involved in cell metabolism such as cystathionine (beta)-synthase (CysM2), GMP synthase (GuaA), malate dehydrogenase (Mdh) and phosphoglycerate mutase I (Gpm), appear to be unique to intracellularly growing Mtb, and may represent essential pathways required by Mtb for intracellular survival.42 In a recent study, our laboratory performed the first characterization of the proteome of Mtb during in vivo infections using the guinea pig model. Individual protein levels varied throughout the course of the infection, supporting the fact that Mtb has different requirements depending on the disease stage. However, several proteins, such as those involved in nitrogen assimilation and cation transport were found to be present during the entire infection. In addition, we were able to determine the 10 most abundant Mtb proteins during the first 30 days postinfection. This information provides the first step toward understanding the actual physiological status of the bacilli in the host and sets the basis for development of novel diagnostic targets and drug designs.43 All of these studies have set the current state of proteomic analysis for Mtb while increasing our understanding of protein
’ FUNCTIONAL PROTEOMICS IN MTB In contrast to descriptive and basic expression-profiling proteomics in which global proteome analysis is performed to identify increases or decreases in protein abundance by comparing two or more samples, functional proteomics focuses on the characterization of protein activities, complexes, and signaling pathways.5658 Tandem MS/MS is generally the preferred method used to identify proteins as part of a functional proteomic workflow. In particular, nanoflow liquid chromatographymass spectrometry (LCMS) applications provide higher sensitivity and low solvent and sample consumption when compared with traditional LCMS.44 The capability of using a low sample concentration is particularly advantageous in the field of human pathogens, decreasing the amount of initial material such as cells, culture filtrate, or infected tissue needed for analysis. After separation of peptide mixtures by nano-LC, peptides are analyzed in the mass spectrometer and identification is performed by matching the measured fragment ion spectra of peptides with theoretical spectra calculated from known DNA or protein sequences.45 Tandem mass spectrometry is based on the retention of precursor ions (i.e., peptides), ionization, fragmentation, and analysis of spectra resulting in information about each peptide at the amino acid level.46,47 Functional and activity based proteomic studies are only emerging as tools to define and annotate Mtb proteins. However, the few studies that have been performed in this area confirm the potential for proteomics to fully characterize Mtb proteins that are relevant to the physiology and pathogenesis of the bacilli.48,5964,70 Identifying Protein Function and Complexes
More than 25% of the Mtb genome is still annotated as conserved hypothetical proteins without known function65 (Figure 1). As functional proteomic studies become more widespread, the annotation of function and activity of Mtb proteins, including conserved hypotheticals and putative proteins, will be improved and our understanding of their role in the Mtb physiology and interactions with the host will increase. For instance, Chavadi and colleages (2009) used 2D-HPLC followed by LCMS analysis using a liquid chromatography quadrupole (LCQ) instrument to understand the implications in the signaling pathway of Mtb metabolism of null mutations in ald (alanine dehydrogenase) and pykA (pyruvate kinase) using isogenic strains of both M. bovis and Mtb.48 PykA is the major enzyme responsible for the production of pyruvate during glycolysis while Ald catalyzes the reversible conversion of pyruvate and ammonia into L-alanine. Therefore, it is expected that null mutations in either of these two enzymes may have an impact on downstream pathways requiring pyruvate, such as fatty acid and amino acid biosynthesis. In this study, loss of PykA 18
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Figure 1. Distribution of functional categories in the Mtb genome (information obtained from Tuberculist, ver 2.3 Release 22 (http://tuberculist.epfl.ch/)). The red pie piece corresponds to conserved hypothetical proteins (Category 10), which contains more than 25% of the encoding genes in Mtb.
filtrates of H37RvΔRD1, explaining the lack of recognition of EspC in BCG vaccinated controls. These findings are extremely important, not only in the context of the physiology of the bacilli, by showing that proteins outside of the Esx-1 cluster may also be part of this secretion system, but also demonstrating that diagnostic specificity can be dependent on antigen secretion, which in turn has relevant implications for vaccine and diagnostics development.
resulted in down-regulation of ald, suggesting a discrete relationship between the activity of these two enzymes. Their proteomics findings also showed that loss of PykA resulted in upregulation of fatty acid catabolism and downregulation of fatty acid biosynthesis in Mtb, and further analysis of fatty acid metabolism in the mutants and wild type confirmed that upon loss of PykA, Mtb metabolism is geared principally to using fatty acids for energy production, while decreasing synthesis of Isocitrate lyase (Icl) and increasing β-oxidation. These findings contribute to our understanding on the regulation and networking of metabolic pathways in Mtb. Mechanisms of immunogenicity can also be addressed using functional proteomics approaches. For example, Millington and colleagues59 studied the immune responses to the Esx-1 substrate protein C (EspC). EspC is a small protein similar to the 10KDa Culture filtrate antigen (Cfp10) and the 6KDa Early secretory antigenic target (Esat-6), both of which are two of the most immunodominant proteins in Mtb.66 These proteins are also exported by the type VII secretion system, known as Esx-1 in Mtb.67,68 Given the similarities of EspC to Esat-6 and Cfp10, the authors hypothesized that EspC could also be an important T-cell antigen. As expected, they found EspC to be as immunodominant as Esat-6 and Cfp10 in both active and latent TB patients. BCG vaccinated controls, on the other hand, failed to generate a T-cell response toward EspC. BCG is divergent from Mtb in several ways. Notably, it is known to contain a deletion (region of difference 1, RD1) that results in loss of the Cfp10 and Esat6 homologues, along with the Esx1 machinery.69 The authors used a quantitative proteomics analysis by metabolic labeling of H37Rv wild type and compared the differential expression of labeled culture filtrate proteins to H37RvΔRD1 (unlabeled) CFP. After off-line fractionation of the samples, peptides were analyzed in a Linear Trap Quadrupole-Furier Transform (LTQFT) mass spectrometer and the effects resulting from the lack of RD1 (region of difference 1) in the secretion of EspC and other Esx-1 dependent proteins were studied. This quantitative proteomic approach revealed that EspC, as well as Esat-6, Cfp10, and Esx-1 dependent proteins EspA and EspB, were absent in the
Characterization of Modified Proteins
Perhaps one of the most important, and yet challenging, subjects in the field of functional proteomics is the high throughput identification and characterization of protein modifications. Thanks to new developments in mass spectrometry instrumentation and proteomic data analysis, the characterization of post-translational modifications such as phosphorylation and protein turnover are now possible. Global identification of Mtb phosphorylated events under several different conditions was addressed by Prisic and colleages.70 Following phosphopeptide enrichment, more than 150 samples were analyzed by liquid chromatographytandem mass spectrometry (LCMS/MS) in a LTQ mass spectrometer. Protein identification with an emphasis on detection of phosphorylation sites was performed using two independent algorithms, ProteinPilot (Applied Biosystems) and Mascot (Matrix Science), and for those hits for which multiple phosphorylation sites were possible, data was further analyzed using the Ascore program.71 Using this strategy, the authors were able to identify more than 500 phosphorylation sites, mapping to 301 Mtb proteins. Proteomics findings were confirmed by in vitro phosphorylation assays, which not only provided additional evidence of phosphorylation but also resulted in the identification of a group of peptides that are specifically phosphorylated by only one Mtb serine/threonine protein kinase (STPK) as opposed to multiple STPKs, suggesting specific roles for these kinases in regulation of Mtb cell processes. This study adds to our understanding of the regulation of enzymatic activity by post-translational modification such as phosphorylation and opens the door 19
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to further investigate its role in virulence and pathogenesis of Mtb. Another key post-translational modification that has gained recognition in the past decade is protein cleavage by proteases. Proteolysis affects protein structure, function, life span, and localization. It also regulates intracellular and extracellular signal transduction and may function as a switch to turn on and off the activity of other enzymes in the cell.72 Due to new attention that proteases have received in the past few years, a novel field of study denoted degradomics has been formed. Degradomics seeks to characterize proteases, inhibitors, and protease substrates present in an organism.73,74 This new field is finally making its entrance in the study of mycobacterial physiology and a few, but relevant, studies have been done, providing insights into protease function and substrates in mycobacteria. PepD is member of the HtrA-like serine protease family. This protein family has been shown to be involved in stress responses.75 Using a coimmunoprecipitation (co-IP) assay coupled to identification by LCMS/MS in a high performance linear trap quadrupole mass spectrometer (LTQ XL), White and colleagues identified several proteins that interact with epitope-tagged PepDS317A (a variant with a lower rate of autocatalysis) and that may in turn be involved in the stress response mediated by this protease.60 Four potential PepD substrates were identified from the pool of interacting proteins by incubation with active PepD followed by LCMS/MS analysis, and a model for the regulation by PepD of one of its identified substrates (Rv2744c) under stress conditions was also proposed. The gene encoding Rv2744c had been previously shown to be up-regulated at high temperatures,76 which together with the findings by White and colleagues suggests that protein abundance and function under stress conditions is regulated at both gene expression and post-translational levels. In a different study, substrates targeted to the proteasome machinery by the recently described modification of pupylation were identified using two complementary proteomic approaches.63 Pupylation involves the binding of a small protein termed Pup (Prokaryotic Ubiquitin-Like Protein) to substrates targeted to degradation by the proteasome.62 In the study by Poulsen and colleagues, pull-down assays using Pup expressed in M. smegmatis were performed. Co-eluting proteins were labeled using isobaric tags for relative and absolute quantification (iTRAQ)52 and then identified by 2D-HPLC followed by tandem MS/MS using a quadrupole-time-of-flight (Q-TOF) mass spectrometer, which, due to the high stability of ions generated by collision-induced dissociation (CID) and the capability to amplify low mass fragments in MS/MS mode, improves the signal strength of iTRAQ reporter ions49,50,54,55 and makes this instrument the gold standard for analysis of isobaric labeled peptides using the iTRAQ system.5153 The authors also used 2D-GE as a complement to the shotgun strategy. Using this approach, 41 pupylation targets were identified, adding significantly to previous studies in which only three potential pupylation substrates had been identified.61,62,77 The distribution of pupylation targets within the functional categories of the Mtb genome showed to be significantly higher for proteins grouped in the intermediary metabolism and regulation category. In addition, it appears that proteins that belong to the same gene cluster may also be targeted together for pupylation. Both of these observations suggest that pupylatyion and the subsequent degradation of its targets by the proteasome may be associated with specific cell processes in Mtb.
Given the role of proteases in the proper functioning of the cell, it is not surprising that they play a role in the virulence and pathogenicity of important human pathogens.7882 In a recent study, proteomics and other “omics” techniques were applied to study the regulation of chaperone and protease systems by the regulator ClgR, with a concentration in the importance of this regulatory cross-talk and virulence of Mtb.64 Protein expression profiles of Mtb wild type and ΔclgR were obtained by onedimensional (1D) gel electrophoresis followed by LCMS/MS. Interestingly, Rv2744c, which as mentioned above was identified as a PepD substrate,60 was also differentially identified between wild type and ΔclgR mutant. Other chaperones and proteases were also recognized as being under the regulation of ClgR. Furthermore, the ΔclgR mutant was unable to grow intracellularly in macrophages, suggesting proteases regulated by ClgR may be important for the survival of Mtb in the host. Nonpathogenic Mycobacteria
Functional proteomics approaches have also been applied to other mycobacteria species, particularly in the field of environmental mycobacteria capable of biodegradation of fuel and other toxic compounds. For instance, 1D gel electrophoresis, coupled to nano-LCMS/MS and supported by other “omics” technologies findings, was used to determine the metabolic network associated to polycyclic aromatic hydrocarbons (PCH) in the environmental species Mycobacterium vanbaalenii PYR-1.83 The authors were able to identify more than 100 proteins that presented consistent overexpression after treatment with several different PCHs. Similarly, a nano-LCMS/MS method was also applied to study the enzymes involved in 2-ethylhexyl nitrate (2-EHN) biodegradation in M. autroafricanum IFP 2173.84 In that study, several enzymes that are most likely involved in the degradation of 2-EHN were identified and could have implications for industrial use. Findings from all of these functional studies not only increase our understanding of Mtb physiology but also provide information than can be used for diagnostics and therapeutic purposes. For example, the impact of loss of PykA in Mtb metabolic enzymes, such as the down-regulation of isocitrate lyase,48 which in turn is responsible for the glyoxylate shunt and required for Mtb persistence in mice85 and in vivo growth,86 may be used for rational drug design to specifically target TB latency and persistence. The immunodominance of EspC and the proteomics findings related to its mechanism of secretion59 reflect the important correlation of understanding mycobacterial physiology and the development of diagnostics tools. For instance, lack of EspC secretion in mycobacterial strains lacking the Esx-1 cluster, such as BCG, highlight the potential use of EspC to diagnose TB in BCG vaccinated patients. Finally, findings related to the intricate mechanisms of posttranslational modifications and their role in the physiology of the mycobacteria clearly state the significance of functional proteomics to understand and describe these pathways.60,62,64 Phosphorylation and degradation are important protein modifications that can impact several cellular processes and, as shown by the results described above, can also have the potential to be involved in hostpathogen interactions. Given the fact that Mtb has more than 50 genes encoding potential proteases,87 most of which are still uncharacterized, we believe that functional proteomics studies in the new field of degradomics will increase in the years to come. 20
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Figure 2. MRM detection and quantification of two Mtb peptides from infected lung tissue. (Top) (A) MS/MS spectrum for Rv2244 (AcpM) peptide TVGDVVAYIQK, with y6 and y9 fragment ions chosen as transitions ion for quantification (circled in red). (B) MS/MS spectrum for Rv2244 (AcpM) peptide IPDEDLAGLR, with y8 and y9 fragment ions chosen as transitions (circled in red). (Bottom) SRM data represented as total ion chromatograms (TICs) for the two identified transitions of each peptide.
In summary, all of these studies demonstrate the potential of functional proteomics in Mtb, by answering important physiological questions and describing new network complexes with implications in Mtb metabolism, protein regulation, and secretion.
reliable and precise results regarding the absolute quantity of one or several proteins in highly complex biochemical matrixes. MRM assays (also known as selected reaction monitoring (SRM)) are usually performed in triple quadrupole instruments (QqQ),8890 which are considered one of the most sensitive and precise mass spectrometers.88 When operated in MRM mode, the QqQ is set to filter specific precursor ions in the first quadrupole (Q1) and the selected ion is then induced to fragment in a pressurized collision cell (Q2). Instead of scanning the full MS/MS of all possible fragment ions, specific transition ions are filtered and analyzed in the last quadropole (Q3).91 This strategy results in the selection and quantification of specific peptide targets even in very complex samples with high abundance of nontargeted proteins. In addition, the use of labeled internal standards, such as synthetic labeled peptides, offers the possibility of absolute quantification and normalization in a technique termed (SID)-MRM-MS.9295 Further information regarding MRM applied to proteomics can be found in several recent reviews.9698 Protein/peptide based MRM has been successfully used to quantify cancer biomarkers in human plasma93,94,99 and is slowly making its entrance to the infectious diseases field.100 In Mtb, the application of MRM has great potential to validate and obtain absolute quantification of proteins that have been found to be expressed under conditions that mimic the host environment3336,101 or during intracellular growth.38,41,42
’ TARGETED PROTEOMICS FOR THE STUDY OF THE MTB PROTEOME Descriptive and functional proteomics have the capability to generate large volumes of data, and depending on the approach used, relative quantification, in addition to protein identification, can also be obtained. However, data analyses of global protein expression profiles are time-consuming and require a variety of computing resources. In addition, quantitative values provided by this type of analysis need to be validated by downstream techniques, such as ELISA, and Western blot targeting individual proteins. While these immune-based techniques can provide more precise quantification, they require monoclonal antibodies, method development for new proteins is extensive, and multiplexing is limited. In many cases, the target proteins are present in low amounts and are masked by high quantities of nontargeted proteins complicating its downstream validation. It is in this context that targeted proteomics, such as multiple reaction monitoring (MRM), becomes an essential tool, not only to corroborate findings from global proteomic analysis but also to generate robust and rapid assays that can be used routinely, providing 21
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’ REFERENCES
As mentioned in the introduction, our laboratory was able to characterize the Mtb proteome in vivo, and several Mtb proteins that are predominantly present in infected guinea pig tissue, as well as proteins demonstrating relative abundance changes during the course of infection, were identified.43 These studies not only offer important understanding of the biology of the bacilli in vivo but are also the foundation for the recognition of important biomarker targets and therefore need to be validated. Given the limitations of immune-based techniques, we have explored the use of MRM to obtain quantification of target proteins directly from infected lung tissue (Figure 2). Using a Xevo TQ-S mass spectrometer (Waters), we have also begun to develop assays to identify, quantify, and differentiate important Mtb antigens that bear a high level of homology, such as the Ag85 complex, in complex biological samples. As a proof of principle, we spiked guinea pig lung tissue with recombinant Ag85A and Ag85B and established limits of detection (LOD) of 0.01 and 1 fmol for each protein, respectively.
(1) WHO Global Tuberculosis Control: Surveillance Planning, Financing Geneva; World Health Organization: Switzerland, 2005. (2) WHO Global tuberculosis control: WHO report 2010; World Health Organization: Switzerland, 2010. (3) Bloom, B. R.; Murray, C. J. Tuberculosis: commentary on a reemergent killer. Science 1992, 257, 1055–1064. (4) Dormans, J.; Burger, M.; Aguilar, D.; Hernandez-Pando, R.; Kremer, K.; et al. Correlation of virulence, lung pathology, bacterial load and delayed type hypersensitivity responses after infection with different Mycobacterium tuberculosis genotypes in a BALB/c mouse model. Clin. Exp. Immunol. 2004, 137, 460–468. (5) Brennan, P. J. Structure, function, and biogenesis of the cell wall of Mycobacterium tuberculosis. Tuberculosis (Edinb.) 2003, 83, 91–97. (6) Chan, J.; Fan, X. D.; Hunter, S. W.; Brennan, P. J.; Bloom, B. R. Lipoarabinomannan, a possible virulence factor involved in persistence of Mycobacterium tuberculosis within macrophages. Infect. Immun. 1991, 59, 1755–1761. (7) Indrigo, J.; Hunter, R. L., Jr.; Actor, J. K. Cord factor trehalose 6,60 -dimycolate (TDM) mediates trafficking events during mycobacterial infection of murine macrophages. Microbiology 2003, 149, 2049–2059. (8) Kang, P. B.; Azad, A. K.; Torrelles, J. B.; Kaufman, T. M.; Beharka, A.; et al. The human macrophage mannose receptor directs Mycobacterium tuberculosis lipoarabinomannan-mediated phagosome biogenesis. J. Exp. Med. 2005, 202, 987–999. (9) Okamoto, Y.; Fujita, Y.; Naka, T.; Hirai, M.; Tomiyasu, I; Mycobacterial sulfolipid shows a virulence by inhibiting cord factor induced granuloma formation and TNF-alpha release. Microb. Pathog. 2006, 40, 245–253. (10) Reed, M. B.; Domenech, P.; Manca, C.; Su, H.; Barczak, A. K.; et al. A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response. Nature 2004, 431, 84–87. (11) Hu, Y.; Movahedzadeh, F.; Stoker, N. G.; Coates, A. R. Deletion of the Mycobacterium tuberculosis alpha-Crystallin-like hspX gene causes increased bacterial growth in vivo. Infect. Immun. 2006, 74, 861–868. (12) Kumar, A.; Toledo, J. C.; Patel, R. P.; Lancaster, J. R., Jr.; Steyn, A. J. Mycobacterium tuberculosis DosS is a redox sensor and DosT is a hypoxia sensor. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 11568–11573. (13) Noss, E. H.; Pai, R. K.; Sellati, T. J.; Radolf, J. D.; Belisle, J.; et al. Toll-like receptor 2-dependent inhibition of macrophage class II MHC expression and antigen processing by 19-kDa lipoprotein of Mycobacterium tuberculosis. J. Immunol. 2001, 167, 910–918. (14) Vergne, I.; Chua, J.; Lee, H. H.; Lucas, M.; Belisle, J.; et al. Mechanism of phagolysosome biogenesis block by viable Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4033–4038. (15) Walburger, A.; Koul, A.; Ferrari, G.; Nguyen, L.; PrescianottoBaschong, C.; et al. Protein kinase G from pathogenic mycobacteria promotes survival within macrophages. Science 2004, 304, 1800–1804. (16) Betts, J. C.; Dodson, P.; Quan, S.; Lewis, A. P.; Thomas, P. J.; et al. Comparison of the proteome of Mycobacterium tuberculosis strain H37Rv with clinical isolate CDC 1551. Microbiology 2000, 146 (Pt 12), 3205–3216. (17) Jungblut, P. R.; Schaible, U. E.; Mollenkopf, H. J.; Zimny-Arndt, U.; Raupach, B.; et al. Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens. Mol. Microbiol. 1999, 33, 1103–1117. (18) Mattow, J.; Schaible, U. E.; Schmidt, F.; Hagens, K.; Siejak, F.; et al. Comparative proteome analysis of culture supernatant proteins from virulent Mycobacterium tuberculosis H37Rv and attenuated M. bovis BCG Copenhagen. Electrophoresis 2003, 24, 3405–3420. (19) Rosenkrands, I.; King, A.; Weldingh, K.; Moniatte, M.; Moertz, E.; et al. Towards the proteome of Mycobacterium tuberculosis. Electrophoresis 2000, 21, 3740–3756. (20) Rosenkrands, I.; Weldingh, K.; Jacobsen, S.; Hansen, C. V.; Florio, W.; et al. Mapping and identification of Mycobacterium
’ CONCLUSIONS Proteomics allows for high throughput profiling of the expressed proteins at the cellular and subcellular levels. It complements genomics and classic gene expression studies by showing which proteins are really present during any given condition, taking into account mRNA stability, as well as post-translational modifications and protein degradation. It also offers insights into subcellular protein localization and functional status of a cell in response to environmental stimuli.17,19 In contrast to the genome, the proteome is not static but highly dynamic,102 and it represents a more realistic view of the intricate status of a living cell. Therefore, functional proteomics, which comprise studies aimed at understanding protein function, protein interactions, and post-translational modifications using peptide-based mass spectrometry, is starting to gain popularity among researchers. Information obtained from these type of studies can then be used to model cellular processes, including pathways, cell growth, and cell metabolism.103 Even though functional proteomics is still a nascent approach toward the study of Mtb, the few studies that have been conducted provided new insights toward lipid metabolism, secretion mechanisms, and post-translational modifications.48,5964 The success of these paradigms lends enthusiasm to functional proteomics-based studies to address more challenging questions regarding essential processes utilized by Mtb to successfully establish itself in the host. A different, but complementary, proteomics advance is the development of techniques and instruments that can be used to specifically target proteins of interest to obtain very accurate relative and absolute quantification. As discussed, MRM is revolutionizing the field, and we expect the next couple of years to bring a merger of high-throughput and functional proteomics techniques with targeted proteomics to advance the discovery of biomarkers for tuberculosis as well as explore novel therapeutic and prevention strategies.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Phone: +1 970 491-6549. Fax: +1 970 491-1815. 22
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studied by metabolic labeling and proteome analysis of cellular and extracellular proteins. J. Bacteriol. 2002, 184, 3485–3491. (40) Cho, S. H.; Goodlett, D.; Franzblau, S. ICAT-based comparative proteomic analysis of non-replicating persistent Mycobacterium tuberculosis. Tuberculosis (Edinb.) 2006, 86, 445–460. (41) Lee, B. Y.; Horwitz, M. A. Identification of macrophage and stress-induced proteins of Mycobacterium tuberculosis. J. Clin. Invest. 1995, 96, 245–249. (42) Mattow, J.; Siejak, F.; Hagens, K.; Becher, D.; Albrecht, D.; et al. Proteins unique to intraphagosomally grown Mycobacterium tuberculosis. Proteomics 2006, 6, 2485–2494. (43) Kruh, N. A.; Troudt, J.; Izzo, A.; Prenni, J.; Dobos, K. M. Portrait of a pathogen: the Mycobacterium tuberculosis proteome in vivo. PLoS One 2010, 5, e13938. (44) Schneider, B. B.; Guo, X.; Fell, L. M.; Covey, T. R. Stable gradient nanoflow LC-MS. J. Am. Soc. Mass Spectrom. 2005, 16, 1545– 1551. (45) Boehm, A. M.; Grosse-Coosmann, F.; Sickmann, A. Command line tool for calculating theoretical MS spectra for given sequences. Bioinformatics 2004, 20, 2889–2891. (46) Arnott, D. Basics of Triple-Stage Quadrupole/Ion-Trap Mass Spectrometry: Precursor, Product and neutral loss scanning. Electrospray Ionisation and Nanospray Ionisation; James, P., Ed.; Springer: Germany, 2001. (47) Schwartz, J. C.; Senko, M. W.; Syka, J. E. A two-dimensional quadrupole ion trap mass spectrometer. J. Am. Soc. Mass Spectrom. 2002, 13, 659–669. (48) Chavadi, S.; Wooff, E.; Coldham, N. G.; Sritharan, M.; Hewinson, R. G.; et al. Global effects of inactivation of the pyruvate kinase gene in the Mycobacterium tuberculosis complex. J. Bacteriol. 2009, 191, 7545– 7553. (49) Chernushevich, I. V.; Loboda, A. V.; Thomson, B. A. An introduction to quadrupole-time-of-flight mass spectrometry. J. Mass Spectrom. 2001, 36, 849–865. (50) Morris, H. R.; Paxton, T.; Dell, A.; Langhorne, J.; Berg, M.; et al. High sensitivity collisionally-activated decomposition tandem mass spectrometry on a novel quadrupole/orthogonal-acceleration time-offlight mass spectrometer. Rapid Commun. Mass Spectrom. 1996, 10, 889–896. (51) Lund, T. C.; Anderson, L. B.; McCullar, V.; Higgins, L.; Yun, G. H.; et al. iTRAQ is a useful method to screen for membrane-bound proteins differentially expressed in human natural killer cell types. J. Proteome Res. 2007, 6, 644–653. (52) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 2004, 3, 1154–1169. (53) Mehaffy, C.; Hess, A.; Prenni, J. E.; Mathema, B.; Kreiswirth, B.; et al. Descriptive proteomic analysis shows protein variability between closely related clinical isolates of Mycobacterium tuberculosis. Proteomics 2010, 10, 1966–1984. (54) Griffin, T. J.; Xie, H.; Bandhakavi, S.; Popko, J.; Mohan, A.; et al. iTRAQ reagent-based quantitative proteomic analysis on a linear ion trap mass spectrometer. J. Proteome Res. 2007, 6, 4200–4209. (55) Steen, H.; Kuster, B.; Mann, M. Quadrupole time-of-flight versus triple-quadrupole mass spectrometry for the determination of phosphopeptides by precursor ion scanning. J. Mass Spectrom. 2001, 36, 782–790. (56) Hinsby, A. M.; Olsen, J. V.; Bennett, K. L.; Mann, M. Signaling initiated by overexpression of the fibroblast growth factor receptor-1 investigated by mass spectrometry. Mol. Cell. Proteomics 2003, 2, 29–36. (57) Monti, M.; Orru, S.; Pagnozzi, D.; Pucci, P. Functional proteomics. Clin. Chim. Acta 2005, 357, 140–150. (58) Pandey, A.; Fernandez, M. M.; Steen, H.; Blagoev, B.; Nielsen, M. M.; et al. Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways. J. Biol. Chem. 2000, 275, 38633–38639.
tuberculosis proteins by two-dimensional gel electrophoresis, microsequencing and immunodetection. Electrophoresis 2000, 21, 935–948. (21) Sonnenberg, M. G.; Belisle, J. T. Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry. Infect. Immun. 1997, 65, 4515–4524. (22) Urquhart, B. L.; Cordwell, S. J.; Humphery-Smith, I. Comparison of predicted and observed properties of proteins encoded in the genome of Mycobacterium tuberculosis H37Rv. Biochem. Biophys. Res. Commun. 1998, 253, 70–79. (23) Malen, H.; Berven, F. S.; Fladmark, K. E.; Wiker, H. G. Comprehensive analysis of exported proteins from Mycobacterium tuberculosis H37Rv. Proteomics 2007, 7, 1702–1718. (24) Wolfe, L. M.; Mahaffey, S. B.; Kruh, N. A.; Dobos, K. M. Proteomic definition of the cell wall of Mycobacterium tuberculosis. J. Proteome Res. 2010, 9, 5816–5826. (25) Mawuenyega, K. G.; Forst, C. V.; Dobos, K. M.; Belisle, J. T.; Chen, J.; et al. Mycobacterium tuberculosis functional network analysis by global subcellular protein profiling. Mol. Biol. Cell 2005, 16, 396–404. (26) Schmidt, F.; Donahoe, S.; Hagens, K.; Mattow, J.; Schaible, U. E.; et al. Complementary analysis of the Mycobacterium tuberculosis proteome by two-dimensional electrophoresis and isotope-coded affinity tag technology. Mol. Cell. Proteomics 2004, 3, 24–42. (27) Gu, S.; Chen, J.; Dobos, K. M.; Bradbury, E. M.; Belisle, J. T.; et al. Comprehensive Proteomic Profiling of the Membrane Constituents of a Mycobacterium tuberculosis Strain. Mol. Cell. Proteomics 2003, 2, 1284– 1296. (28) Malen, H.; De Souza, G. A.; Pathak, S.; Softeland, T.; Wiker, H. G. Comparison of membrane proteins of Mycobacterium tuberculosis H37Rv and H37Ra strains. BMC Microbiol. 2011, 11, 18. (29) Sinha, S.; Kosalai, K.; Arora, S.; Namane, A.; Sharma, P.; et al. Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics. Microbiology 2005, 151, 2411–2419. (30) Xiong, Y.; Chalmers, M. J.; Gao, F. P.; Cross, T. A.; Marshall, A. G. Identification of Mycobacterium tuberculosis H37Rv integral membrane proteins by one-dimensional gel electrophoresis and liquid chromatography electrospray ionization tandem mass spectrometry. J. Proteome Res. 2005, 4, 855–861. (31) de Souza, G. A.; Wiker, H. G. A proteomic view of mycobacteria. Proteomics 2011, 11, 3118–3127. (32) Jagusztyn-Krynicka, E. K.; Roszczenko, P.; Grabowska, A. Impact of proteomics on anti-Mycobacterium tuberculosis (MTB) vaccine development. Pol. J. Microbiol. 2009, 58, 281–287. (33) Betts, J. C.; Lukey, P. T.; Robb, L. C.; McAdam, R. A.; Duncan, K. Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol. Microbiol. 2002, 43, 717–731. (34) Starck, J.; Kallenius, G.; Marklund, B. I.; Andersson, D. I.; Akerlund, T. Comparative proteome analysis of Mycobacterium tuberculosis grown under aerobic and anaerobic conditions. Microbiology 2004, 150, 3821–3829. (35) Boon, C.; Li, R.; Qi, R.; Dick, T. Proteins of Mycobacterium bovis BCG induced in the Wayne dormancy model. J. Bacteriol. 2001, 183, 2672–2676. (36) Yuan, Y.; Crane, D. D.; Barry, C. E., 3rd Stationary phaseassociated protein expression in Mycobacterium tuberculosis: function of the mycobacterial alpha-Crystallin homolog. J. Bacteriol. 1996, 178, 4484–4492. (37) Florczyk, M. A.; McCue, L. A.; Stack, R. F.; Hauer, C. R.; McDonough, K. A. Identification and characterization of mycobacterial proteins differentially expressed under standing and shaking culture conditions, including Rv2623 from a novel class of putative ATP-binding proteins. Infect. Immun. 2001, 69, 5777–5785. (38) Monahan, I. M.; Betts, J; Banerjee, D. K.; Butcher, P. D. Differential expression of mycobacterial proteins following phagocytosis by macrophages. Microbiology 2001, 147, 459–471. (39) Rosenkrands, I.; Slayden, R. A.; Crawford, J.; Aagaard, C.; Barry, C. E., 3rd; et al. Hypoxic response of Mycobacterium tuberculosis 23
dx.doi.org/10.1021/pr2008658 |J. Proteome Res. 2012, 11, 17–25
Journal of Proteome Research
REVIEWS
(78) Brondsted, L.; Andersen, M. T.; Parker, M.; Jorgensen, K.; Ingmer, H. The HtrA protease of Campylobacter jejuni is required for heat and oxygen tolerance and for optimal interaction with human epithelial cells. Appl. Environ. Microbiol. 2005, 71, 3205–3212. (79) Darwin, K. H.; Ehrt, S.; Gutierrez-Ramos, J. C.; Weich, N.; Nathan, C. F. The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide. Science 2003, 302, 1963–1966. (80) Gaillot, O.; Pellegrini, E.; Bregenholt, S.; Nair, S.; Berche, P. The ClpP serine protease is essential for the intracellular parasitism and virulence of Listeria monocytogenes. Mol. Microbiol. 2000, 35, 1286–1294. (81) Lewis, C.; Skovierova, H.; Rowley, G.; Rezuchova, B.; Homerova, D.; et al. Salmonella enterica Serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection. Microbiology 2009, 155, 873–881. (82) Webb, C.; Moreno, M.; Wilmes-Riesenberg, M.; Curtiss, R., 3rd; Foster, J. W. Effects of DksA and ClpP protease on sigma S production and virulence in Salmonella typhimurium. Mol. Microbiol. 1999, 34, 112–123. (83) Kweon, O.; Kim, S. J.; Holland, R. D.; Chen, H.; Kim, D. W.; et al. Polycyclic Aromatic Hydrocarbon Metabolic Network in Mycobacterium vanbaaleniiPYR-1. J. Bacteriol. 2011, 193, 4326–4337. (84) Nicolau, E.; Kuhn, L.; Marchal, R.; Jouanneau, Y. Proteomic investigation of enzymes involved in 2-ethylhexyl nitrate biodegradation in Mycobacterium austroafricanum IFP 2173. Res. Microbiol. 2009, 160, 838–847. (85) McKinney, J. D.; Honer zu Bentrup, K.; Munoz-Elias, E. J.; Miczak, A.; Chen, B. Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 2000, 406, 735–738. (86) Munoz-Elias, E. J.; McKinney, J. D. Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat. Med. 2005, 11, 638–644. (87) Ribeiro-Guimaraes, M. L.; Pessolani, M. C. Comparative genomics of mycobacterial proteases. Microb. Pathog. 2007, 43, 173–178. (88) Bluemlein, K.; Ralser, M. Monitoring protein expression in whole-cell extracts by targeted label- and standard-free LC-MS/MS. Nat. Protoc. 2011, 6, 859–869. (89) Drabovich, A. P.; Diamandis, E. P. Combinatorial peptide libraries facilitate development of multiple reaction monitoring assays for low-abundance proteins. J. Proteome Res. 2010, 9, 1236–1245. (90) Prakash, A.; Rezai, T.; Krastins, B.; Sarracino, D.; Athanas, M.; et al. Platform for establishing interlaboratory reproducibility of selected reaction monitoring-based mass spectrometry peptide assays. J. Proteome Res. 2010, 9, 6678–6688. (91) Lange, V.; Picotti, P.; Domon, B.; Aebersold, R. Selected reaction monitoring for quantitative proteomics: a tutorial. Mol. Syst. Biol. 2008, 4, 222. (92) Addona, T. A.; Abbatiello, S. E.; Schilling, B.; Skates, S. J.; Mani, D. R.; et al. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma. Nat. Biotechnol. 2009, 27, 633–641. (93) Anderson, L.; Hunter, C. L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol. Cell. Proteomics 2006, 5, 573–588. (94) Keshishian, H.; Addona, T.; Burgess, M.; Kuhn, E.; Carr, S. A. Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution. Mol. Cell. Proteomics 2007, 6, 2212–2229. (95) Kuhn, E.; Wu, J.; Karl, J.; Liao, H.; Zolg, W.; et al. Quantification of C-reactive protein in the serum of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and 13C-labeled peptide standards. Proteomics 2004, 4, 1175–1186. (96) Calvo, E.; Camafeita, E.; Fernandez-Gutierrez, B.; Lopez, J. A. Applying selected reaction monitoring to targeted proteomics. Expert Rev. Proteomics 2011, 8, 165–173. (97) Elschenbroich, S.; Kislinger, T. Targeted proteomics by selected reaction monitoring mass spectrometry: applications to systems biology and biomarker discovery. Mol. Biosyst. 2011, 7, 292–303.
(59) Millington, K. A.; Fortune, S. M.; Low, J.; Garces, A.; HingleyWilson, S. M.; et al. Rv3615c is a highly immunodominant RD1 (Region of Difference 1)-dependent secreted antigen specific for Mycobacterium tuberculosis infection. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 5730– 5735. (60) White, M. J.; Savaryn, J. P.; Bretl, D. J.; He, H.; Penoske, R. M.; et al. The HtrA-like serine protease PepD interacts with and modulates the Mycobacterium tuberculosis 35-kDa antigen outer envelope protein. PLoS One 2011, 6, e18175. (61) Pearce, M. J.; Arora, P.; Festa, R. A.; Butler-Wu, S. M.; Gokhale, R. S.; et al. Identification of substrates of the Mycobacterium tuberculosis proteasome. EMBO J. 2006, 25, 5423–5432. (62) Pearce, M. J.; Mintseris, J.; Ferreyra, J.; Gygi, S. P.; Darwin, K. H. Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis. Science 2008, 322, 1104–1107. (63) Poulsen, C.; Akhter, Y.; Jeon, A. H.; Schmitt-Ulms, G.; Meyer, H. E.; et al. Proteome-wide identification of mycobacterial pupylation targets. Mol. Syst. Biol. 2010, 6, 386. (64) Estorninho, M.; Smith, H.; Thole, J.; Harders-Westerveen, J.; Kierzek, A.; et al. ClgR regulation of chaperone and protease systems is essential for Mycobacterium tuberculosis parasitism of the macrophage. Microbiology 2010, 156, 3445–3455. (65) Lew, J. M.; Kapopoulou, A.; Jones, L. M.; Cole, S. T. TubercuList - 10 years after. Tuberculosis (Edinb.) 2011, 91, 1–7. (66) Lalvani, A.; Pathan, A. A.; McShane, H.; Wilkinson, R. J.; Latif, M.; et al. Rapid detection of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. Am. J. Respir. Crit. Care Med. 2001, 163, 824–828. (67) Brodin, P.; Majlessi, L.; Marsollier, L.; de Jonge, M. I.; Bottai, D.; et al. Dissection of ESAT-6 system 1 of Mycobacterium tuberculosis and impact on immunogenicity and virulence. Infect. Immun. 2006, 74, 88–98. (68) Guinn, K. M.; Hickey, M. J.; Mathur, S. K.; Zakel, K. L.; Grotzke, J. E.; et al. Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Mol. Microbiol. 2004, 51, 359–370. (69) Pym, A. S.; Brodin, P.; Brosch, R.; Huerre, M.; Cole, S. T. Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Mol. Microbiol. 2002, 46, 709–717. (70) Prisic, S.; Dankwa, S.; Schwartz, D.; Chou, M. F.; Locasale, J. W.; et al. Extensive phosphorylation with overlapping specificity by Mycobacterium tuberculosis serine/threonine protein kinases. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 7521–7526. (71) Beausoleil, S. A.; Villen, J.; Gerber, S. A.; Rush, J.; Gygi, S. P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 2006, 24, 1285–1292. (72) Doucet, A.; Butler, G. S.; Rodriguez, D.; Prudova, A.; Overall, C. M. Metadegradomics: toward in vivo quantitative degradomics of proteolytic post-translational modifications of the cancer proteome. Mol. Cell. Proteomics 2008, 7, 1925–1951. (73) Overall, C. M.; Tam, E. M.; Kappelhoff, R.; Connor, A.; Ewart, T.; et al. Protease degradomics: mass spectrometry discovery of protease substrates and the CLIP-CHIP, a dedicated DNA microarray of all human proteases and inhibitors. Biol. Chem. 2004, 385, 493–504. (74) Lopez-Otin, C.; Overall, C. M. Protease degradomics: a new challenge for proteomics. Nat. Rev. Mol. Cell. Biol. 2002, 3, 509–519. (75) Kim, D. Y.; Kim, K. K. Structure and function of HtrA family proteins, the key players in protein quality control. J. Biochem. Mol. Biol. 2005, 38, 266–274. (76) Stewart, G. R.; Wernisch, L.; Stabler, R.; Mangan, J. A.; Hinds, J.; et al. Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays. Microbiology 2002, 148, 3129–3138. (77) Burns, K. E.; Liu, W. T.; Boshoff, H. I.; Dorrestein, P. C.; Barry, C. E., 3rd Proteasomal protein degradation in Mycobacteria is dependent upon a prokaryotic ubiquitin-like protein. J. Biol. Chem. 2009, 284, 3069–3075. 24
dx.doi.org/10.1021/pr2008658 |J. Proteome Res. 2012, 11, 17–25
Journal of Proteome Research
REVIEWS
(98) Gallien, S.; Duriez, E.; Domon, B. Selected reaction monitoring applied to proteomics. J. Mass Spectrom. 2011, 46, 298–312. (99) Kuzyk, M. A.; Smith, D.; Yang, J.; Cross, T. J.; Jackson, A. M.; et al. Multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma. Mol. Cell. Proteomics 2009, 8, 1860–1877. (100) Lange, V.; Malmstrom, J. A.; Didion, J.; King, N. L.; Johansson, B. P.; et al. Targeted quantitative analysis of Streptococcus pyogenes virulence factors by multiple reaction monitoring. Mol. Cell. Proteomics 2008, 7, 1489–1500. (101) Garbe, T. R.; Hibler, N. S.; Deretic, V. Response of Mycobacterium tuberculosis to reactive oxygen and nitrogen intermediates. Mol. Med. 1996, 2, 134–142. (102) Boehm, A. M.; Putz, S.; Altenhofer, D.; Sickmann, A.; Falk, M. Precise protein quantification based on peptide quantification using iTRAQ. BMC Bioinform. 2007, 8, 214. (103) Cavalieri, D.; De Filippo, C. Bioinformatic methods for integrating whole-genome expression results into cellular networks. Drug Discovery Today 2005, 10, 727–734.
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