Implementation of Freeman-Wimley prediction algorithm in a web-based application for in silico identification of beta-barrel membrane proteins

Subcellular fractionation of proteins is a preferred method of choice for detection and identification of proteins from complex mixtures such as bacterial cells. To characterize the membrane proteins of the Antarctic bacterium Pseudomonas syringae Lz4W, the membrane fractions were prepared using three different methods, namely Triton X-100 solubilization, sucrose density gradient, and carbonate extraction methods. The proteins were separated on one-dimensional polyacrylamide gels and analyzed using a combination of liquid chromatography-coupled electrospray ionization-MS. The membrane proteins that were prepared by carbonate extraction were separated on two-dimensional PAGE in different pI ranges using the detergent 2% amidosulfobetaine (ASB). The proteins were then subjected to matrix-assisted laser desorption ionization-time-of-flight/time-of-flight for analysis and identification. Because the genome sequence of P. syringae Lz4W is not known, the proteins were identified by using the relevant sequence databases of the Pseudomonas sp available at National Centre for Biotechnology Information (NCBI). The sequence identification of some tryptic peptides were validated by de novo sequencing and others by chemical modification and mass spectrometry. The peptide sequences of P. syringae Lz4W were then matched with the sequences of the peptides from different Pseudomonas sp. by similarity search of the proteins from different species using clustal W2 program. Thus by using a combination of the methods, we have been able to identify large number of proteins of this bacterial strain, which include most of the outer membrane proteins.

Application of Freeman-Wimley prediction algorithm to computational identification of beta-barrel membrane proteins in mycoplasma predicted proteomes: comparison with other methods

The eggs of oviparous animals are storehouses of maternal proteins required for embryonic development. Identification and molecular characterization of such proteins will provide much insight into the regulation of embryonic development. We previously analyzed soluble proteins in the eggs of the black widow spider (Latrodectus tredecimguttatus), and report here on the extraction and mass spectrometric identification of the egg membrane proteins. Comparison of different lysis solutions indicated that the highest extraction of the membrane proteins was achieved with 3%-4% sodium laurate in 40 mmol/L Tris-HCl buffer containing 4% CHAPS and 2% DTT (pH 7.4). SDS-PAGE combined with nLC-MS/MS identified 39 proteins with membrane-localization annotation, including those with structural, catalytic, and regulatory activities. Nearly half of the identified membrane proteins were metabolic enzymes involved in various cellular processes, particularly energy metabolism and biosynthesis, suggesting that relevant metabolic processes were active during the embryonic development of the eggs. Several identified cell membrane proteins were involved in the special structure formation and function of the egg cell membranes. The present proteomic analysis of the egg membrane proteins provides new insight into the molecular mechanisms of spider embryonic development.

Two-dimensional gel electrophoresis (2-D) of proteins combining with microsequencing analysis provides a powerful approach for the identification of thylakoid membrane proteins and the N-terminal sequence information for cloning of corresponding genes [see Shi-Gui Yu et al., Photosyn. Res. 41(1994) 475-486]. In order to purify and to characterize protein spots in the basic range of 2-D, we have modified the non-equilibrium pH-gradient electrophoresis (NEPHGE) gel system and adapted it to the membrane protein analysis. The 2-D protein pattern of spinach thylakoid membranes from the NEPHGE analysis shows about 50 polypeptide spots in the basic Ip area. On the basis of microsequence data and sequence comparison through databases, it has been found that a protein having a molecular size of 40 kDa and focusing at pH range of 7.8 is a novel one and its N-terminal 18 amino acid sequence is VSLPKEQLVTSLTQVEQT. This newly discovered protein has 66.7% identity and 100% similarity with the N-terminal region of a protein sequence deduced from EST (Expressed Sequence Tag) and the DNA sequence of the chromosome 5 from Arabidopsis thaliana. The molecular mass estimated from the amino acid sequence of this nuclei-encoded protein is also about 40 kDa which fits well with the novel one. The secondary structure analysis of this protein demonstrates a typical membrane spanning domain in its N-terminal region. Protein analysis for thylakoid fragments derived from different parts of the spinach thylakoids reveals that the novel protein is localized in both the stroma and the grana lamellae of chloroplasts.

Transmembrane beta barrel proteins (TMBs) play a major role in the normal functioning of the cell and are an important constituent of the translocation machinery of the outer membrane proteins in bacteria, mitochondria and chloroplast. Currently there are only around 36 experimental 3D structures available for TMBs in PDB (at 30% sequence identity). Furthermore, with large amounts of sequence data available from high-throughput methods, it is imperative to develop accurate and fast computational methods for their identification and topology prediction. Here, we present a concept for a fast method to identify TMBs and predict their topology. The method uses sparse encoded amino acid data as input and employs a Support Vector Machine (SVM) and a Hidden Markov Model (HMM) to generate accurate topologies. The topologies in the training phase are divided into pre-barrel state, outer-loop state, inner-loop state and the transmembrane beta-strand state. In the first stage, 4 separate SVMs are used to predict the local state preference for each residue. A profile generated from the probabilities thus obtained is used as input to the HMM stage to determine the overall topology. If the number of predicted strands is between 8 and 24, then the given sequence is identified as a TMB. We see the application of our method in the proteome-wide topology prediction of TMBs, where current methods might have a limitation due to the time consuming homologous sequence search step. Funding Christoph Peters: Travel Fellowship awarded by ISCB Student Council with financial support from EMBO, 2012 J-A Ekstroms travel stipend, 2012 A fast and accurate method for large-scale transmembrane beta barrel topology prediction

Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥ 2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.

Objective To clone, express and purify Chlamydia trachomatis polymorphic membrane protein (Pmp G), and to identify its immunogenicity. Methods The Pmp G gene of C. trachomatis serotype E was amplified by PCR, cloned into prokaryotic expression vector PET30a (+). The positive recombinant was transformed into the bacterium E coli (BL-21), identified by enzyme digestion, PCR amplification and gene sequencing. Then, it was induced to express followed by the identification of expression product with SDS-PAGE and Western blotting. The purified protein was used to immunize BALB/C mice to test its immunogenicity. Results PCR produced a 1092 bp-sized DNA fragment, which had a sequence consistent with that of PmpG gene of C. trachomatis E type in the GenBank database. The molecular weight of expression product was 55 kD, which was proved to be the expected size, and Western Blotting confirmed it to be the specific protein. Moreover, special antibodies to PmpG were induced to be generated by mice immunized with the purified protein. Conclusions The constructed prokaryotic expression vector for PmpG is expressed successfully in E. coli, and the expression product shows immunogenicity. Key words: Chlamydia trachomatis; Bacterial outer membrane proteins; Cloning, molecular; Recombinant proteins

Membrane proteins are attractive targets for proteomics research because of their vital roles in numerous processes of the cell including: cell adhesion, immune response, metabolism and signal transduction... At the start of proteomics research, two-dimensional gel electrophoresis (2D-PAGE) was routinely used to separate complex proteomic samples. However, this method faces difficulties in separating membrane proteins due to their hydrophobicity. In this study, mouse brain membrane fractions were prepared using carbonate extraction and ultracentrifugation. The separation and identification of the membrane proteins by using a gel-based approach in combination with two-dimensional nano liquid chromatography coupled online with tandem mass spectrometry (2DNanoLC-Q-TOF-MS/MS) were presented. In total, 298 identified membrane proteins from mouse brain tissues were verified and predicted by UniProt database, SOSUI and TMHMM algorithms. Among them, 129 (43.3%) proteins that have at least one transmembrane domain were predicted by SOSUI and TMHMM. Furthermore, the function, subcellular location and hydrophobicity value of the identified membrane proteins were also categorized.

The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological mechanisms that regulate proliferation and differentiation. The comparison of their membrane proteomes will help unravel the biological principles of pluripotency, and the identification of biomarker proteins in their plasma membranes is considered a crucial step to fully exploit pluripotent cells for therapeutic purposes. For these tasks, membrane proteomics is the method of choice, but as indicated by the scarce identification of membrane and plasma membrane proteins in global proteomic surveys it is not an easy task. In this minireview, we first describe the general challenges of membrane proteomics. We then review current sample preparation steps and discuss protocols that we found particularly beneficial for the identification of large numbers of (plasma) membrane proteins in human tumour- and embryo-derived stem cells. Our optimized assembled protocol led to the identification of a large number of membrane proteins. However, as the composition of cells and membranes is highly variable we still recommend adapting the sample preparation protocol for each individual system.

The core prerequisites for an efficient proteome-scale analysis of mammalian membrane proteins are effective isolation, solubilization, digestion and multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). This protocol is for analysis of the mammalian membrane proteome that relies on solubilization and tryptic digestion of membrane proteins in a buffer containing 60% (vol/vol) methanol. Tryptic digestion is followed by strong cation exchange (SCX) chromatography and reversed phase (RP) chromatography coupled online with MS/MS for protein identification. The use of a methanol-based buffer eliminates the need for reagents that interfere with chromatographic resolution and ionization of the peptides (e.g., detergents, chaotropes, inorganic salts). Sample losses are minimized because solubilization and digestion are carried out in a single tube avoiding any sample transfer or buffer exchange between these steps. This protocol is compatible with stable isotope labeling at the protein and peptide level, enabling identification and quantitation of integral membrane proteins. The entire procedure--beginning with isolated membrane fraction and finishing with MS data acquisition--takes 4-5 d.

ABSTRACTMembers of the genus Cronobacter are responsible for severe infections in infants and immunosuppressed individuals. Although several virulence factors have been described, many proteins involved in the pathogenesis of such infections have not yet been mapped. This study is the first to fractionate Cronobacter sakazakii cells into outer membrane, inner membrane, periplasmic, and cytosolic fractions as the basis for improved proteome mapping. A novel method was designed to prepare the fractionated samples for protein identification. The identification was performed via one-dimensional electrophoresis-liquid chromatography electrospray ionization tandem mass spectrometry. To determine the subcellular localization of the identified proteins, we developed a novel Python-based script (Subcelloc) that combines three web-based tools, PSORTb 3.0.2, CELLO 2.5, and UniProtKB. Applying this approach enabled us to identify 1,243 C. sakazakii proteins, which constitutes 28% of all predicted proteins and 49% of all theoretically expressed outer membrane proteins. These results represent a significant improvement on previous attempts to map the C. sakazakii proteome and could provide a major step forward in the identification of Cronobacter virulence factors.IMPORTANCE Cronobacter spp. are opportunistic pathogens that can cause rare and, in many cases, life-threatening infections, such as meningitis, necrotizing enterocolitis, and sepsis. Such infections are mainly linked to the consumption of contaminated powdered infant formula, with Cronobacter sakazakii clonal complex 4 considered the most frequent agent of serious neonatal infection. However, the pathogenesis of diseases caused by these bacteria remains unclear; in particular, the proteins involved throughout the process have not yet been mapped. To help address this, we present an improved method for proteome mapping that emphasizes the isolation and identification of membrane proteins. Specific focus was placed on the identification of the outer membrane proteins, which, being exposed to the surface of the bacterium, directly participate in host-pathogen interaction.

Membrane proteins play a large variety of functions in life and represent 30% of all genomes sequenced. Due to their hydrophobic nature, they are tightly bound to their biological membrane, and detergents are always required to extract and isolate them before identification by mass spectrometry (MS). The latter, however remains difficult. Peptide mass fingerprinting methods using techniques such as MALDI-TOF MS, for example, have become an important analytical tool in the identification of proteins. However, PMF of membrane proteins is a real challenge for at least three reasons. First, membrane proteins are naturally present at low levels; second, most of the detergents strongly inhibit proteases and have deleterious effects on MALDI spectra; and third, despite the presence of detergent, membrane proteins are unstable and often aggregate. We took the mitochondrial uncoupling protein 1 (UCP1) as a model and showed that differential acetonitrile extraction of tryptic peptides combined with the use of polystirene Bio-Beads triggered high resolution of the MALDI-TOF identification of mitochondrial membrane proteins solubilized either with Triton-X100 or CHAPS detergents.

Proteomics is potentially a powerful technology for elucidating brain function and neurodegenerative diseases. So far, the brain proteome has generally been analyzed by two-dimensional gel electrophoresis, which usually leads to the complete absence of membrane proteins. We describe a proteomic approach for profiling of plasma membrane proteins from mouse brain. The procedure consists of a novel method for extraction and fractionation of membranes, on-membrane digestion, diagonal separation of peptides, and high-sensitivity analysis by advanced MS. Breaking with the classical plasma membrane fractionation approach, membranes are isolated without cell compartment isolation, by stepwise depletion of nonmembrane molecules from entire tissue homogenate by high-salt, carbonate, and urea washes followed by treatment of the membranes with sublytic concentrations of digitonin. Plasma membrane is further enriched by of density gradient fractionation and protein digested on-membrane by endoproteinase Lys-C. Released peptides are separated, fractions digested by trypsin, and analyzed by LC-MS/MS. In single experiments, the developed technology enabled identification of 862 proteins from 150 mg of mouse brain cortex. Further development and miniaturization allowed analysis of 15 mg of hippocampus, revealing 1,685 proteins. More that 60% of the identified proteins are membrane proteins, including several classes of ion channels and neurotransmitter receptors. Our work now allows in-depth study of brain membrane proteomes, such as of mouse models of neurological disease.

Functional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes. Nevertheless, structural studies on this part of the proteome are limited. The present review attempts to cover the vast array of methods that have appeared in the last few years for separation and identification of photosynthetic proteins of thylakoid membranes present in chloroplasts, a good model for setting up analytical methods suitable for membrane proteins. The two major methods for the separation of thylakoid membrane proteins are gel electrophoresis and liquid chromatography. Isoelectric focusing in a first dimension followed by denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a second dimension is an effective way to resolve large numbers of soluble and peripheral membrane proteins. However, it is not applicable for isolation of native protein complexes or for the separation of highly hydrophobic membrane proteins. High-performance liquid chromatography (HPLC), on the other hand, is highly suitable for any type of membrane protein separation due to its compatibility with detergents that are necessary to keep the hydrophobic proteins in solution. With regard to the identification of the separated proteins, several methods are available, including immunological and mass spectrometric methods. Besides immunological identification, peptide mass fingerprinting, peptide fragment fingerprinting or intact molecular mass determination by electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been shown to be very sensitive and effective. In particular, identification of proteins by their intact molecular mass is advantageous for the investigation of numerous biological problems, because it is rapid and reflects the full sequence of the protein and all its posttranslational modifications. However, intact molecular mass determinations of gel-separated membrane proteins are hampered due to the difficulties in extracting the hydrophobic proteins from the gel, whereas HPLC on-line interfaced with ESI-MS enables the rapid and accurate determination of intact molecular masses and consequently an unequivocal protein identification. This strategy can be viewed as a multidimensional separation technique distinguishing between hydrophobicity in the first dimension and between different mass-to-charge ratios in the second dimension, allowing the separation and identification even of isomeric forms.

Over the last decade, several artificial devices, imitating functionalities found in nature, have emerged in the field of synthetic biology. Often they resemble cellular vesicles which carry out a defined function and where molecular transport is mediated via specific membrane proteins. This work describes the creation of a framework for the reconstitution of membrane proteins into synthetic membranes. The study of membrane proteins in terms of their structure (e.g. protein crystallization) and their detailed functionality requires the isolation and re-insertion into a non-native environment. A process called reconstitution which is considered delicate. Beside the commonly used phospholipids, which are part of the natural cell membrane, a membrane environment can be created by the use of amphiphilic block copolymers. Driven by self-assembly, these molecules can be used as a platform for nano-devices, as they can be decorated with active molecular compounds and the resulting membrane can incorporate membrane proteins. Various factors and their interplay and dependencies affect the outcome of the reconstitution of membrane proteins into synthetic membranes. Identifying the key factors and predicting their effect a priori is a challenging task. Reliable and systematic approaches are available for lipid based systems but, up to now, not for polymeric ones. A well-established method in the fields of chemical and process engineering is design of experiments. This statistical tool provides a way to do experimental planning systematically and assess the effects and interactions of factors on a measurable response. Within this thesis, this framework was applied to the reconstitution of the light-driven proton pump proteorhodopsin into membranes for the first time. As proteorhodopsin provides a vectorial transport of protons across a membrane, its orientation is critical for its use as an energy generator in a synthetic system. Six factors were studied: the polarization of the membrane, the pH value during reconstitution, the lipid to protein ratio, the salt concentration in the buffer, the amount of detergent used and the effect of the addition of the ionophore valinomycin. Two insertion pathways were identified for proteorhodopsin: i) charge assisted and ii) detergent mediated. Both of them result in functional proteoliposomes which exhibit the formation of a proton gradient upon illumination. The conditions of the reconstitution decide which path will be taken, as detergent concentrations around 0.5 % will induce the detergent mediated pathway and the combination of a polarized membrane together with higher detergent concentrations around 1 % will induce the charge assisted pathway. It is noteworthy that this study provides evidence that the detergent mediated one is dominant, as at 0.5 % detergent, an increased membrane charge does not affect the result. Transferring the knowledge gained towards polymeric systems, the second part of this study aims to investigate and compare the reconstitution of proteorhodopsin into polymer and lipid vesicles. As data from successful reconstitutions into polymersomes is rare, a lipid based system was used as a benchmark. Similar to the former chapter presented here, statistical modeling takes a significant part. Efficient one-step screening and optimization designs were employed to examine the assembly process of both membrane types together with proteorhodopsin. It could be revealed that both systems react differently to changing parameter combinations. The assembly of proteopolymersomes has stronger pH dependency compared to proteoliposomes and the addition of detergent does not show the membrane saturation effect known from liposomes. Probing the resulting proteovesicles for proton pumping activities, it was revealed that their performance is comparable, even though polymer membranes are not able to host the same numbers of proteorhodopsin molecules as lipid ones. Due to the applied statistical modeling, the derived equations could be used for mathematical optimization which predicted a set of parameters for reconstitution which are predicted to yield large, uniform and highly functional proteovesicles. Indeed, the results obtained from the verification of these factor settings were close to the predictions. The study provides experimental and modeling evidence for different reconstitution mechanisms depending on the membrane type. By making use of them, proteorhodopsin can be used to provide energy in an artificially created vesicular environment. Depending on the desired application, the membrane base can be composed of biocompatible lipids or robust block copolymers, providing a novel flexibility to researchers. Altogether, this thesis serves as an example of thoroughly designed procedure which fulfills the requirements of reproducibility and predictability. It can pave the way for creation of a toolbox which makes the expansion into the field of hybrid materials (lipid/polymer/protein) as well as more complex systems as molecular factories possible.