Proteome of the Escherichia coli envelope and technological challenges in membrane proteome analysis

The envelope of Escherichia coli is a complex organelle composed of the outer membrane, periplasm-peptidoglycan layer and cytoplasmic membrane. Each compartment has a unique complement of proteins, the proteome. Determining the proteome of the envelope is essential for developing an in silico bacterial model, for determining cellular responses to environmental alterations, for determining the function of proteins encoded by genes of unknown function and for development and testing of new experimental technologies such as mass spectrometric methods for identifying and quantifying hydrophobic proteins. The availability of complete genomic information has led several groups to develop computer algorithms to predict the proteome of each part of the envelope by searching the genome for leader sequences, β-sheet motifs and stretches of α-helical hydrophobic amino acids. In addition, published experimental data has been mined directly and by machine learning approaches. In this review we examine the somewhat confusing available literature and relate published experimental data to the most recent gene annotation of E. coli to describe the predicted and experimental proteome of each compartment. The problem of characterizing integral versus membrane-associated proteins is discussed. The E. coli envelope proteome provides an excellent test bed for developing mass spectrometric techniques for identifying hydrophobic proteins that have generally been refractory to analysis. We describe the gel based and solution based proteome analysis approaches along with protein cleavage and proteolysis methods that investigators are taking to tackle this difficult problem.     

Comparative analysis of envelope proteomes in Escherichia coli B and K-12 strains

Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane beta-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.     

Exploring the membrane proteome—Challenges and analytical strategies

The analysis of proteins in biological membranes forms a major challenge in proteomics. Despite continuous improvements and the development of more sensitive analytical methods, the analysis of membrane proteins has always been hampered by their hydrophobic properties and relatively low abundance. In this review, we describe recent successful strategies that have led to in-depth analyses of the membrane proteome. To facilitate membrane proteome analysis, it is essential that biochemical enrichment procedures are combined with special analytical workflows that are all optimized to cope with hydrophobic polypeptides. These include techniques for protein solubilization, and also well-matched developments in protein separation and protein digestion procedures. Finally, we discuss approaches to target membrane–protein complexes and lipid–protein interactions, as such approaches offer unique insights into function and architecture of cellular membranes.     

Proteomics of total membranes and subcellular membranes

Membrane proteins are key molecules in the cell and are important targets for drug development. Much effort has, therefore, been directed towards research of this group of proteins, but their hydrophobic nature can make working with them challenging. Here we discuss methodologies used in the study of the membrane proteome, specifically discussing approaches that circumvent technical issues specific to the membrane. In addition, we review several techniques used for visualization, qualification, quantitation and localization of membrane proteins. The combination of the techniques we describe holds great promise to allow full characterization of the membrane proteome and to map the dynamic changes within it essential for cellular function.     

Proteomics of total membranes and subcellular membranes

Membrane proteins are key molecules in the cell and are important targets for drug development. Much effort has, therefore, been directed towards research of this group of proteins, but their hydrophobic nature can make working with them challenging. Here we discuss methodologies used in the study of the membrane proteome, specifically discussing approaches that circumvent technical issues specific to the membrane. In addition, we review several techniques used for visualization, qualification, quantitation and localization of membrane proteins. The combination of the techniques we describe holds great promise to allow full characterization of the membrane proteome and to map the dynamic changes within it essential for cellular function.     

Genome-wide identification of the subcellular localization of the Escherichia coli B proteome using experimental and computational methods

Escherichia coli K-12 and B strains have most widely been employed for scientific studies as well as industrial applications. Recently, the complete genome sequences of two representative descendants of E. coli B strains, REL606 and BL21(DE3), have been determined. Here, we report the subproteome reference maps of E. coli B REL606 by analyzing cytoplasmic, periplasmic, inner and outer membrane, and extracellular proteomes based on the genome information using experimental and computational approaches. Among the total of 3487 spots, 651 proteins including 410 non-redundant proteins were identified and characterized by 2-DE and LC-MS/MS; they include 440 cytoplasmic, 45 periplasmic, 50 inner membrane, 61 outer membrane, and 55 extracellular proteins. In addition, subcellular localizations of all 4205 ORFs of E. coli B were predicted by combined computational prediction methods. The subcellular localizations of 1812 (43.09%) proteins of currently unknown function were newly assigned. The results of computational prediction were also compared with the experimental results, showing that overall precision and recall were 92.16 and 92.16%, respectively. This work represents the most comprehensive analyses of the subproteomes of E. coli B, and will be useful as a reference for proteome profiling studies under various conditions. The complete proteome data are available online (http://ecolib.kaist.ac.kr).     

Consequences of depletion of the signal recognition particle in Escherichia coli

Thus far, the role of the Escherichia coli signal recognition particle (SRP) has only been studied using targeted approaches. It has been shown for a handful of cytoplasmic membrane proteins that their insertion into the cytoplasmic membrane is at least partially SRP-dependent. Furthermore, it has been proposed that the SRP plays a role in preventing toxic accumulation of mistargeted cytoplasmic membrane proteins in the cytoplasm. To complement the targeted studies on SRP, we have studied the consequences of the depletion of the SRP component Fifty-four homologue (Ffh) in E. coli using a global approach. The steady-state proteomes and the proteome dynamics were evaluated using one- and two-dimensional gel analysis, followed by mass spectrometry-based protein identification and immunoblotting. Our analysis showed that depletion of Ffh led to the following: (i) impaired kinetics of the biogenesis of the cytoplasmic membrane proteome; (ii) lowered steady-state levels of the respiratory complexes NADH dehydrogenase, succinate dehydrogenase, and cytochrome bo(3) oxidase and lowered oxygen consumption rates; (iii) increased levels of the chaperones DnaK and GroEL at the cytoplasmic membrane; (iv) a σ(32) stress response and protein aggregation in the cytoplasm; and (v) impaired protein synthesis. Our study shows that in E. coli SRP-mediated protein targeting is directly linked to maintaining protein homeostasis and the general fitness of the cell.     

Efficient subfractionation of gram-negative bacteria for proteomics studies

Proteomics studies of pathogenic bacteria are an important basis for biomarker discovery and for the development of antimicrobial drugs and vaccines. Especially where vaccines are concerned, it is of great interest to explore which bacterial factors are exposed on the bacterial cell surface and thus can be directly accessed by the immune system. One crucial step in proteomics studies of bacteria is an efficient subfractionation of their cellular compartments. We set out to compare and improve different protocols for the fractionation of proteins from Gram-negative bacteria into outer membrane, cytoplasmic membrane, periplasmic, and cytosolic fractions, with a focus on the outer membrane. Overall, five methods were compared, three methods for the fast isolation of outer membrane proteins and two methods for the fractionation of each cellular compartment, using Escherichia coli BL21 as a model organism. Proteins from the different fractions were prepared for further mass spectrometric analysis by SDS gel electrophoresis and consecutive in-gel tryptic digestion. Most published subfractionation protocols were not explicitly developed for proteomics applications. Thus, we evaluated not only the separation quality of the five methods but also the suitability of the samples for mass spectrometric analysis. We could obtain high quality mass spectrometry data from one-dimensional SDS-PAGE, which greatly reduces experimental time and sample amount compared to two-dimensional electrophoresis methods. We then applied the most specific fractionation technique to different Gram-negative pathogens, showing that it is efficient in separating the subcellular proteomes independent of the species and that it is capable of producing high-quality proteomics data in electrospray ionization mass spectrometry.     

Hiding behind hydrophobicity. Transmembrane segments in mass spectrometry

Proteomics of membrane proteins is essential for the understanding of cellular function. However, mass spectrometric analysis of membrane proteomes has been less successful than the proteomic determination of soluble proteins. To elucidate the mystery of transmembrane proteins in mass spectrometry, we present a detailed statistical analysis of experimental data derived from chloroplast membranes. This approach was further accomplished by the analysis of the Arabidopsis thaliana proteome after in silico digestion. We demonstrate that both the length and the hydrophobicity of the proteolytic fragments containing transmembrane segments are major determinants for detection by mass spectrometry. Based on a comparative analysis, we discuss possibilities to overcome the problem and provide possible protocols to shift the hydrophobicity of transmembrane segment-containing peptides to facilitate their detection.     

Protein identification in complex mixtures

This paper investigates the prospects of successful mass spectrometric protein identification based on mass data from proteolytic digests of complex protein mixtures. Sets of proteolytic peptide masses representing various numbers of digested proteins in a mixture were generated in silico. In each set, different proteins were selected from a protein sequence collection and for each protein the sequence coverage was randomly selected within a particular regime (15-30% or 30-60%). We demonstrate that the Probity algorithm, which is characterized by an optimal tolerance for random interference, employed in an iterative procedure can correctly identify >95% of proteins at a desired significance level in mixtures composed of hundreds of yeast proteins under realistic mass spectrometric experimental constraints. By using a model of the distribution of protein abundance, we demonstrate that the very high efficiency of identification of protein mixtures that can be achieved by appropriate choices of informatics procedures is hampered by limitations of the mass spectrometric dynamic range. The results stress the desire to choose carefully experimental protocols for comprehensive proteome analysis, focusing on truly critical issues such as the dynamic range, which potentially limits the possibilities of identifying low abundance proteins.     

Combining experimental and predicted datasets for determination of the subcellular location of proteins in Arabidopsis

Substantial experimental datasets defining the subcellular location of Arabidopsis (Arabidopsis thaliana) proteins have been reported in the literature in the form of organelle proteomes built from mass spectrometry data (approximately 2,500 proteins). Subcellular location for specific proteins has also been published based on imaging of chimeric fluorescent fusion proteins in intact cells (approximately 900 proteins). Further, the more diverse history of biochemical determination of subcellular location is stored in the entries of the Swiss-Prot database for the products of many Arabidopsis genes (approximately 1,800 proteins). Combined with the range of bioinformatic targeting prediction tools and comparative genomic analysis, these experimental datasets provide a powerful basis for defining the final location of proteins within the wide variety of subcellular structures present inside Arabidopsis cells. We have analyzed these published experimental and prediction data to answer a range of substantial questions facing researchers about the veracity of these approaches to determining protein location and their interrelatedness. We have merged these data to form the subcellular location database for Arabidopsis proteins (SUBA), providing an integrated understanding of protein location, encompassing the plastid, mitochondrion, peroxisome, nucleus, plasma membrane, endoplasmic reticulum, vacuole, Golgi, cytoskeleton structures, and cytosol (www.suba.bcs.uwa.edu.au). This includes data on more than 4,400 nonredundant Arabidopsis protein sequences. We also provide researchers with an online resource that may be used to query protein sets or protein families and determine whether predicted or experimental location data exist; to analyze the nature of contamination between published proteome sets; and/or for building theoretical subcellular proteomes in Arabidopsis using the latest experimental data.     

Effect of mutation in phoB and phoM genes for the positive control of alkaline phosphatase biosynthesis on Escherichia coli envelope proteins

The suppression of some envelope proteins, localized in both the periplasm and the outer and inner membranes was shown in phoB and phoM phoR mutants of E. coli. Among these proteins are the proteins of the phosphate regulon and also those not pertaining them. As a result of phoB and phoM phoR mutations, the cytoplasmic membrane was found to be lacking in minor protein of 28,000 Mr, which belongs to the phosphate regulon. Besides, the phoM phoR mutation leads to the loss of protein of 55,000 Mr of the outer membranes, whereas phoB mutation causes loss of protein 37 000 Mr, identified as outer membrane protein OmpT. A damage in the phoB mutant of the protein proteolytic modification, probably determining the suppression of the biosynthesis of E. coli envelope secreted proteins is suggested.     

Systematic identification of the subproteome of Escherichia coli cell envelope reveals the interaction network of membrane proteins and membrane-associated peripheral proteins

Membrane proteins of Gram-negative bacteria are key molecules that interface the cells with the environment. Despite recent proteomic identification of numerous oligomer proteins in the Escherichia coli cell envelope, the protein complex of E. coli membrane proteins and their peripherally associated proteins remain ill-defined. In the current study, we systematically analyze the subproteome of E. coli cell envelope enriched in sarcosine-insoluble fraction (SIF) and sarcosine-soluble fraction (SSF) by using proteomic methodologies. One hundred and four proteins out of 184 spots on 2D electrophoresis gels are identified, which includes 31 outer membrane proteins (OMPs). Importantly, our further proteomic studies reveal a number of previously unrecognized membrane-interacting protein complexes, such as the complex consisting of OmpW and fumarate reductase. This established complete proteomic profile of E. coli envelope also sheds new insight into the function(s) of E. coli outer envelope.     

Fractionation of soluble proteins in Escherichia coli using DEAE-, SP-, and phenyl sepharose chromatographies.

In an effort to simplify a complex mixture of soluble proteins from Escherichia coli, methods to fractionate the samples prior to two-dimensional (2D) gel electrophoresis were developed. These methods involve the use of DEAE-Sepharose, SP-Sepharose, and phenyl Sepharose chromatographic columns and the fractionation of the protein mixtures based on differential anionic, cationic, and hydrophobic properties of the proteins, respectively. Fractionation of the soluble proteins from an E. coli extract with DEAE-Sepharose resulted in a threefold increase in the number of detectable 2D gel spots. These gel spots were amenable to protein identification by using in-gel trypsin digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and peptide mass fingerprinting. Significantly, the DEAE-Sepharose column fractionation effectively partitioned the soluble proteins from the cell extracts. Similarly, an SP-Sepharose column was used to fractionate the soluble proteins from E. coli and resulted in over a twofold increase in the number of detectable gel spots. Lastly, fractionation of the cell extract with the phenyl Sepharose column resulted in a threefold increase in the number of detectable 2D gel spots. This work describes an easy, inexpensive way to fractionate the soluble proteins in E. coli and a way to better profile the E. coli proteome.     

Profiling the membrane proteome of Shewanella oneidensis MR-1 with new affinity labeling probes

The membrane proteome plays a critical role in electron transport processes in Shewanella oneidensis MR-1, a bacterial organism that has great potential for bioremediation. Biotinylation of intact cells with subsequent affinity-enrichment has become a useful tool for characterization of the membrane proteome. As opposed to these commonly used, water-soluble commercial reagents, we here introduce a family of hydrophobic, cell-permeable affinity probes for extensive labeling and detection of membrane proteins. When applied to S. oneidensis cells, all three new chemical probes allowed identification of a substantial proportion of membrane proteins from total cell lysate without the use of specific membrane isolation method. From a total of 410 unique proteins identified, approximately 42% are cell envelope proteins that include outer membrane, periplasmic, and inner membrane proteins. This report demonstrates the first application of this intact cell biotinylation method to S. oneidensis and presents the results of many identified proteins that are involved in metal reduction processes. As a general labeling method, all chemical probes we introduced in this study can be extended to other organisms or cell types and will help expedite the characterization of membrane proteomes.     

Characterization of the Salmonella typhimurium proteome by semi-automated two dimensional HPLC-mass spectrometry: detection of proteins implicated in multiple antibiotic resistance

The proteome of Salmonella enterica serovar Typhimurium was characterized by 2-dimensional HPLC mass spectrometry to provide a platform for subsequent proteomic investigations of low level multiple antibiotic resistance (MAR). Bacteria (2.15 +/- 0.23 x 10(10) cfu; mean +/- s.d.) were harvested from liquid culture and proteins differentially fractionated, on the basis of solubility, into preparations representative of the cytosol, cell envelope and outer membrane proteins (OMPs). These preparations were digested by treatment with trypsin and peptides separated into fractions (n = 20) by strong cation exchange chromatography (SCX). Tryptic peptides in each SCX fraction were further separated by reversed-phase chromatography and detected by mass spectrometry. Peptides were assigned to proteins and consensus rank listings compiled using SEQUEST. A total of 816 +/- 11 individual proteins were identified which included 371 +/- 33, 565 +/- 15 and 262 +/- 5 from the cytosolic, cell envelope and OMP preparations, respectively. A significant correlation was observed (r2 = 0.62 +/- 0.10; P < 0.0001) between consensus rank position for duplicate cell preparations and an average of 74 +/- 5% of proteins were common to both replicates. A total of 34 outer membrane proteins were detected, 20 of these from the OMP preparation. A range of proteins (n = 20) previously associated with the mar locus in E. coli were also found including the key MAR effectors AcrA, TolC and OmpF.     

Structural elucidation of critical residues involved in binding of human monoclonal antibodies to hepatitis C virus E2 envelope glycoprotein

Human monoclonal antibodies derived from B cells of HCV-infected individuals provide information on the immune response to native HCV envelope proteins as they are recognized during infection. Monoclonal antibodies have been useful in the determination of the function and structure of specific immunogenic domains of proteins and should also be useful for the structure/function characterization of HCV E1 and E2 envelope glycoproteins. The HCV E2 envelope glycoprotein has at least three immunodistinctive conformation domains, designated A, B, and C. Conformational epitopes within domain B and C are neutralizing antibody targets on HCV pseudoparticles as well as from infectious cell culture virus. In this study, a combination of differential surface modification and mass spectrometric limited proteolysis followed by alanine mutagenesis was used to provide insight into potential conformational changes within the E2 protein upon antibody binding. The arginine guanidine groups in the E2 protein were modified with CHD in both the affinity bound and free states followed by mass spectrometric analysis, and the regions showing protection upon antibody binding were identified. This protection can arise by direct contact between the residues and the monoclonal antibody, or by antibody-induced conformational changes. Based on the mass spectrometric data, site-directed mutagenesis experiments were performed which clearly identified additional amino acid residues on E2 distant from the site of antibody interaction, whose change to alanine inhibited antibody recognition by inducing conformational changes within the E2 protein.     

Mass spectrometric analysis of integral membrane proteins at the subpicomolar level: application to rhodopsin

Integral membrane proteins are among the most interesting molecules for biomedical research, as some of the most important cellular functions are inherently tied to biological membranes. One such example is the vast expanse of receptors on cell surfaces. However, due to difficulties in the biochemical purification and structure/function analysis of membrane proteins, caused by their hydrophobic or amphophilic nature, membrane proteins are still much less studied than soluble proteins. Our laboratory has successfully developed and applied a methodology for the mass spectrometric analysis of integral membrane proteins. Here, we present an improvement in the sensitivity of detection made possible by the advancement of mass spectrometric instrumentation and refinement of the chromatographic analysis. Subpicomolar samples of bovine rhodopsin purified from native membranes were successfully analyzed, obtaining complete sequence coverage and the detection and localization of posttranslational modifications. Therefore, it is demonstrated that the detection limits and sequence coverage for soluble and membrane proteins can be comparable. The methodology presented here allows mass spectrometric analysis of subpicomolar levels of photopigments or other integral membrane proteins either from their native membranes or as products of expression systems.     

An efficient strategy for high throughput screening of recombinant integral membrane protein expression and stability

Membrane proteins account for about 30% of the genomes sequenced to date and play important roles in a variety of cellular functions. However, determining the three-dimensional structures of membrane proteins continues to pose a major challenge for structural biologists due to difficulties in recombinant expression and purification. We describe here a high throughput pipeline for Escherichia coli based membrane protein expression and purification. A ligation-independent cloning (LIC)-based vector encoding a C-terminal green fluorescence protein (GFP) tag was used for cloning in a high throughput mode. The GFP tag facilitated expression screening in E. coli through both cell culture fluorescence measurements and in-gel fluorescence imaging. Positive candidates from the GFP screening were subsequently sub-cloned into a LIC-based, GFP free vector for further expression and purification. The expressed, C-terminal His-tagged membrane proteins were purified via membrane enrichment and Ni-affinity chromatography. Thermofluor technique was applied to screen optimal buffers and detergents for the purified membrane proteins. This pipeline has been successfully tested for membrane proteins from E. coli and can be potentially expanded to other prokaryotes.