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Mini Review
Global analysis of bacterial membrane proteins and their modifications Boumediene Soufi ∗ , Boris Macek ∗ Proteome Center Tuebingen, Interfaculty Institute for Cell Biology, University of Tuebingen, Auf der Morgenstelle 15, 72076 Tuebingen, Germany
a r t i c l e
i n f o
Keywords: Shotgun proteomics Membrane proteins Posttranslational modifications Bacteria Virulence
a b s t r a c t Membrane proteins are situated at the interface of bacterial cell and its environment, and are therefore involved in vital physiological processes such as nutrient exchange, signal transduction and virulence. Due to their distinct biophysical properties, especially hydrophobicity, they are difficult subjects to study. Classical proteomics technologies have relied on multidimensional separation of proteins on gels, which largely limited the choice of detergents and made the development of specialized enrichment protocols for membrane proteins necessary. Shotgun proteomic approaches, based on the digestion of whole proteomes and subsequent analysis of peptides by LC–MS, has largely circumvented these problems due to its compatibility with potent detergents. Here we briefly present and discuss the major developments in bacterial membrane proteomics and argue that recent developments in biochemical sample preparation and high resolution mass spectrometry have the potential to comprehensively identify and quantify membrane proteins without the need for specific enrichment procedures prior to LC–MS analysis. © 2014 Elsevier GmbH. All rights reserved.
Introduction Bacterial membrane proteins play an essential role in many biological areas such as in signal transduction and pathogenicity. Membrane proteins represent approximately 20 to 30% of the entire genetic complement of a bacterial cell. Despite their importance, there is still a general lack of knowledge of many bacterial proteins due to the fact that many are of low abundance, insoluble and of a hydrophobic nature hence making them difficult to identify and characterize (Poetsch and Wolters, 2008). Proteomics has established itself as a critical tool in many areas in biology. This is largely due to the advances made in mass spectrometry (MS)-based technologies, which provides high accuracy, sensitivity and resolution in a highly efficient and high throughput manner. MS-based proteomics is revolutionizing biomedical research, significantly impacting the microbiology field. Bacteria are amenable for global quantitative gene expression analyses, due to their relatively simple proteomes compared to their more complex eukaryotic counterparts. The successful development of MS-based quantitative proteomics has allowed researchers
∗ Corresponding authors. Tel.: +49 7071 29 70556; fax: +49 7071 29 5779. E-mail addresses: [email protected] (B. Soufi), [email protected] (B. Macek).
to produce numerous studies that identify and characterize membrane proteins in bacteria (Table 1). Historically, proteomic studies of bacterial proteins have been performed using a technique known as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by MS analysis in order to identify the proteins present (Santoni et al., 2000). In this approach proteins are successively separated based on their isoelectric points and molecular weights and then visualized by specific stains in the form of protein spots on the gel. This technique has the capability to resolve thousands of proteins, and is very advantageous when studying certain protein features like isoforms. Numerous 2D-PAGE studies have assisted in advancing the identification and knowledge of bacterial membrane proteins as well as the proteomes of many bacterial species in general (Curreem et al., 2012; Li et al., 2007; Peng et al., 2005; Yun et al., 2008); However, this technique faces inherent problems: it is relatively time consuming and it is difficult to identify certain protein classes (e.g. proteins of extreme sizes or PI values). Utilizing 2D-PAGE to identify bacterial membrane proteins poses additional problems. The intrinsic hydrophobic and low abundant nature of membrane proteins presents an extreme challenge with respect to solubilizing them in order to be compatible with isoelectric focusing techniques used in 2D-PAGE. This ultimately results in the reduction of the transfer efficiency of the membrane proteins to the second dimension gel. Moreover, 2-D PAGE is not suitable when studying certain classes of membrane proteins such as integral membrane proteins
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Please cite this article in press as: Soufi, B., Macek, B., Global analysis of bacterial membrane proteins and their modifications. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2014.12.017
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Table 1 Selected proteomics based studies that have focused on bacterial membrane proteins within the last decade. Topic
Method
Year
Reference
Vibrio cholera OMVs Helicobacter pylori outer membranes Bacillus subtilis global membrane profiling Neisseria meningitides OMVs Bacillus subtilis stress conditions Synechocystis sp. membrane complexes Escherichia coli membrane proteins Escherichia coli OMVs Escherichia coli envelope proteins Staphylococcus aureus global profiling Streptococcus pyogenes surface proteins Mycobacterium tuberculosis membrane Escherichia coli membrane methods Meningococcal OMVs Bacteria general membrane proteins Vibrio alginolyticus outer membranes Shigella flexneri outer membranes Neisseria gonorrhoeae envelope proteins
Shotgun 2D-PAGE Shotgun 2D-PAGE Shotgun 2D-PAGE 2D-PAGE Shotgun 2D-PAGE Both Shotgun Shotgun Shotgun 2D-PAGE 2D-PAGE 2D-PAGE 2D-PAGE Shotgun
2014 2004 2004 2006 2010 2004 2004 2007 2006 2005 2007 2005 2013 2006 2004 2005 2005 2014
Altindis et al. (2014) Baik et al. (2004) Eymann et al. (2004) Ferrari et al. (2006) Hahne et al. (2010) Herranen et al. (2004) Lai et al. (2004) Lee et al. (2007) Lok et al. (2006) Scherl et al. (2005) Severin et al. (2007) Sinha et al. (2005) Tanca et al. (2013) Vipond et al. (2006) Wilmes and Bond (2004) Xu et al. (2005) Ying et al. (2005) Zielke et al. (2014)
(Santoni et al., 2000). Several improvements have been made in an attempt to alleviate these issues with the 2D-PAGE approach as well as improving the coverage of membrane proteins in general. For example, using various detergents such as zwitterionic or anionic detergents have been shown to improve the solubility of membrane proteins (Vuckovic et al., 2013). Other sample preparation methods, such as cell surface and membrane shaving (Solis and Cordwell, 2011), biotinylation (Macher and Yen, 2007), use of various organic solvents such as methanol (Blonder et al., 2006), and synthetic membrane systems known as nanodiscs (Yan et al., 2011) can be utilized to increase the efficiency and purification of bacterial membrane proteins prior to MS analysis. These and other membrane protein extraction techniques are extensively covered in a recent review (Vuckovic et al., 2013). Despite several improvements made to the 2D-PAGE MS proteomics approach, the overall limitations with respect to analysis of membrane proteins lead to the recent application of gel-free MS based approaches, better known as “shotgun” proteomics. It is very well suited to identify proteins in complex mixtures in a very robust and high throughput manner. This mini-review will focus on these gel-free MS based approaches that have provided new information about bacterial membrane proteins especially within the context of quantitative proteomics, post translational modifications (PTMs) and pathogenicity.
example, this approach identified a large number of insoluble ABC transporters (protein family of integral membrane proteins) in Geobacillus thermoleovorans and Oceanobacillus iheyensis (Graham et al., 2006, 2007). Importantly, these workflows have the possibility to produce a comprehensive coverage of bacterial membrane proteins even without specialized enrichment procedures. To demonstrate this, for the purpose of this review we re-analyzed our recently published proteogenomics study in Escherichia coli, which with 2626 identified proteins represents one of the largest proteome datasets of this organism to date (Krug et al., 2013). We predicted how many membrane proteins were identified in this study, using a Hidden Markov Model approach as previously described (Krogh et al., 2001). The criterion was that each protein must have been predicted with at least one transmembrane domain, and that at least 18 amino acids or more are predicted to be located inside the membrane. This resulted in 503 hits out of a potential 898 total hits to be predicted as membrane proteins in the E. coli proteome, corresponding to nearly a 60% coverage of membrane proteins without any specialized enrichment. It is noteworthy to mention that other similar proteomics approaches previously performed could potentially show this same trend of membrane protein coverage without specialized membrane protein extraction protocols (Macek et al., 2007, 2008; Ravikumar et al., 2014; Soares et al., 2013; Soufi et al., 2008, 2010; Wolff et al., 2007).
MS-based shotgun proteomics Quantitative proteomics A typical shotgun MS-based proteomics workflow is explained in detail in Fig. 1. One critical advantage of this approach is that the entire protein content of a cell can be extracted utilizing a variety of methods depending on the type of sample and analysis that is required. For example, in the context of bacterial membrane proteins, detergent based extraction methods have been widely employed as they serve as an efficient means to isolate many insoluble and low abundant targets (Vuckovic et al., 2013). One such detergent known as sodium dodecyl sulfate (SDS) is very popular in large scale global proteomics studies. Specific techniques that are compatible with the shotgun proteomics platform, such as filteraided sample preparation (FASP) (Wisniewski et al., 2009), have been developed in order to utilize SDS and subsequently remove it prior to protein digestion, as the presence of even minor amounts of SDS can cause problems with chromatography as well as the inhibition of many proteases such as trypsin. These workflows can be universally employed to virtually any type of biological organism or material, and have been utilized for identification of many bacterial membrane proteins. For
One important advantage of the Shotgun MS-based proteomics approaches is that they have the ability to adapt quantitative proteomics strategies that can compare two or more perturbations or conditions in a biological system in a proteome-wide global approach. Several methodologies exist each with their respective advantages as well as limitations. One common approach is to utilize stable (non-radioactive) isotope labelling to quantify the relative abundance of a protein from different samples directly from the acquired mass spectral data. Two major strategies for introducing a stable isotope label into the proteome exist: chemical and metabolic labelling. One strategy is known as stable isotope labelling by amino acids in cell culture (SILAC) (Ong et al., 2002), which has been successfully applied to many bacterial systems (Ravikumar et al., 2014; Soufi et al., 2010). SILAC metabolically (in vivo) incorporates the label, which is typically introduced in form of labelled lysine, arginine or another amino acid. Protein samples from two or three states are labelled with either unlabeled (“light”) or labelled (“heavy”) version of the amino acid, and can
Please cite this article in press as: Soufi, B., Macek, B., Global analysis of bacterial membrane proteins and their modifications. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2014.12.017
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Fig. 1. Quantitative proteomics workflow. (A) Cells are grown in culture (solid or liquid) and lysed using various methods such as the SDS detergent based approach which is advantageous for membrane protein coverage. (B) Protein extracts are digested using either in-solution or via 1-D SDS–PAGE approaches by various endoproteinases such as LysC or trypsin, followed by enrichment of various PTMs (if applicable). (C) For proteome quantitation, peptide digests can be separated according to their pI-value by iso-electric focusing or 1-D SDS PAGE. (D) Resulting peptide mixtures are separated on nanoflow HPLC and directly measured in a mass spectrometer capable of high resolution. (E) Relative peptide quantitation is based on the first stage of mass spectrometry (Full-scan MS) (F) Peptide identification is achieved by fragmentation in the second stage of mass spectrometry (MS/MS). (G) Data is processed using various bioinformatic workflows. The resulting data is utilized in the interpretation and ultimately the answer to the specific biological topic in question.
be relatively quantified in terms of protein abundance between them. In order to ensure full amino acid incorporation, the cell must not be able to synthesize it endogenously. This implies that some bacterial species must be rendered auxotrophic for a specific amino acid by knocking out the genes involved its biosynthetic pathway, which could present a possible limitation if the required mutagenesis cannot be performed. The labelled and unlabeled samples are mixed in equimolar amounts and digested using a specific endoprotease prior to MS measurement, in which intensities and fragmentation patterns of peptides are measured and used for subsequent quantification and identification of differently regulated proteins in the two samples. In terms of bacterial membrane studies, the SILAC approach was successfully employed in combination with nanodisc-based technology to study soluble interaction partners of E. coli membrane proteins in global and quantitative fashion (Zhang et al., 2012). Moreover, SILAC was also performed in Bifidobacterium longum in order to achieve a better understanding of bile tolerance in this organism (Ruiz et al., 2009). To this end, the authors identified 141 membrane proteins in a quantitative fashion, thus far the largest cell-envelope proteome obtained in B. longum. One limitation to these approaches is that they do not provide any insights into the absolute molar amount or concentration of a particular protein per cell. Recently, numerous techniques were developed to address this issue that are compatible with relative quantitation approaches, providing further valuable biological information. These include Absolute QUAntification (AQUA) (Gerber et al., 2003), FlexiQuant (Singh et al., 2009), protein standard absolute quantification (PSAQ) (Brun et al., 2007), absolute quantification using protein epitope signature tags (PrEST)
(Zeiler et al., 2012), and intensity based absolute quantification (iBAQ) (Schwanhausser et al., 2011). All of these methodologies can be applied in bacteria, and can be used to elucidate the absolute amounts of bacterial membrane proteins compared to for example their cytoplasmic protein counterparts, or to better understand the stoichiometry of membrane protein complexes. It is quite clear that these techniques will allow a better understanding of many biological processes in a global systems biology approach. Proteomics and posttranslational modifications Post-translational modifications (PTMs) of proteins are employed by all living cells as a very efficient means of signal transduction and regulation. Historically, characterizing and identifying PTMs in bacteria was extremely challenging and complex compared to their eukaryotic counterparts. This is largely due to the fact that generally PTMs are difficult to detect due to their low abundance and stoichiometry. Due to advances is MS instrumentation and biochemical sample preparation methods, which can now efficiently enrich for modified peptides from extremely complex peptide mixtures, many of these past issues have been circumvented which paved the way for many PTMs to be studied in bacteria. These include but are not limited to phosphorylation, acetylation, AMPylation, glucosylation, ADP ribosylation, and deamidation. One such case where this technology better known as gel-free, site-specific, quantitative phosphoproteomics has significantly advanced our knowledge with regards to bacterial modifications is in the area of serine/threonine/tyrosine (Ser/Thr/Tyr) phosphorylation. Despite early evidence that this modification may play
Please cite this article in press as: Soufi, B., Macek, B., Global analysis of bacterial membrane proteins and their modifications. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2014.12.017
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important roles in bacterial physiology (Dadssi and Cozzone, 1990; Forchhammer and Tandeau de Marsac, 1995; Kirstein et al., 2005; Reizer et al., 1996), Ser/Thr/Tyr phosphorylation has been thought to occur predominantly in eukaryotes and to be not functionally significant in bacteria. However due to advances in MS based proteomics, combined with powerful phosphorylation enrichment strategies such as phosphopeptide immunoprecipitation (Rush et al., 2005), immobilized metal ion affinity chromatography (IMAC) (Villen and Gygi, 2008) or titanium dioxide chromatography (TiO2 ) (Pinkse et al., 2004) have led to many important discoveries in bacterial Ser/Thr/Tyr phosphorylation (Cousin et al., 2013; Macek and Mijakovic, 2011). Many of these studies also detected a large portion of modified membrane proteins. The knowledge of the existence of such PTMs could elucidate a better understanding of the function of bacterial membrane proteins. For example, a recent phosphoproteomic study in Helicobacter Pylori employed the TiO2 enrichment strategy to perform a global site specific enrichment of Ser/Thr/Tyr phosphorylated proteins (Ge et al., 2011). To this end, authors found that many of these phosphorylation events were overrepresented in numerous membrane proteins, indicating that these proteins and their respective phosphorylation events could be an important mechanism in virulence control. Moreover, several US, EU and national consortia, such as the Comprehensive Research Centre (CRC) 766 in Germany, are currently applying state of the art (phospho) proteomics technologies towards projects involving diverse bacterial species such as Streptomyces coelicolor, Staphylococcus aureus and Synechocystis sp. PCC 6803, in order to elucidate the potential role of phosphorylation on both the cell envelope and membrane components. For example, the PII nitrogen regulation proteins of cyanobacterium Synechocystis are dephosphorylated by the PphA phosphatase which affects the binding properties towards different protein interactors (Kloft et al., 2005). Within CRC766 we are currently establishing a MS based phosphoproteomics workflow in order to identify the phosphorylation dependent interaction partners and potentially the modifying kinase of PII (Spät, Forchhammer, unpublished data). In addition, we could show that the serine/threonine kinase PkaI (SCO4778) controls activity of the Streptomyces Spore wall Synthesizing Complex (SSSC) in S. coelicolor by phosphorylating MreC and PBP2 (Ladwig et al., in preparation). We are currently applying phosphoproteomics workflows towards elucidating the role of PkaI during morphological differentiation and identify additional target proteins of PkaI.
Proteomics of membrane proteins involved in virulence Situated at the interface between the bacterial cell and its environment, many bacterial membrane proteins play a crucial role in disease. Gel free MS based quantitative and qualitative Shotgun proteomics approaches have made significant contributions towards discoveries in the involvement of bacterial membrane proteins roles in virulence and pathogenicity. As previously mentioned, the bacterial cell envelope is a crucial component in maintaining cellular shape and homeostasis. It also plays an important role in antibiotic resistance, and has developed resistance mechanisms against many antibiotics including but not limited to beta-lactams, methicillin, and fosfomycin (Bush, 2012; Hempel et al., 2011). Resistance to beta-lactam and macrolide antibiotics has been observed in many bacteria, especially those of the Gram negative species (Poole, 2001). Many outer membrane proteins (OMP) as well as porins (ß-barrel proteins that extend across the outer membrane) play an important part of these resistance mechanisms by controlling the membrane permeability of the cell. In a shotgun-based proteomic study an altered outer membrane protein profile was found in clarithromycin resistant
Helicobacter pylori (Smiley et al., 2013). Further proteomic based studies describing the importance of OMPs is extensively described elsewhere (Lima et al., 2013). Besides elucidating the important roles of bacterial antibiotic resistance mechanisms, Gel-free MS based shotgun proteomics has eluded to the importance of membrane proteins and disease in many other aspects. For example, many bacterial strains are used to produce probiotics for human consumption which are associated with numerous health benefitting properties. A recent shotgun proteomics approach was applied to the popular probiotic strain Bifidobacterium animalis subsp. Lactis BB-12 in an attempt to characterize the membrane proteome in order to gain a better understanding of oligosaccharide transport in this bacterium (Gilad et al., 2012). The authors identified 248 predicted membrane proteins, of which 90 were predicted to contain at least one transmembrane segment, and further showed that a large proportion of them were implicated to be involved in the transport of many compounds such as amino acids, oligosaccharides and nucleotides. MS based proteomics has also provided many important aspects with regards to those bacterial pathogens that depend on successful adherence in order to influence their pathogenic effects on their hosts. For example, adherence is necessary in order for secretion systems such as Type III secretion systems to act, which are very commonly utilized in the virulence of Gram negative bacterial pathogens. A recent study performed a global quantitative proteomics analysis employing the SILAC approach to study these Type III secretion systems in different mutated strains of enteropathogenic Escherichia coli (EPEC) (Deng et al., 2012). The authors identified many known as well as putative periplasmic and outer membrane proteins suggesting that these Type III secretion systems in EPEC may be composed of more membrane proteins as well as other uncharacterized proteins than initially thought, providing new insights into the potential virulence and pathogenic roles that these membrane proteins might play. Conclusions and future perspectives Modern Gel-free based shotgun proteomics approaches have evolved within the last decade into a powerful research platform for many different applications in bacteria such as comprehensive analysis of proteomes and sub-proteomes (e.g. membrane proteome) as well as relative and absolute quantitation of proteins and their modifications. We believe that the improvements in MS instrumentation (sensitivity, accuracy, speed) combined with numerous advances towards the enrichment protocols of bacterial membrane proteins will lead to routine identification and characterization of bacterial membrane proteins in a systematic and robust fashion. The major technological advances in MS-based quantitative proteomics are therefore transforming the research in the field of microbiology, providing new insights in bacterial virulence mechanisms, signal transduction as well as providing an essential connection to the rapidly evolving and dynamic area of systems biology. Acknowledgement Authors wish to acknowledge German Research Foundation (DFG, SFB766) for funding. References Altindis, E., Fu, Y., Mekalanos, J.J., 2014. Proteomic analysis of Vibrio cholerae outer membrane vesicles. Proc. Natl. Acad. Sci. U.S.A. 111, E1548–E1556. Baik, S.C., Kim, K.M., Song, S.M., Kim, D.S., Jun, J.S., Lee, S.G., Song, J.Y., Park, J.U., Kang, H.L., Lee, W.K., Cho, M.J., Youn, H.S., Ko, G.H., Rhee, K.H., 2004. Proteomic analysis of the sarcosine-insoluble outer membrane fraction of Helicobacter pylori strain 26695. J. Bacteriol. 186, 949–955.
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Mini Review
Global analysis of bacterial membrane proteins and their modifications Boumediene Soufi ∗ , Boris Macek ∗ Proteome Center Tuebingen, Interfaculty Institute for Cell Biology, University of Tuebingen, Auf der Morgenstelle 15, 72076 Tuebingen, Germany
a r t i c l e
i n f o
Keywords: Shotgun proteomics Membrane proteins Posttranslational modifications Bacteria Virulence
a b s t r a c t Membrane proteins are situated at the interface of bacterial cell and its environment, and are therefore involved in vital physiological processes such as nutrient exchange, signal transduction and virulence. Due to their distinct biophysical properties, especially hydrophobicity, they are difficult subjects to study. Classical proteomics technologies have relied on multidimensional separation of proteins on gels, which largely limited the choice of detergents and made the development of specialized enrichment protocols for membrane proteins necessary. Shotgun proteomic approaches, based on the digestion of whole proteomes and subsequent analysis of peptides by LC–MS, has largely circumvented these problems due to its compatibility with potent detergents. Here we briefly present and discuss the major developments in bacterial membrane proteomics and argue that recent developments in biochemical sample preparation and high resolution mass spectrometry have the potential to comprehensively identify and quantify membrane proteins without the need for specific enrichment procedures prior to LC–MS analysis. © 2014 Elsevier GmbH. All rights reserved.
Introduction Bacterial membrane proteins play an essential role in many biological areas such as in signal transduction and pathogenicity. Membrane proteins represent approximately 20 to 30% of the entire genetic complement of a bacterial cell. Despite their importance, there is still a general lack of knowledge of many bacterial proteins due to the fact that many are of low abundance, insoluble and of a hydrophobic nature hence making them difficult to identify and characterize (Poetsch and Wolters, 2008). Proteomics has established itself as a critical tool in many areas in biology. This is largely due to the advances made in mass spectrometry (MS)-based technologies, which provides high accuracy, sensitivity and resolution in a highly efficient and high throughput manner. MS-based proteomics is revolutionizing biomedical research, significantly impacting the microbiology field. Bacteria are amenable for global quantitative gene expression analyses, due to their relatively simple proteomes compared to their more complex eukaryotic counterparts. The successful development of MS-based quantitative proteomics has allowed researchers
∗ Corresponding authors. Tel.: +49 7071 29 70556; fax: +49 7071 29 5779. E-mail addresses: [email protected] (B. Soufi), [email protected] (B. Macek).
to produce numerous studies that identify and characterize membrane proteins in bacteria (Table 1). Historically, proteomic studies of bacterial proteins have been performed using a technique known as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by MS analysis in order to identify the proteins present (Santoni et al., 2000). In this approach proteins are successively separated based on their isoelectric points and molecular weights and then visualized by specific stains in the form of protein spots on the gel. This technique has the capability to resolve thousands of proteins, and is very advantageous when studying certain protein features like isoforms. Numerous 2D-PAGE studies have assisted in advancing the identification and knowledge of bacterial membrane proteins as well as the proteomes of many bacterial species in general (Curreem et al., 2012; Li et al., 2007; Peng et al., 2005; Yun et al., 2008); However, this technique faces inherent problems: it is relatively time consuming and it is difficult to identify certain protein classes (e.g. proteins of extreme sizes or PI values). Utilizing 2D-PAGE to identify bacterial membrane proteins poses additional problems. The intrinsic hydrophobic and low abundant nature of membrane proteins presents an extreme challenge with respect to solubilizing them in order to be compatible with isoelectric focusing techniques used in 2D-PAGE. This ultimately results in the reduction of the transfer efficiency of the membrane proteins to the second dimension gel. Moreover, 2-D PAGE is not suitable when studying certain classes of membrane proteins such as integral membrane proteins
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Please cite this article in press as: Soufi, B., Macek, B., Global analysis of bacterial membrane proteins and their modifications. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2014.12.017
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Table 1 Selected proteomics based studies that have focused on bacterial membrane proteins within the last decade. Topic
Method
Year
Reference
Vibrio cholera OMVs Helicobacter pylori outer membranes Bacillus subtilis global membrane profiling Neisseria meningitides OMVs Bacillus subtilis stress conditions Synechocystis sp. membrane complexes Escherichia coli membrane proteins Escherichia coli OMVs Escherichia coli envelope proteins Staphylococcus aureus global profiling Streptococcus pyogenes surface proteins Mycobacterium tuberculosis membrane Escherichia coli membrane methods Meningococcal OMVs Bacteria general membrane proteins Vibrio alginolyticus outer membranes Shigella flexneri outer membranes Neisseria gonorrhoeae envelope proteins
Shotgun 2D-PAGE Shotgun 2D-PAGE Shotgun 2D-PAGE 2D-PAGE Shotgun 2D-PAGE Both Shotgun Shotgun Shotgun 2D-PAGE 2D-PAGE 2D-PAGE 2D-PAGE Shotgun
2014 2004 2004 2006 2010 2004 2004 2007 2006 2005 2007 2005 2013 2006 2004 2005 2005 2014
Altindis et al. (2014) Baik et al. (2004) Eymann et al. (2004) Ferrari et al. (2006) Hahne et al. (2010) Herranen et al. (2004) Lai et al. (2004) Lee et al. (2007) Lok et al. (2006) Scherl et al. (2005) Severin et al. (2007) Sinha et al. (2005) Tanca et al. (2013) Vipond et al. (2006) Wilmes and Bond (2004) Xu et al. (2005) Ying et al. (2005) Zielke et al. (2014)
(Santoni et al., 2000). Several improvements have been made in an attempt to alleviate these issues with the 2D-PAGE approach as well as improving the coverage of membrane proteins in general. For example, using various detergents such as zwitterionic or anionic detergents have been shown to improve the solubility of membrane proteins (Vuckovic et al., 2013). Other sample preparation methods, such as cell surface and membrane shaving (Solis and Cordwell, 2011), biotinylation (Macher and Yen, 2007), use of various organic solvents such as methanol (Blonder et al., 2006), and synthetic membrane systems known as nanodiscs (Yan et al., 2011) can be utilized to increase the efficiency and purification of bacterial membrane proteins prior to MS analysis. These and other membrane protein extraction techniques are extensively covered in a recent review (Vuckovic et al., 2013). Despite several improvements made to the 2D-PAGE MS proteomics approach, the overall limitations with respect to analysis of membrane proteins lead to the recent application of gel-free MS based approaches, better known as “shotgun” proteomics. It is very well suited to identify proteins in complex mixtures in a very robust and high throughput manner. This mini-review will focus on these gel-free MS based approaches that have provided new information about bacterial membrane proteins especially within the context of quantitative proteomics, post translational modifications (PTMs) and pathogenicity.
example, this approach identified a large number of insoluble ABC transporters (protein family of integral membrane proteins) in Geobacillus thermoleovorans and Oceanobacillus iheyensis (Graham et al., 2006, 2007). Importantly, these workflows have the possibility to produce a comprehensive coverage of bacterial membrane proteins even without specialized enrichment procedures. To demonstrate this, for the purpose of this review we re-analyzed our recently published proteogenomics study in Escherichia coli, which with 2626 identified proteins represents one of the largest proteome datasets of this organism to date (Krug et al., 2013). We predicted how many membrane proteins were identified in this study, using a Hidden Markov Model approach as previously described (Krogh et al., 2001). The criterion was that each protein must have been predicted with at least one transmembrane domain, and that at least 18 amino acids or more are predicted to be located inside the membrane. This resulted in 503 hits out of a potential 898 total hits to be predicted as membrane proteins in the E. coli proteome, corresponding to nearly a 60% coverage of membrane proteins without any specialized enrichment. It is noteworthy to mention that other similar proteomics approaches previously performed could potentially show this same trend of membrane protein coverage without specialized membrane protein extraction protocols (Macek et al., 2007, 2008; Ravikumar et al., 2014; Soares et al., 2013; Soufi et al., 2008, 2010; Wolff et al., 2007).
MS-based shotgun proteomics Quantitative proteomics A typical shotgun MS-based proteomics workflow is explained in detail in Fig. 1. One critical advantage of this approach is that the entire protein content of a cell can be extracted utilizing a variety of methods depending on the type of sample and analysis that is required. For example, in the context of bacterial membrane proteins, detergent based extraction methods have been widely employed as they serve as an efficient means to isolate many insoluble and low abundant targets (Vuckovic et al., 2013). One such detergent known as sodium dodecyl sulfate (SDS) is very popular in large scale global proteomics studies. Specific techniques that are compatible with the shotgun proteomics platform, such as filteraided sample preparation (FASP) (Wisniewski et al., 2009), have been developed in order to utilize SDS and subsequently remove it prior to protein digestion, as the presence of even minor amounts of SDS can cause problems with chromatography as well as the inhibition of many proteases such as trypsin. These workflows can be universally employed to virtually any type of biological organism or material, and have been utilized for identification of many bacterial membrane proteins. For
One important advantage of the Shotgun MS-based proteomics approaches is that they have the ability to adapt quantitative proteomics strategies that can compare two or more perturbations or conditions in a biological system in a proteome-wide global approach. Several methodologies exist each with their respective advantages as well as limitations. One common approach is to utilize stable (non-radioactive) isotope labelling to quantify the relative abundance of a protein from different samples directly from the acquired mass spectral data. Two major strategies for introducing a stable isotope label into the proteome exist: chemical and metabolic labelling. One strategy is known as stable isotope labelling by amino acids in cell culture (SILAC) (Ong et al., 2002), which has been successfully applied to many bacterial systems (Ravikumar et al., 2014; Soufi et al., 2010). SILAC metabolically (in vivo) incorporates the label, which is typically introduced in form of labelled lysine, arginine or another amino acid. Protein samples from two or three states are labelled with either unlabeled (“light”) or labelled (“heavy”) version of the amino acid, and can
Please cite this article in press as: Soufi, B., Macek, B., Global analysis of bacterial membrane proteins and their modifications. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2014.12.017
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Fig. 1. Quantitative proteomics workflow. (A) Cells are grown in culture (solid or liquid) and lysed using various methods such as the SDS detergent based approach which is advantageous for membrane protein coverage. (B) Protein extracts are digested using either in-solution or via 1-D SDS–PAGE approaches by various endoproteinases such as LysC or trypsin, followed by enrichment of various PTMs (if applicable). (C) For proteome quantitation, peptide digests can be separated according to their pI-value by iso-electric focusing or 1-D SDS PAGE. (D) Resulting peptide mixtures are separated on nanoflow HPLC and directly measured in a mass spectrometer capable of high resolution. (E) Relative peptide quantitation is based on the first stage of mass spectrometry (Full-scan MS) (F) Peptide identification is achieved by fragmentation in the second stage of mass spectrometry (MS/MS). (G) Data is processed using various bioinformatic workflows. The resulting data is utilized in the interpretation and ultimately the answer to the specific biological topic in question.
be relatively quantified in terms of protein abundance between them. In order to ensure full amino acid incorporation, the cell must not be able to synthesize it endogenously. This implies that some bacterial species must be rendered auxotrophic for a specific amino acid by knocking out the genes involved its biosynthetic pathway, which could present a possible limitation if the required mutagenesis cannot be performed. The labelled and unlabeled samples are mixed in equimolar amounts and digested using a specific endoprotease prior to MS measurement, in which intensities and fragmentation patterns of peptides are measured and used for subsequent quantification and identification of differently regulated proteins in the two samples. In terms of bacterial membrane studies, the SILAC approach was successfully employed in combination with nanodisc-based technology to study soluble interaction partners of E. coli membrane proteins in global and quantitative fashion (Zhang et al., 2012). Moreover, SILAC was also performed in Bifidobacterium longum in order to achieve a better understanding of bile tolerance in this organism (Ruiz et al., 2009). To this end, the authors identified 141 membrane proteins in a quantitative fashion, thus far the largest cell-envelope proteome obtained in B. longum. One limitation to these approaches is that they do not provide any insights into the absolute molar amount or concentration of a particular protein per cell. Recently, numerous techniques were developed to address this issue that are compatible with relative quantitation approaches, providing further valuable biological information. These include Absolute QUAntification (AQUA) (Gerber et al., 2003), FlexiQuant (Singh et al., 2009), protein standard absolute quantification (PSAQ) (Brun et al., 2007), absolute quantification using protein epitope signature tags (PrEST)
(Zeiler et al., 2012), and intensity based absolute quantification (iBAQ) (Schwanhausser et al., 2011). All of these methodologies can be applied in bacteria, and can be used to elucidate the absolute amounts of bacterial membrane proteins compared to for example their cytoplasmic protein counterparts, or to better understand the stoichiometry of membrane protein complexes. It is quite clear that these techniques will allow a better understanding of many biological processes in a global systems biology approach. Proteomics and posttranslational modifications Post-translational modifications (PTMs) of proteins are employed by all living cells as a very efficient means of signal transduction and regulation. Historically, characterizing and identifying PTMs in bacteria was extremely challenging and complex compared to their eukaryotic counterparts. This is largely due to the fact that generally PTMs are difficult to detect due to their low abundance and stoichiometry. Due to advances is MS instrumentation and biochemical sample preparation methods, which can now efficiently enrich for modified peptides from extremely complex peptide mixtures, many of these past issues have been circumvented which paved the way for many PTMs to be studied in bacteria. These include but are not limited to phosphorylation, acetylation, AMPylation, glucosylation, ADP ribosylation, and deamidation. One such case where this technology better known as gel-free, site-specific, quantitative phosphoproteomics has significantly advanced our knowledge with regards to bacterial modifications is in the area of serine/threonine/tyrosine (Ser/Thr/Tyr) phosphorylation. Despite early evidence that this modification may play
Please cite this article in press as: Soufi, B., Macek, B., Global analysis of bacterial membrane proteins and their modifications. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2014.12.017
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important roles in bacterial physiology (Dadssi and Cozzone, 1990; Forchhammer and Tandeau de Marsac, 1995; Kirstein et al., 2005; Reizer et al., 1996), Ser/Thr/Tyr phosphorylation has been thought to occur predominantly in eukaryotes and to be not functionally significant in bacteria. However due to advances in MS based proteomics, combined with powerful phosphorylation enrichment strategies such as phosphopeptide immunoprecipitation (Rush et al., 2005), immobilized metal ion affinity chromatography (IMAC) (Villen and Gygi, 2008) or titanium dioxide chromatography (TiO2 ) (Pinkse et al., 2004) have led to many important discoveries in bacterial Ser/Thr/Tyr phosphorylation (Cousin et al., 2013; Macek and Mijakovic, 2011). Many of these studies also detected a large portion of modified membrane proteins. The knowledge of the existence of such PTMs could elucidate a better understanding of the function of bacterial membrane proteins. For example, a recent phosphoproteomic study in Helicobacter Pylori employed the TiO2 enrichment strategy to perform a global site specific enrichment of Ser/Thr/Tyr phosphorylated proteins (Ge et al., 2011). To this end, authors found that many of these phosphorylation events were overrepresented in numerous membrane proteins, indicating that these proteins and their respective phosphorylation events could be an important mechanism in virulence control. Moreover, several US, EU and national consortia, such as the Comprehensive Research Centre (CRC) 766 in Germany, are currently applying state of the art (phospho) proteomics technologies towards projects involving diverse bacterial species such as Streptomyces coelicolor, Staphylococcus aureus and Synechocystis sp. PCC 6803, in order to elucidate the potential role of phosphorylation on both the cell envelope and membrane components. For example, the PII nitrogen regulation proteins of cyanobacterium Synechocystis are dephosphorylated by the PphA phosphatase which affects the binding properties towards different protein interactors (Kloft et al., 2005). Within CRC766 we are currently establishing a MS based phosphoproteomics workflow in order to identify the phosphorylation dependent interaction partners and potentially the modifying kinase of PII (Spät, Forchhammer, unpublished data). In addition, we could show that the serine/threonine kinase PkaI (SCO4778) controls activity of the Streptomyces Spore wall Synthesizing Complex (SSSC) in S. coelicolor by phosphorylating MreC and PBP2 (Ladwig et al., in preparation). We are currently applying phosphoproteomics workflows towards elucidating the role of PkaI during morphological differentiation and identify additional target proteins of PkaI.
Proteomics of membrane proteins involved in virulence Situated at the interface between the bacterial cell and its environment, many bacterial membrane proteins play a crucial role in disease. Gel free MS based quantitative and qualitative Shotgun proteomics approaches have made significant contributions towards discoveries in the involvement of bacterial membrane proteins roles in virulence and pathogenicity. As previously mentioned, the bacterial cell envelope is a crucial component in maintaining cellular shape and homeostasis. It also plays an important role in antibiotic resistance, and has developed resistance mechanisms against many antibiotics including but not limited to beta-lactams, methicillin, and fosfomycin (Bush, 2012; Hempel et al., 2011). Resistance to beta-lactam and macrolide antibiotics has been observed in many bacteria, especially those of the Gram negative species (Poole, 2001). Many outer membrane proteins (OMP) as well as porins (ß-barrel proteins that extend across the outer membrane) play an important part of these resistance mechanisms by controlling the membrane permeability of the cell. In a shotgun-based proteomic study an altered outer membrane protein profile was found in clarithromycin resistant
Helicobacter pylori (Smiley et al., 2013). Further proteomic based studies describing the importance of OMPs is extensively described elsewhere (Lima et al., 2013). Besides elucidating the important roles of bacterial antibiotic resistance mechanisms, Gel-free MS based shotgun proteomics has eluded to the importance of membrane proteins and disease in many other aspects. For example, many bacterial strains are used to produce probiotics for human consumption which are associated with numerous health benefitting properties. A recent shotgun proteomics approach was applied to the popular probiotic strain Bifidobacterium animalis subsp. Lactis BB-12 in an attempt to characterize the membrane proteome in order to gain a better understanding of oligosaccharide transport in this bacterium (Gilad et al., 2012). The authors identified 248 predicted membrane proteins, of which 90 were predicted to contain at least one transmembrane segment, and further showed that a large proportion of them were implicated to be involved in the transport of many compounds such as amino acids, oligosaccharides and nucleotides. MS based proteomics has also provided many important aspects with regards to those bacterial pathogens that depend on successful adherence in order to influence their pathogenic effects on their hosts. For example, adherence is necessary in order for secretion systems such as Type III secretion systems to act, which are very commonly utilized in the virulence of Gram negative bacterial pathogens. A recent study performed a global quantitative proteomics analysis employing the SILAC approach to study these Type III secretion systems in different mutated strains of enteropathogenic Escherichia coli (EPEC) (Deng et al., 2012). The authors identified many known as well as putative periplasmic and outer membrane proteins suggesting that these Type III secretion systems in EPEC may be composed of more membrane proteins as well as other uncharacterized proteins than initially thought, providing new insights into the potential virulence and pathogenic roles that these membrane proteins might play. Conclusions and future perspectives Modern Gel-free based shotgun proteomics approaches have evolved within the last decade into a powerful research platform for many different applications in bacteria such as comprehensive analysis of proteomes and sub-proteomes (e.g. membrane proteome) as well as relative and absolute quantitation of proteins and their modifications. We believe that the improvements in MS instrumentation (sensitivity, accuracy, speed) combined with numerous advances towards the enrichment protocols of bacterial membrane proteins will lead to routine identification and characterization of bacterial membrane proteins in a systematic and robust fashion. The major technological advances in MS-based quantitative proteomics are therefore transforming the research in the field of microbiology, providing new insights in bacterial virulence mechanisms, signal transduction as well as providing an essential connection to the rapidly evolving and dynamic area of systems biology. Acknowledgement Authors wish to acknowledge German Research Foundation (DFG, SFB766) for funding. References Altindis, E., Fu, Y., Mekalanos, J.J., 2014. Proteomic analysis of Vibrio cholerae outer membrane vesicles. Proc. Natl. Acad. Sci. U.S.A. 111, E1548–E1556. Baik, S.C., Kim, K.M., Song, S.M., Kim, D.S., Jun, J.S., Lee, S.G., Song, J.Y., Park, J.U., Kang, H.L., Lee, W.K., Cho, M.J., Youn, H.S., Ko, G.H., Rhee, K.H., 2004. Proteomic analysis of the sarcosine-insoluble outer membrane fraction of Helicobacter pylori strain 26695. J. Bacteriol. 186, 949–955.
Please cite this article in press as: Soufi, B., Macek, B., Global analysis of bacterial membrane proteins and their modifications. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2014.12.017
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