Two-dimensional Protein Patterns Research Articles

Signal Propagation between Neuronal Populations Controlled by Micropatterning.

The central nervous system consists of an unfathomable number of functional networks enabling highly sophisticated information processing. Guided neuronal growth with a well-defined connectivity and accompanying polarity is essential for the formation of these networks. To investigate how two-dimensional protein patterns influence neuronal outgrowth with respect to connectivity and functional polarity between adjacent populations of neurons, a microstructured model system was established. Exclusive cell growth on patterned substrates was achieved by transferring a mixture of poly-l-lysine and laminin to a cell-repellent glass surface by microcontact printing. Triangular structures with different opening angle, height, and width were chosen as a pattern to achieve network formation with defined behavior at the junction of adjacent structures. These patterns were populated with dissociated primary cortical embryonic rat neurons and investigated with respect to their impact on neuronal outgrowth by immunofluorescence analysis, as well as their functional connectivity by calcium imaging. Here, we present a highly reproducible technique to devise neuronal networks in vitro with a predefined connectivity induced by the design of the gateway. Daisy-chained neuronal networks with predefined connectivity and functional polarity were produced using the presented micropatterning method. Controlling the direction of signal propagation among populations of neurons provides insights to network communication and offers the chance to investigate more about learning processes in networks by external manipulation of cells and signal cascades.

Engineering connectivity by multiscale micropatterning of individual populations of neurons.

Functional networks are the basis of information processing in the central nervous system. Essential for their formation are guided neuronal growth as well as controlled connectivity and information flow. The basis of neuronal development is generated by guiding cues and geometric constraints. To investigate the neuronal growth and connectivity of adjacent neuronal networks, two-dimensional protein patterns were created. A mixture of poly-L-lysine and laminin was transferred onto a silanized glass surface by microcontact printing. The structures were populated with dissociated primary cortical embryonic rat neurons. Triangular structures with diverse opening angles, height, and design were chosen as two-dimensional structures to allow network formation with constricted gateways. Neuronal development was observed by immunohistochemistry to pursue the influence of the chosen structures on the neuronal outgrowth. Neurons were stained for MAP2, while poly-L-lysine was FITC labeled. With this study we present an easy-to-use technique to engineer two-dimensional networks in vitro with defined gateways. The presented micropatterning method is used to generate daisy-chained neuronal networks with predefined connectivity. Signal propagation among geometrically constrained networks can easily be monitored by calcium-sensitive dyes, providing insights into network communication in vitro.

Multivariate Data Analysis of Two-Dimensional Gel Electrophoresis Protein Patterns from Few Samples

One application of 2D gel electrophoresis is to reveal differences in protein pattern between two or more groups of individuals, attributable to their group membership. Multivariate data analytical methods are useful in pinpointing the spots relevant for discrimination by focusing not only on single spot differences, but on the covariance structure between proteins. However, their outcome is dependent on data scaling, and they may fail in producing valid multivariate models due to the much higher number of "irrelevant" spots present in the gels. The case where only few gels are available and where the aim is to find as many as possible of the group-dependent proteins seems particularly difficult to handle. The present paper investigates such a case regarding the effect of scaling and of prefiltering by univariate nonparametric statistics on the selection of spots. Besides, a modified 'autoscaling' of the full data set based on within-group standard deviations is introduced and shown to be advantageous in revealing potential group-dependent proteins additional to those found by prefiltering.

Proteome profiles of mucosal immunoglobulin uptake in inflamed porcine gut

Acquisition of passive immunity by endocytosis of intact immunoglobulins (Ig) from colostrum is critical for prevention of intestinal and systemic diseases in neonatal mammals. We compared proteome patterns of healthy and inflamed gut tissues from pre-term piglets to investigate the effect of inflammation on acquisition of passive immunity. A clear difference in the two-dimensional gel electrophoresis protein patterns between healthy and inflamed intestinal tissues was observed, suggesting that inflamed tissues failed to absorb and transfer Ig from colostrum to epithelial cells. We have mapped and identified the Ig proteins that are taken up by healthy intestinal tissues, and found that isoforms of the IgA and IgG heavy chain and Ig kappa and lambda light chains were internalized. Our results indicate that colostrum protein uptake in the porcine gut is a selective process that is obstructed in inflamed pre-term gut.

Lysosome membrane lipid microdomains: novel regulators of chaperone-mediated autophagy

Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins in lysosomes. The limiting step of this type of autophagy is the binding of substrates to the lysosome-associated membrane protein type 2A (LAMP-2A). In this work, we identify a dynamic subcompartmentalization of LAMP-2A in the lysosomal membrane, which underlies the molecular basis for the regulation of LAMP-2A function in CMA. A percentage of LAMP-2A localizes in discrete lysosomal membrane regions during resting conditions, but it exits these regions during CMA activation. Disruption of these regions by cholesterol-depleting agents or expression of a mutant LAMP-2A excluded from these regions enhances CMA activity, whereas loading of lysosomes with cholesterol significantly reduces CMA. Organization of LAMP-2A into multimeric complexes, required for translocation of substrates into lysosomes via CMA, only occurs outside the lipid-enriched membrane microdomains, whereas the LAMP-2A located within these regions is susceptible to proteolytic cleavage and degradation. Our results support that changes in the dynamic distribution of LAMP-2A into and out of discrete microdomains of the lysosomal membrane contribute to regulate CMA.

Correlation analysis of two-dimensional gel electrophoretic protein patterns and biological variables.

BackgroundTwo-dimensional gel electrophoresis (2DE) is a powerful technique to examine post-translational modifications of complexly modulated proteins. Currently, spot detection is a necessary step to assess relations between spots and biological variables. This often proves time consuming and difficult when working with non-perfect gels. We developed an analysis technique to measure correlation between 2DE images and biological variables on a pixel by pixel basis. After image alignment and normalization, the biological parameters and pixel values are replaced by their specific rank. These rank adjusted images and parameters are then put into a standard linear Pearson correlation and further tested for significance and variance.ResultsWe validated this technique on a set of simulated 2DE images, which revealed also correct working under the presence of normalization factors. This was followed by an analysis of p53 2DE immunoblots from cancer cells, known to have unique signaling networks. Since p53 is altered through these signaling networks, we expected to find correlations between the cancer type (acute lymphoblastic leukemia and acute myeloid leukemia) and the p53 profiles. A second correlation analysis revealed a more complex relation between the differentiation stage in acute myeloid leukemia and p53 protein isoforms.ConclusionThe presented analysis method measures relations between 2DE images and external variables without requiring spot detection, thereby enabling the exploration of biosignatures of complex signaling networks in biological systems.

Isoelectric focusing in serial immobilized pH gradient gels to improve protein separation in proteomic analysis

We previously demonstrated the separation of proteins by isoelectric focusing (IEF) over pH 4-8 immobilized pH gradients (IPGs) over 54 cm (Poland et al., Electrophoresis 2003, 24, 1271). Here we show that similar results can be conveniently achieved using commercially available IPGs of appropriate pH ranges positioned end-on-end in series during electrophoresis, which we term "daisy chain IEF". Proteins efficiently electrophorese from one IPG to another during IEF by traversing buffer-filled porous bridges between the serial IPGs. A variety of materials can function as bridges, including paper, polyacrylamide gels or even IPGs. The quality of two-dimensional (2-D) protein patterns is not apparently worse than that generated by conventional IEF using the same individual IPGs. A major advantage of this method is that sample is consumed efficiently, without the requirement for preliminary steps, such as chamber IEF. This advantage is pronounced when working with extremely limited sources of samples, such as with clinical biopsies or cellular subfractions. The present study was limited by the commercial availability of suitable pH gradients. Proteomics analyses could be further improved if commercial vendors would manufacture IPGs with suitable pH ranges to achieve high resolution (approximately 100 cm) IEF separation of proteins in one electrophoretic step over the pH range 2-12.

The influence of agr and sigmaB in growth phase dependent regulation of virulence factors in Staphylococcus aureus.

The expression of many virulence determinants in Staphylococcus aureus is tightly coordinated generally by global regulatory elements such as accessory gene regulator (agr), staphylococcal accessory regulator and the alternative sigma factor sigmaB. We have compared the two-dimensional (2-D) protein pattern of extracellular protein extracts of wild-type cells with the 2-D patterns of the respective regulatory mutants in order to identify proteins whose amount is influenced by a mutation in agr or sigB. In order to quantify changes in the level of interesting proteins we used the Ettan-fluorescence difference gel electrophoresis technique (Amersham Biosciences). As in most bacteria, the amount of extracellular proteins was strongly regulated and increased mainly in the stationary phase of growth at high cell densities. By comparing the extracellular protein pattern of the RN6390 rsbU strain with that of an isogenic agr mutant RN6911 we show that the level of about 70 protein spots changed in the mutant. To analyze the role of sigmaB in virulence gene expression an RsbU+ (RN6390 RsbU+) derivative was included in this study. The protein pattern of the RsbU+ strain (RN6390 RsbU+) was very similar to that of the Deltaagr/DeltarsbU mutant strain (RN6911) indicating an opposing effect of agr and rsbU on the expression of the same genes.

Proteome analysis reveals the significant alternations of cardiac small heat shock proteins expression in heart failure

Purpose: Considering that the number of functional gene productions far exceeds the number of genes that encode them, the causes of heart failure (HF) are likely to results from alternations in cardiac protein expression. The aim of our study was to use a proteomics approach to investigate global alternations in protein expression in tachycardia induced HF dogs. Method and Results: We compared the two-dimensional (2D) gel electrophoresis protein patterns of left ventricular samples from the normal and failing myocardium. Differentially expressed cardiac proteins showed as many as 200 cardiac protein spots. Seven protein spots were significantly upregulated in HF compared to the normals. Those spots were identified as alpha B crystalline, HSP 20, and HSP27 by MALDI-TOF/MS. They are members of the small heat shock protein (HSP) family and exert maintaining the morphological and functional integrity of the cardiac contractile apparatus and increase a tolerance against apoptosis. Since phosphorylation is one of the most important post-translational modifications, we evaluated whether the overexpressed HSPs were phosphorylated in HF. 2D-phosphoprotein staining and western blotting demonstrated phosphorylated forms of those small HSPs were significantly increased compared to the normals. Conclusion: Proteomics studies are anticipated to provide new insights into cellular mechanisms involved in HF and post-translational modification of small HSPs may be a therapeutic targets for improving the function of failing cardiomyocytes.

Mechanisms of hydrazine toxicity in rat liver investigated by proteomics and multivariate data analysis.

A proteomics approach combined with multivariate data analysis was used to examine the hepatotoxic effect of hydrazine in 30 male Sprague Dawley rats, assigned to four treatment groups and two control groups. Liver samples from the individual animals were resolved by two-dimensional differential gel electrophoresis (2-D DIGE) and protein patterns from the 2-D gels were analyzed by principal component analysis (PCA) and partial least squares regression (PLSR). The PCA plot was able to describe the variation in the protein expression related to dose and time, by separation or clustering of different animal groups. PLSR followed by variable selection (Jack-knifing) was used to select proteins that varied significantly in relation to the dose related response of the hydrazine treatment. The 10 up-regulated and 10 down-regulated proteins with highest rank in the PLSR model were identified by mass spectrometry. Hydrazine treatment induced altered expression of proteins related to lipid metabolism, Ca(2+) homeostasis, thyroid hormone pathways and stress response. Several of the identified proteins have not previously been implicated in hydrazine toxicity and may thus be regarded as new potential biomarkers of induced liver toxicity.

Two-Dimensional Protein Pattern Analysis of Extracellular Proteins Secreted by Embryogenic and Non-Embryogenic Suspension Cultures of Dactylis Glomerata L.

ABSTRACTProteins from the medium of orchardgrass (Dactylis glomerata L.) embryogenic suspension culture during defined stages of somatic embryogenesis were compared with those of non-embryogenic suspension culture during unorganized cell proliferation. After separation on two-dimensional polyacrylamide gel electrophoresis, we were able to classify 40 proteins in each of 6 groups: a group common to both embryogenic and non-embryogenic suspension cultures, a group specific only for embryogenic, respectively non-embryogenic suspension cultures and 3 groups of microcluster-, proembryogenic masses- and somatic embryo-specific proteins. One of the groups is particularly interesting as it corresponds to polypeptides that are related to the earliest stages of somatic embryo-genesis-the transition of microclusters into proembryogenic masses when no morphological changes are visible yet. Using this experimental approach, we identified the major extracellular proteins involved in somatic embryo development and we discuss on the possibility for their use as early markers for somatic embryogenesis.

A colloidal silver staining–destaining method for precise assignment of immunoreactive spots in two-dimensional protein patterns

The characterization of protein expression patterns by two-dimensional gel electrophoresis depends on efficient and reliable identification strategies for target spots. In addition to sophisticated techniques, such as microsequencing and peptide mass spectrometry, immunodetection of membrane-immobilized proteins is a valuable method with which to identify the corresponding spots for a given set of candidate proteins. To precisely assign immunoreactive spots, this approach requires specific immunodetection and staining of total protein to be performed on the same membrane. Here, we describe a highly sensitive, colloidal silver-based method for the assignment of immunoreactive spots in two-dimensional protein patterns. This simple and rapid procedure involves a destaining step after staining of nitrocellulose-bound proteins with colloidal silver. We show that destaining of proteins is a prerequisite for subsequent immunodetection using enhanced chemiluminescence. Several types of antibodies were successfully employed for antigen detection after the staining–destaining procedure. Our results demonstrate that the colloidal silver-based method is generally applicable for the unambiguous identification of candidate proteins in complex two-dimensional patterns.

Short- and long-term changes in proteome composition and kinetic properties in a culture of Escherichia coli during transition from glucose-excess to glucose-limited growth conditions in continuous culture and vice versa.

To investigate the ability of Escherichia coli K12 MG1655 to cope with excess and limitation of a carbon and energy source, we studied the changes in kinetic properties and two-dimensional (2D) gel protein patterns of an E. coli culture. The population was transferred from glucose-excess batch to glucose-limited continuous culture (D = 0.3 h(-1)), in which it was cultivated for 500 h (217 generations) and then transferred back to glucose-excess batch culture. Two different stages to glucose-limitation were recognized: a short-term physiological adaptation characterized by a general effort in enhancing the cell's substrate scavenging ability and mutations resulting in a population exhibiting increased glucose affinity. Physiological short-term adaptation to glucose-limitation was achieved by upregulation of 12 proteins, namely MglB, MalE, ArgT, DppA, RbsB, YdcS, LivJ (precursor), UgpB (precursor), AceA, AldA, AtpA and GatY. Eight of these proteins are periplasmic binding proteins of ABC transporters. Most of them are not involved in glucose transport regulons, but rather in chemotaxis and transport of other substrates, whereas MalE and MglB have previously been shown to belong to transport systems important in glucose transport under glucose-limited conditions. Evolution under low glucose concentration led to an up to 10-fold increase in glucose affinity (from a K(s) of 366 +/- 36 microg l(-1) at the beginning to 44 +/- 7 microg l(-1)). The protein pattern of a "500-h-old" continuous culture showed a highly increased expression of MglB and MalE as well as of the regulator protein MalI. When adapted cells taken from the "500-h-old" continuous culture were transferred to batch culture, an increased expression of MalE was observed, compared with cells from un-adapted batch-grown cells. Otherwise, no significant changes were observed in the protein pattern of batch-grown populations before and after 500 h of evolution in the glucose-limited continuous culture.

A reference map of human lung MRC-5 fibroblast proteins using immobilized pH gradient-isoelectric focusing-based two-dimensional electrophoresis.

We report the first protein map of human adult lung MRC-5 fibroblasts using isoelectric focusing-immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis. MRC-5 is an immortalised cell line used in a wide range of investigations. The two-dimensional gel pattern of proteins generated from any given cell system provides a fingerprint that is unique to those cells. Therefore, the establishment of a protein map for a particular cell system provides a useful reference tool as a "master map" for subsequent studies using those cells. In this map a total of 98 protein spots were identified by comparative searches of the nucleotide and protein database using peptide masses obtained by matrix-assisted laser desorption/ionization time of flight following trypsin digestion. To increase the utility of the reference map, cells were cultured in both Dulbecco's modified Eagle medium (DMEM), the standard medium, and Roswell Park Memorial Institute (RPMI)-1640. Two-dimensional gel protein patterns of MRC-5 cultures were shown to be largely unaffected by the use of RPMI compared to DMEM, respectively. In combination with the reference map, the standardised protocol described provides a tool for comparative studies involving MRC-5 cells in which nonspecific variation is minimized.

A reference map of human lung MRC-5 fibroblast proteins using immobilized pH gradient-isoelectric focusing-based two-dimensional electrophoresis

We report the first protein map of human adult lung MRC-5 fibroblasts using isoelectric focusing-immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis. MRC-5 is an immortalised cell line used in a wide range of investigations. The two-dimensional gel pattern of proteins generated from any given cell system provides a fingerprint that is unique to those cells. Therefore, the establishment of a protein map for a particular cell system provides a useful reference tool as a “master map” for subsequent studies using those cells. In this map a total of 98 protein spots were identified by comparative searches of the nucleotide and protein database using peptide masses obtained by matrix-assisted laser desorption/ionization time of flight following trypsin digestion. To increase the utility of the reference map, cells were cultured in both Dulbecco's modified Eagle medium (DMEM), the standard medium, and Roswell Park Memorial Institute (RPMI)-1640. Two-dimensional gel protein patterns of MRC-5 cultures were shown to be largely unaffected by the use of RPMI compared to DMEM, respectively. In combination with the reference map, the standardised protocol described provides a tool for comparative studies involving MRC-5 cells in which nonspecific variation is minimized.

Large-scale monitoring of pleiotropic regulation of gene expression by the prokaryotic nucleoid-associated protein, H-NS.

Despite many years of intense work investigating the function of nucleoid-associated proteins in prokaryotes, their role in bacterial physiology remains largely unknown. The two-dimensional protein patterns were compared and expression profiling was carried out on H-NS-deficient and wild-type strains of Escherichia coli K-12. The expression of approximately 5% of the genes and/or the accumulation of their protein was directly or indirectly altered in the hns mutant strain. About one-fifth of these genes encode proteins that are involved in transcription or translation and one-third are known to or were in silico predicted to encode cell envelope components or proteins that are usually involved in bacterial adaptation to changes in environmental conditions. The increased expression of several genes in the mutant resulted in a better ability of this strain to survive at low pH and high osmolarity than the wild-type strain. In particular, the putative regulator, YhiX, plays a central role in the H-NS control of genes required in the glutamate-dependent acid stress response. These results suggest that there is a strong relationship between the H-NS regulon and the maintenance of intracellular homeostasis.

Characterization of the sigma(B) regulon in Staphylococcus aureus.

The sigma(B)-dependent stress regulon in gram-positive bacteria might fulfill a physiological role in stress response and virulence similar to that of the sigma(S) regulon in Escherichia coli and other gram-negative bacteria. In order to obtain evidence for the function of the sigma(B) regulon of Staphylococcus aureus, especially in virulence control, sigma(B)-dependent stress genes were identified. The two-dimensional protein pattern of wild-type cells of S. aureus COL was compared with that of an isogenic sigB mutant. By this approach, we found that the synthesis of about 27 cytoplasmic proteins seemed to be under the positive control of sigma(B). N-terminal sequencing of 18 proteins allowed the identification of their genes on the almost finished genome sequence of S. aureus COL and the analysis of the promoter structure. Transcriptional analyses of 11 of these genes confirmed their sigma(B) dependency, and moreover, about 7 additional sigma(B)-dependent genes were found which are cotranscribed with the newly detected genes, forming operons. Altogether, we identified 23 sigma(B)-dependent genes and their corresponding proteins. Among them are proteins probably involved in the generation of NADH or in membrane transport mechanisms. Furthermore, at least one clpC-homologous gene was localized on the S. aureus sequence solely transcribed by sigma(B). In contrast, a second clpC-homologous gene in S. aureus forming an operon with ctsR, yacH, and yacI was sigma(B) independently expressed.

Luteinizing hormone induces mouse vas deferens protein expression in the murine ovary.

The aim of our study was to isolate and identify novel proteins that are involved in the process of ovulation. To achieve this goal we used the technique of proteome analysis. Comparison of ovary protein patterns, obtained by high resolution two-dimensional gel electrophoresis from recombinant FSH (rFSH)- and rFSH + human CG (hCG)-treated mice, showed significant differences in protein spot positions and intensities. Subsequent analysis of one of these proteins was performed by mass spectrometry, resulting in the identification of the mouse vas deferens protein (MVDP). MVDP, which was absent in the two-dimensional gel electrophoresis protein pattern of rFSH-primed mice and appeared 3 h after the hCG surge, is a member of the aldo-keto reductase superfamily and was originally identified in the mouse vas deferens. This is the first study describing MVDP expression and regulation by LH in the ovary. Northern blot analysis of female mice tissues showed that mvdp messenger RNA (mRNA) was only present in adrenal glands and in hCG-treated ovaries. In situ hybridization studies localized the mvdp mRNA unequivocally to ovarian thecal and interstitial cells with an expression profile starting already 1.5 h, and decreasing 24 h, after LH treatment. In the adrenal glands, mvdp mRNA was not regulated by LH and localized in the cells of the zona fasciculata. In murine adrenocortical cells, a recent study proposed a detoxifying role of MVDP. MVDP might fulfill the same function in the ovary; however, because of its strong and early transcriptional induction by LH, it is also possible that MVDP catalyses another important step during the cascade of events occurring at the time of ovulation.

Expression of a new cold shock protein of 21.5 kDa and of the major cold shock protein by Streptococcus thermophilus after cold shock.

Streptococcus thermophilus is widely used in food fermentations; it commonly suffers diverse stress challenges during manufacturing. This study investigated the cold shock response of S. thermophilus when the cell culture temperature shifted from 42 degrees C to 15 degrees C or 20 degrees C. The growth of cells was affected more drastically after cold shock at 15 degrees C than at 20 degrees C. The generation time was increased by a factor of 19 when the temperature was lowered from 42 degrees to 20 degrees C, and by a factor of 72 after a cold shock at 15 degrees C. The two-dimensional electrophoretic protein patterns of S. thermophilus under cold shock conditions were compared with the reference protein pattern when cells were grown at optimal temperature. Two proteins of 21.5 and 7.5 kDa synthesized in response to cold shock were characterized. N-terminal sequencing and sequence homology searches have shown that the 7.5-kDa protein belonged to the family of the major cold shock proteins, while no homology was found for the new cold shock protein of 21.5 kDa.

Immobiline-based two-dimensional gel electrophoresis: an optimised protocol for resolution of human colonic mucosal proteins.

An optimised protocol for the production of two-dimensional protein patterns of human colonic mucosal cells using immobilised pH gradients in the first dimension is presented. We tested a wide variety of solubilising agents and electrophoretic parameters, separately and in combination. Protein solubilisation was found to be best using a lysis solution containing 9 M urea, 2% Triton X-100, 2% 2-mercaptoethanol, 0.8% Pharmalyte pH 3-10 and 8 mM phenylmethylsulfonyl fluoride (PMSF). Horizontal streaking of basic proteins in the first dimension was virtually eliminated by a combination of washing Immobiline strips in 100 mM asorbic acid, pH 4.5, for 24 h before use and isoelectric focusing samples at 40 degrees C. The presence of 2% glycerol in the first dimension resulted in tighter resolution throughout the entire pH range. The use of these conditions may prove to have broad applicability to the generation of optimally resolved Immobiline-based two-dimensional protein patterns of many other tissues.