The development of a membrane-based screening method to detect antibodies to intermediate filament proteins.

Autoantibodies directed against the intermediate filament proteins (IF) arise in a variety of disease states. The authors have investigated the binding of the IF to solid membrane supports in a dot blot format in an attempt to develop a simple procedure to detect antibodies (ab) to IF. Commercially obtained, purified IF were utilized. These were: vimentin (VIM), cytokeratin 8 (CYK), glial fibrillary acidic protein (GFA), desmin (DES), and the neurofilament triplet proteins (68, 160, and 200 KDa, respectively designated LMW, MMW, and HMW). Murine monoclonal antibody (mAb) probes were used to detect the presence and immunoreactivity of IF. The mAb were visualized with HRP-anti-mouse conjugates using alpha-chloronaphthol/H 2O 2 as substrate. The membranes studied were nitrocellulose (NC), and two of modified nylon. Nitrocellulose provided the most reproducible binding; no advantage was found to ensue from the use of the other membranes with regard either to quantitative binding or improved capping. Among the IF studied, VIM, GFA, LMW, MMW, and HMW bound well to NC; optimal mass/dot was 1 mug. Filtered, non-fat dry milk is a better capping agent than either albumin or fetal calf serum, but interferes with ab binding to GFA. Binding of CYK and DES is weak at neutral pH. Standard densitometric techniques provide the possibility of quantitation. We conclude that dot and slot blot assays may be practical methods to detect ab to IF antigens.     

Improved selection of LMW over HMW proteins from human plasma by mesoporous silica particles with external modification

Selective extraction of low molecular weight (LMW) proteins and peptides from complex biological samples plays an important role in the discovery of useful biomarkers and signaling molecules. Various methods, such as solid-phase extraction (SPE), ultrafiltration, and size-exclusion chromatography have been developed for such extraction purpose. In this study, we present, to our knowledge, the first demonstration of alkyl-diol@SiO₂ mesoporous material MCM-41 (alkyl-diol group on the external surface of mesoporous material) for selective extraction of LMW proteins and peptides from complex biological samples. The adsorption kinetics of LMW proteins, the influence of pH on adsorption and the desorption recovery by different elution solvents were investigated by using standard proteins as model samples. It was demonstrated that the modification of alkyl-diol group on the external surface could efficiently decrease the adsorption of HMW protein and increase the desorption recovery of LMW protein. Furthermore, the mesoporous materials were applied to selectively extract LMW proteins and peptides (<10 kDa) from crude human plasma. And the modified MCM-41 material had much better extraction selectivity and efficiency for LMW proteins and peptides than unmodified one.     

Characterization and use of crystalline bacterial cell surface layers

Crystalline bacterial cell surface layers (S-layers) are one of the most common outermost cell envelope components of prokaryotic organisms (archaea and bacteria). S-layers are monomolecular arrays composed of a single protein or glycoprotein species and represent the simplest biological membranes developed during evolution. S-layers as the most abundant of prokaryotic cellular proteins are appealing model systems for studying the structure, synthesis, genetics, assembly and function of proteinaceous supramolecular structures. The wealth of information existing on the general principle of S-layers have revealed a broad application potential. The most relevant features exploited in applied S-layer research are: (i) pores passing through S-layers show identical size and morphology and are in the range of ultrafiltration membranes; (ii) functional groups on the surface and in the pores are aligned in well-defined positions and orientations and accessible for chemical modifications and binding functional molecules in very precise fashion; (iii) isolated S-layer subunits from a variety of organisms are capable of recrystallizing as closed monolayers onto solid supports (e.g., metals, polymers, silicon wafers) at the air–water interface, on lipid films or onto the surface of liposomes; (iv) functional domains can be incorporated in S-layer proteins by genetic engineering. Thus, S-layer technologies particularly provide new approaches for biotechnology, biomimetics, molecular nanotechnology, nanopatterning of surfaces and formation of ordered arrays of metal clusters or nanoparticles as required for nanoelectronics.     

Affinity purification of viral protein having heterogeneous quaternary structure: Modeling the impact of soluble aggregates on chromatographic performance

Prokaryote-expressed polyomavirus structural protein VP1 with an N-terminal glutathione-S-transferase tag (GST-VP1) self-assembles into pentamer structures that further organize into soluble aggregates of variable size (3.4 × 10²–1.8 × 10⁴ kDa) [D.I. Lipin, L.H.L. Lua, A.P.J. Middelberg, J. Chromatogr. A 1190 (2008) 204]. The adsorption mechanism for the full range of GST-VP1 soluble aggregates was described assuming a dual-component model [T.Y. Gu, G.J. Tsai, G.T. Tsao, AICHE J. 37 (1991) 1333], with components differentiated by size, and hence pore accessibility, rather than by protein identity. GST-VP1 protein was separated into two component groups: aggregates small enough to access resin pores (LMW: 3.4 × 10²–1.4 × 10³ kDa) and aggregates excluded from the resin pores (HMW: 9.0 × 10²–1.8 × 10⁴ kDa). LMW aggregates bound to resin at a higher saturation concentration (29.7 g L⁻¹) than HMW aggregates (13.3 g L⁻¹), while the rate of adsorption of HMW aggregates was an order of magnitude higher than for LMW aggregates. The model was used to predict both batch and packed bed adsorption of GST-VP1 protein in solutions with known concentrations of HMW and LMW aggregates to Glutathione Sepharose HP resin. Asymmetrical flow field flow fractionation with UV absorbance was utilized in conjunction with adsorption experimentation to show that binding of HMW aggregates to the resin was strong enough to withstand model-predicted displacement by LMW aggregates. High pore concentrations of LMW aggregates were also found to significantly inhibit the diffusion rate of further protein in the resin pores. Additional downstream processing experimentation showed that enzymatic cleavage of LMW aggregates to remove GST tags yields more un-aggregated VP1 pentamers than enzymatic cleavage of HMW aggregates. This model can be used to enhance the chromatographic capture of GST-VP1, and suggests an approach for modeling chromatographic purification of proteins that have a range of quaternary structures, including soluble aggregates.     

Characterization of wheat gluten proteins by hplc and MALDI TOF mass spectrometry

We have performed a detailed characterization and identification of wheat gluten proteins obtained from the Teal variety of Canadian hard red spring wheat. RP-HPLC separation of the sample into 35 fractions has reduced the spectral complexity; this was followed by MALDI mass spectrometry (MS), which showed the presence of six or fewer resolved protein components above 20 kDa in each RP-HPLC fraction, giving a total of 93 MS resolved peaks. These included 17 peaks in the ω-gliadin fractions (F1–4), 12 in the high molecular weight (HMW) glutenin subunit fractions (F5–8), 59 in the α- and β-gliadins and low molecular weight (LMW) glutenin subunit fractions (F9–31) and 5 peaks in the γ-gliadin fractions (F32–35). Peptide maps of tryptic digests of HPLC fractions were obtained from a tandem quadrupole time-of-flight mass spectrometer (MALDI QqTOF MS) and were submitted to the ProFound search engine. HMW glutenin subunits including Ax2*, Dx5, Bx7, and Dy10 (consistent with the known profile of Teal), and LMW glutenin subunits including six from group 3 type II and 1 from group 2 type I, were identified with reasonable sequence coverage from HPLC fraction 5, 7, 17, and 18. The identities of the peptides attributed to selected gluten proteins were confirmed using MS/MS with BioMultiView to match the predicted and measured partial amino acid sequences. Because of incomplete wheat DNA databases, many wheat gluten proteins could not be identified. These results suggest that the combination of RP-HPLC with MS and MS/MS techniques is a promising approach for the characterization of wheat gluten proteins.     

The role of peptides and proteins in melanoidin formation

High‐molecular‐weight (HMW) coloured compounds called melanoidins are widely distributed, particularly in foods. It has been proposed that they originate through the Maillard reaction, a non‐enzymatic browning reaction, due to the interaction between protein or peptide amino groups and carbohydrates. The melanoidin structure is not definitively known, and they have been generally defined as HMW nitrogen‐containing brown polymers. In order to gain information on the nature of melanoidins, a simple in vitro model was chosen to investigate the products of the reactions between sugars and peptide/proteins. This approach would elucidate whether melanoidin formation is due to the binding of different sugar units to a peptide/protein or vice versa. With this aim, the reactivity of two different peptides, EPK177 and physalaemin, and a low‐molecular‐weight (LMW) protein, lysozyme, was tested towards different saccharides (glucose, maltotriose (MT), maltopentaose and dextran 1000) in aqueous solutions at different temperatures. The incubation mixtures were analysed at different reaction times by MALDI/MS. Furthermore, in order to verify the possible role of sugar pyrolysis products in melanoidin formation, the products arising from the thermal treatment at 200 °C of MT were incubated with lysozyme, and the reaction products were analysed by the same MS approach. The obtained results allowed the establishment of some general views: melanoidins cannot simply originate by reactions of sugar moieties with proteins. In fact, the reaction easily occurs, but it does not lead to any coloured product, as melanoidins have been described to be; melanoidins cannot originate from the thermal degradation products of glycated proteins. In fact, the thermal treatment of glycated lysozyme leads to a severe degradation of the protein with the formation of LMW species, far from the view of melanoidins as HMW compounds; experimental evidence has been gained on the melanoidin formation through reaction of intact protein with the pyrolysis products of MT. This hypothesis has been supported either from MALDI measurements or from spectroscopic data that show an absorption band in the range 300–600 nm, typical of melanoidins. Copyright © 2009 John Wiley & Sons, Ltd.     

Influence of surface chemistry and protein concentration on the adsorption rate and S-layer crystal formation

Bacterial crystalline surface layers (S-layers) are the outermost envelope of prokaryotic organisms representing the simplest biological membranes developed during evolution. In this context, the bacterial protein SbpA has already shown its intrinsic ability to reassemble on different substrates forming protein crystals of square lattice symmetry. In this work, we present the interaction between the bacterial protein SbpA and five self-assembled monolayers carrying methyl (CH(3)), hydroxyl (OH), carboxylic acid (COOH) and mannose (C(6)H(12)O(6)) as functional groups. Protein adsorption and S-layer formation have been characterized by atomic force microscopy (AFM) while protein adsorption kinetics, mass uptake and the protein layer viscoelastic properties were investigated with quartz crystal microbalance with dissipation monitoring (QCM-D). The results indicate that the protein adsorption rate and crystalline domain area depend on surface chemistry and protein concentration. Furthermore, electrostatic interactions tune different protein rate adsorption and S-layer recrystallization pathways. Electrostatic interactions induce faster adsorption rate than hydrophobic or hydrophilic interactions. Finally, the shear modulus and the viscosity of the recrystallized S-layer on CH(3)C(6)S, CH(3)C(11)S and COOHC(11)S substrates were calculated from QCM-D measurements. Protein-protein interactions seem to play a main role in the mechanical stability of the formed protein (crystal) bilayer.     

Direct detection of fatty acid ethyl esters using low temperature plasma (LTP) ambient ionization mass spectrometry for rapid bacterial differentiation

Low temperature plasma mass spectrometry (LTP-MS) was employed to detect fatty acid ethyl esters (FAEE) from bacterial samples directly. Positive ion mode FAEE mass spectrometric profiles of sixteen different bacterial samples were obtained without extraction or other sample preparation. In the range m/z 200-300, LTP mass spectra show highly reproducible and characteristic patterns. To identify the FAEE's associated with the characteristic peaks, accurate masses were recorded in the full scan mode using an LTQ/Orbitrap instrument, and tandem mass spectrometry was performed. Data were examined by principal component analysis (PCA) to determine the degree of differentiation possible amongst different bacterial species. Gram-positive and gram-negative bacteria are readily distinguished, and 11 out of 13 Salmonella strains show distinctive patterns. Growth media effects are observed but do not interfere with species recognition based on the PCA results.     

Composite sequence proteomic analysis of protein biomarkers of Campylobacter coli, C. lari and C. concisus for bacterial identification

Protein biomarkers observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) of cell lysates of three strains of Campylobacter coli, two strains of C. lari and one strain of C. concisus have been identified by 'bottom-up' proteomic techniques. The significant findings are as follows. First, the protein biomarkers identified were: PhnA-related protein, 4-oxalocrotonate tautomerase (DmpI)-related protein, NifU-like protein, cytochrome c, DNA-binding protein HU, 10 kDa chaperonin, thioredoxin, as well as several conserved hypothetical and ribosomal proteins. Second, variations in the biomarker ion m/z in MALDI-TOF-MS spectra across species and strains are the result of variations in the amino acid sequence of the protein due to non-synonymous mutations of the biomarker gene. Third, the most common post-translational modifications (PTMs) were the removal of the N-terminal methionine and N-terminal signal peptides. However, in the case of the NifU protein (an iron-sulfur cluster transport protein), post-translational cleavage occurred from the C-terminus. Fourth, only the genomes of the C. coli strain RM2228 and C. lari strain RM2100 have been sequenced; thus, proteomic identification of the proteins of the other strains in this study relied upon sequence homology to the genomic sequence of these strains as well as the genomes of sequences of other Campylobacter strains. In some cases, the determination of the full amino acid sequence of a protein biomarker from a genomically non-sequenced strain was accomplished by combining non-overlapping partial sequences from proteomic identifications of genomically-sequenced strains that were of the same species (or of a different species) to that of the non-sequenced strain. The accuracy of this composite sequence was confirmed by both MS and MS/MS. It was necessary, in some cases, to perform de novo sequencing on 'gaps' in the composite sequence that were not homologous to any genomically-sequenced strain. In order to validate the composite sequence approach, composite sequences were further confirmed by subsequent DNA sequencing of the biomarker gene. Thus, using the composite sequence approach, it was possible to determine the full amino acid sequence of an unknown protein from a genomically non-sequenced bacterial strain without the necessity of either sequencing the biomarker gene or performing full de novo MS/MS sequencing. The sequence obtained could then be used as a strain-specific biomarker for analysis by 'top-down' proteomics techniques.     

Comparative Analysis of the Distribution of Copper in Green and Brown Brazilian Propolis Extracts

We proposed here a novel analytical procedure for copper speciation in green and brown propolis extracts using SEC—HPLC—GFAAS with 0.5% m v^?1 SDS in 2.5 m mol L^?1 Tris–HCl (pH 7.4) as the mobile phase buffer solution. Both basic (0.05 mol L^?1 NaOH) and acid (0.05 mol L^?1 HCl) conditions were evaluated for sample extraction. Depending on the extraction procedure, differences in copper distribution were identified. Copper was mainly associated with high-molecular-weight (HMW) fractions in green propolis extract when extracted with basic solution, whereas with acid extraction solution, only low-molecular-weight (LMW) fractions were obtained in both samples. Furthermore, combined analysis of results obtained using SEC-UV and GF AAS confirmed the association of copper with LMW and HMW species.     

Monomolecular reassembly of a crystalline bacterial cell surface layer (S-layer) on untreated and modified silicon surfaces

Scanning force microscopy was used to investigate the recrystallization of isolated bacterial cell surface layer (S-layer) proteins of Bacillus stearothermophilus NRS 2004/3a variant V1 on untreated, cleaned, silanized and photoresist-coated silicon wafers. With the exception of the hydrophilic surface of cleaned wafers, all other surfaces showed hydrophobic surface characteristics. Recrystallization occurred only at the hydrophobic surfaces, and, with respect to the bacterial cell, the S-layer was always oriented with its more hydrophobic outer face against the interface. Monolayer formation was initiated by crystal growth from several distant randomly oriented nucleation points and terminated by neighbouring, also growing, crystalline areas. The size of the individual crystalline domains formed in this way was in the range of 5–10 μm in diameter. The entire silicon surface was covered by a coherent monolayer after a recrystallization time of approximately 1 h.     

Ultrafiltration membranes prepared from crystalline bacterial cell surface layers as model systems for studying the influence of surface properties on protein adsorption

Crystalline bacterial cell surface layers (S-layers) were used for the preparation of the active filtration layer of ultrafiltration membranes (S-layer ultrafiltration membranes; SUMs). Since the S-layer is uniform in its pore size and morphology and its functional groups are aligned in well-defined positions, the SUMs provide ideal model systems for studying protein adsorption and membrane fouling. Due to the presence of surface-located carboxyl groups the standard SUMs have the net negative charge but exhibit basically a hydrophobic character. In order to change the net charge, the charge density and the accessibility of charged groups of the SUMs as well as their hydrophobicity, free carboxyl groups of the S-layer protein were modified with selected low molecular weight nucleophiles under conditions of preserving the crystalline lattice structure. SUMs with 1.6 to 7 charged or functional groups exposed per nm² of the membrane area were used for adsorption experiments. After solutions of differently sized and charged test proteins were filtered, the relative flux losses of distilled particle free water were measured. The results showed that the adsorption capacity of the SUMs increased with the extent of their hydrophobicity. Test proteins showed their own specific adsorption characteristics, which clearly demonstrated the difficulties in determining parameters controlling the membrane fouling. Independent of the net charge of the test proteins and that of the SUMs, the flux loss of SUMs increased with the increased charge density and an improved accessibility of the charged groups on the S-layer surface. No essential differences in the adsorption characteristics were observed between the zwitterionic SUMs of slightly surplus of free carboxyl groups and the standard SUMs of net negative charge.     

Low-molecular-weight human serum proteome using ultrafiltration, isoelectric focusing, and mass spectrometry

Identification of the serum proteome is a daunting analytical task due to the complex nature of the sample which has an extremely large dynamic range of protein components. This report addresses this issue by using centrifugal ultrafiltration to enrich the low-molecular-weight (LMW) serum proteome while decreasing the amount of abundant high-molecular-weight proteins. Reduction of the complex nature of the sample was achieved by fractionation of the LMW serum proteins using solution-phase isoelectric focusing (IEF). Multiple enzyme digestions are performed and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analysis of the tandem mass spectra resulted in the identification of 262 proteins belonging to LMW serum proteome. Our results demonstrate the effectiveness of this methodology to isolate and identify LMW proteins with improved confidence in the MS data acquired. In addition, our methodology can be combined with other multidimensional chromatography techniques performed on the peptide level to increase the number of identified proteins.     

血清中低分子量蛋白质的提取和鉴定

采用改进的圆盘凝胶电泳提取人血清中低分子量蛋白质, 去除了血清中分子量大于3×104的蛋白质, 将提取的低分子量蛋白质热变性后直接在溶液中酶解成肽, 经液相色谱-质谱分析, 并进行Mascot数据库检索, 确认出人血清中97种蛋白质.     

Role of lactobacillus cell surface hydrophobicity as probed by AFM in adhesion to surfaces at low and high ionic strength

The S-layer present at the outermost cell surface of some lactobacillus species is known to convey hydrophobicity to the lactobacillus cell surface. Yet, it is commonly found that adhesion of lactobacilli to solid substrata does not proceed according to expectations based on cell surface hydrophobicity. In this paper, the role of cell surface hydrophobicity of two lactobacillus strains with and without a surface layer protein (SLP) layer has been investigated with regard to their adhesion to hydrophobically or hydrophilically functionalized glass surfaces under well-defined flow conditions and in low and high ionic strength suspensions. Similarly, the interaction of the lactobacilli with similarly functionalized atomic force microscope (AFM) tips was measured. In a low ionic strength suspension, both lactobacillus strains show higher initial deposition rates to hydrophobic glass than to hydrophilic glass, whereas in a high ionic strength suspension no clear influence of cell surface hydrophobicity on adhesion is observed. Independent of ionic strength, however, AFM detects stronger interaction forces when both bacteria and tip are hydrophobic or hydrophilic than when bacteria and tip have opposite hydrophobicities. This suggest that the interaction develops in a different way when a bacterium is forced into contact with the tip surface, like in AFM, as compared with contacts developing between a cell surface and a macroscopic substratum under flow. In addition, the distance dependence of the total Gibbs energy of interaction could only be qualitatively correlated with bacterial deposition and desorption in the parallel plate flow chamber.     

Hyaluronic acid‐dependent protection against alkali‐burned human corneal cells

Hyaluronic acid (HA) is a high‐molecular‐weight glycosaminoglycan and extracellular matrix component that promotes cell proliferation. This study aimed to evaluate the effects of HA on alkali‐injured human corneal epithelial cells in vitro, and to elucidate the mechanisms by which HA mediates corneal cell protection. A human corneal epithelial cell line (HCE‐2) was treated with sodium hydroxide before incubation with low‐molecular‐weight HA (LMW‐HA, 127 kDa) or high‐molecular‐weight HA (HMW‐HA, 1525 kDa). A global proteomic analysis was then performed. Our data indicated that HA treatment protects corneal epithelial cells from alkali injury, and that the molecular weight of HA is a crucial factor in determining its effects. Only HMW‐HA reduced NaOH‐induced cytotoxic effects in corneal cells significantly and increased their migratory and wound healing ability. Results from 2D‐DIGE and MALDI‐TOF/TOF MS analyses indicated that HMW‐HA modulates biosynthetic pathways, cell migration, cell outgrowth, and protein degradation to stimulate wound healing and prevent cell death. To our knowledge, our study is the first to report the possible mechanisms by which HMW‐HA promotes repair in alkali‐injured human corneal epithelial cells.     

Bulk Metathesis Polymerization of Cyclooctene

The bulk ring-opening metathesis polymerization (ROMP) of cyclooctene initiated with the WCI₆/Sn(CH₃)₄ catalytic system was investigated at 40, 100, and 160°C using high vacuum techniques. The polymerizations were followed over a period of several days. Detailed analyses of the polymerization products by gel permeation chromatography (GPC) and ¹H and ¹³C NMR were carried out. Along with unsaturated high molecular weight (HMW) polymer (polyoctenamer), low molecular weight (LMW) polymer was found, the proportion of the latter increasing with time. The LMW fraction contains saturated LMW polymer together with ring polymer. The results are explained in terms of kinetic and thermody-namic arguments.     

Prolonged stochastic single ion channel recordings in S-layer protein stabilized lipid bilayer membranes

S-layer proteins are commonly found in bacteria and archaea as two-dimensional monomolecular crystalline arrays as the outermost cell membrane component. These proteins have the unique property that following disruption by chemical agents, monomers of the protein can re-assemble to their original lattice structure. This unique property makes S-layers interesting for utilization in bio-nanotechnological applications. Here, we show that the addition of S-layer proteins to bilayer lipid membranes increases the lifetime and the stability of the bilayer. M2delta ion channels were functionally incorporated into these S-layer stabilized membranes and we were able to record their activity for up to 20 h. Transmission electron microscopy (TEM) was used to visualize the 2D crystalline pattern of the S-layer and the M2delta ion channel characteristics in bilayer lipid membrane's were compared in the presence and absence of S-layers.     

Analysis of subcellular surface structure, function and dynamics

Analytics of single biological cells allows quantitative investigation from a structural, functional and dynamical point of view and opens novel possibilities to an unamplified subcellular analysis. In this article, we report on three different experimental methods and their applications to single cellular systems with a subcellular sensitivity down to the single molecule level. First, the subcellular surface structure of living bacteria (Corynebacterium glutamicum) was investigated with atomic force microscopy (AFM) at the resolution of individual surface layer (S-layer) proteins; discrimination of bacterial strains that lack the expression of hexagonally packed surface layer proteins was possible. Second, quantitative measurement of individual recognition events of membrane-bound receptors on living B-cells was achieved in single cell manipulation and probing experiments with optical tweezers (OT) force spectroscopy. And third, intracellular dynamics of translocating photoactivatable GFP in plant protoplasts (Nicotiana tabacum BY-2) was quantitatively monitored by two-photon laser scanning microscopy (2PLSM).     

A robust new strategy for high-molecular-weight proteome research: A 2-hydroxyethyl agarose/polyacrylamide gel enhanced separation and ZnO-PMMA nanobeads assisted identification

A new mass spectrometry based analysis strategy has been established here for high-molecular-weight (HMW) proteome research. First, a 2-hydroxyethyl agarose/polyacrylamide (HEAG/PAM) electrophoresis gel was designed for the first time to realize an easy-handling separation method with high spatial resolution for HMW proteins, good reproducibility and mass spectrometry-compatible sliver staining. Second, ZnO-polymethyl methacrylate (ZnO-PMMA) nanobeads were applied here for enriching and desalting the peptides from the HMW proteins. Third, the peptides were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) with the presence of the ZnO-PMMA nanobeads, and their MS signals were enhanced markedly. The success rate of identification for HMW proteins was significantly increased due to high enriching efficiency and salt tolerance capability as well as signal enhancing capability of the ZnO-PMMA nanobeads. We believe that this analysis strategy will inspire and accelerate the HMW proteome studies.