High-resolution Two-dimensional Polyacrylamide Gel Research Articles

Differential Expression of Middle Silk Gland Proteins Caused by Cold Stress in Philosamia ricini

This study reports differentially expressed proteins in the middle silkglands of normal and cold stress treated 5th instar Philosamia ricini larvae by using high resolution two-dimensional polyacrylamide gel electrophoresis. About 300 protein spots were resolved in both type of the larvae. Most of the proteins were shown to be distributed in the area from 20 to 70 kDa ranging between pH 4 and 8. Six proteins showed considerably decreased expression in cold stress treated samples. Three proteins displayed increased level of expression after cold stress treatment, while two other proteins were expressed exclusively in cold stress treated silkgland tissues.

Amidation inhibitors 4-phenyl-3-butenoic acid and 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester are novel HDAC inhibitors with anti-tumorigenic properties.

4-Phenyl-3-butenoic acid (PBA) is an inhibitor of peptidylglycine alpha-amidating monooxygenase with anti-inflammatory properties that has been shown to inhibit the growth of ras-mutated epithelial and human lung carcinoma cells. In this report, we show that PBA also increases the acetylation levels of selected histone subtypes in a dose and time dependent manner, an effect that is attributable to the inhibition of histone deacetylase (HDAC) enzymes. Comparison studies with the known HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) using high resolution two-dimensional polyacrylamide gels and Western analysis provide evidence that PBA acts as an HDAC inhibitor within cells. PBA and a more potent amidation inhibitor, 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester (AOPHA-Me), inhibit HDAC enzymes in vitro at micromolar concentrations, with IC50 values approximately 30 fold lower for AOPHA-Me than PBA for selected HDAC isoforms. Overall, these results indicate that PBA and AOPHA-Me are novel anti-tumorigenic HDAC inhibitors.

Soluble Alpha-APP (sAPPalpha) Regulates CDK5 Expression and Activity in Neurons

A growing body of evidence suggests a role for soluble alpha-amyloid precursor protein (sAPPalpha) in pathomechanisms of Alzheimer disease (AD). This cleavage product of APP was identified to have neurotrophic properties. However, it remained enigmatic what proteins, targeted by sAPPalpha, might be involved in such neuroprotective actions. Here, we used high-resolution two-dimensional polyacrylamide gel electrophoresis to analyze proteome changes downstream of sAPPalpha in neurons. We present evidence that sAPPalpha regulates expression and activity of CDK5, a kinase that plays an important role in AD pathology. We also identified the cytoprotective chaperone ORP150 to be induced by sAPPalpha as part of this protective response. Finally, we present functional evidence that the sAPPalpha receptor SORLA is essential to mediate such molecular functions of sAPPalpha in neurons.

Ascaris lumbricoides and Ascaris suum: Comparative proteomic studies using 2-DE coupled with mass spectrometry

Roundworms of the genus Ascaris comprise species of medical (A. lumbricoides) and veterinary (A. suum) importance. Despite the economic importance, public health impact, and worldwide prevalence both parasites still have a controversial taxonomy and their proteomes are poorly described in databases. In the present study, high-resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF MS was used to characterize and contrast proteomic profiles of adult female A. lumbricoides and A. suum. Approximately, 3-fold higher protein spots were obtained in the present study compared to traditional method, and more than 630 and 750 protein spots were reproducibly detected among the 6 gels examined (3 gels for each of A. lumbricoides and A. suum). The peptide mass finger printings (PMFs) were acquired successfully for 17 from 22 differential 2-DE spots, corresponding to 12 unique proteins. Among them, 2 Asu-enriched and 3 Alu-specific proteins were found in A. lumbricoides; and 6 Asu-enriched and 1 Alu-specific proteins were found in A. suum. Functional analysis revealed low level of difference among differentially expressed proteins of the two nematode species, while the 1 species-specific protein of A. suum was different from that of A. lumbricoides. The proteomic patterns of A. lumbricoides and A. suum provide proteomic evidence that these two taxa are very closely related. This work indicates that the combined 2-DE and MALDI-TOF/TOF MS approaches employed in the current study allow the identification of proteins whose expression is different between Ascaris species.

An improved plant leaf protein extraction method for high resolution two-dimensional polyacrylamide gel electrophoresis and comparative proteomics

We report here a simple and universally applicable protocol for extracting high quality proteins from plant leaf tissues. The protocol provides improved resolution and reproducibility of two-dimensional polyacrylamide gel electrophoresis (2-DE) and reduces the time required to analyze samples. Partitioning rubisco by polyethylene glycol (PEG) fractionation provides clearer detection of low-abundance proteins. Co-extraction of interfering substances increases the sample conductivity, which results in poor electrophoretic separation. Re-extraction of PEG-fractionated samples with phenol effectively eliminated interfering substances, which results in optimal conductivity during separation in the first dimension of the isoelectric focusing. Smooth focusing reduces analysis time and provides superior resolution in 2-DE gels. Incubating the samples at −80° C instead of −20° C reduced protein precipitation time to 2–3 h. Removal of nonprotein contaminants and the use of sonication increased protein solubility without additional reagents. These changes enabled loading and separation of maximum amounts of proteins, which permitted improved protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). An immunological approach revealed that little or no ribulose-1, 5-bisphosphte bisphosphate carboxylase oxygenase was present in the PEG supernatant. In addition, low-abundance proteins, such as myelocytomatosis transcription factor (MYC) and alpha subunit of heterotrimeric guanine nucleotide-binding protein complex (Gα), were detected only in the modified PEG supernatant and not in the total protein. These results suggest that our protocol produced high quality proteins and made many low-abundant proteins available for proteomic analysis. The successful application of this protocol for analyzing the leaf proteomes of soybean, Miscanthus sinensis, barley, Chinese cabbage, peanut and tea (Camellia sinensis) suggests that it could be used for comparative proteomic analysis of a wide range of plant leaves.

Differential proteomic analysis of developmental stages of Acca sellowiana somatic embryos

Feijoa (Acca sellowiana, Myrtaceae), a native fruit species from southern Brazil and northern Uruguay, is considered to constitute a reference system for somatic embryogenesis in woody dicots. This in vitro regenerative pathway is an efficient micropropagation method, and a suitable model system for studies in plant developmental physiology. This study attempts to detect and identify proteins that are expressed during the different developmental stages of somatic embryos of A. sellowiana. Using high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE), a high degree of similarity between protein profiles of the assayed somatic embryos was observed. Of the 74 different protein spots extracted for analysis, 60 were identified by means of 2-DE/MALDI-TOF/MS. Twelve proteins were expressed in all the assayed stages. Ten proteins were expressed in the initial stages and 22 proteins were expressed in the mature developmental stages of somatic embryos. Only one protein was expressed exclusively in the torpedo stage, whereas four were expressed in the pre-cotyledonary, and none in the cotyledonary stage. The proteins identified were involved in the synthesis of phenylalanine ammonia-lyase, a conspicuous polyphenol present in the induction of feijoa embryogenic cultures. The presence of essential proteins of nitrogen metabolism, such as the cytosolic glutamine synthetase protein, was also observed. The physiological implications of these findings are discussed.

Selection and Identification of Acute Proteins with Proteomic Techniques in Rhinencephalon of Cyprinus Carpio After Wounded Acutely

A combined available approach of low osmosis and freeze-soluble with liquid nitrogen was used to disrupt the rhinencephalon tissue of Cyprinus carpio (CC). A centrifugal method with low speed was used to extract proteins in rhinencephalon tissue of CC (CCRT), and high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) map with 1200 protein spots was obtained. In this study, several techniques were used for extracting and separating proteome in CCRT. Three lysis solutions were prepared to disrupt the tissue in the fish, and used to extract proteome, water-soluble proteins and fat-soluble proteins, respectively. Both water-soluble proteins and fat-soluble protein from the CCRT were effectively separated by an optimized 2D-PAGE method with high resolution. With treating by 10% acetic acid, six acute proteins in CCRT were selected by proteomic techniques, and further identified by peptide mass fingerprinting (PMF) and database research, respectively. Three of these differential proteins can be primarily defined to be 70 kD heat-shock protein, DNA ligase IV, and β 5 tubulin. It was proposed that these proteins may be used as markers to study the repairing pathway and mechanism in brain once wounded.

Studies on middle and posterior silk glands of silkworm ( Bombyx mori) using two-dimensional electrophoresis and mass spectrometry

Silk glands, present in the larval stage of the silkworm, produce threads of silky material to form the cocoon and are mainly composed of three parts: the anterior, the middle, and the posterior silk glands, each playing different roles in silk secretion. High-resolution two-dimensional polyacrylamide gel electrophoresis and computer-assisted analysis were used to investigate quantitative and qualitative differences between the middle and posterior silk glands. Silver staining revealed over 600 spots for each sample, mostly distributed from 15 to 100 kDa with pH 4–7. Computer-assisted image analysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and post-source decay technology suggested that there were significant differences in spot distribution and expression between the middle and posterior silk glands. In addition, 98 spots from the posterior silk gland were excised and further investigated following trypsin digestion. The results suggested that more than 20% of the 88 proteins identified were related to heat-shock proteins and chaperones. Redox system and DNA replication proteins involved in silk protein synthesis were also detected in the posterior silk gland. Interestingly, two novel serpin proteins were identified in the middle silk gland, and to a lesser extent in the posterior gland, which were presumed to be involved in regulation of proteolytic activity and protection of silk proteins from degradation.

Monitoring of stress responses

New developments in the RNA analysis techniques now enable a comprehensive view on the bacterial physiology under bioprocess conditions. The DNA-chip technology allows a genome wide transcriptional profiling of bacterial cells, whose genome sequence is available. Although the analyses of microbial bioprocesses have still been somewhat limited to date, this technique has already been successfully applied in different laboratories for the investigation of stress responses of selected industrially relevant bacterial hosts. Transcriptome analyses in combination with high resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry have been extensively applied for the description of general and specific stress and starvation responses of Escherichia coli and Bacillus subtilis. The consideration of bacterial stress and starvation responses is of crucial importance for the successful establishment of an industrial large scale bioprocess. Stress genes can be used as marker genes in order to monitor the fitness of industrial bacterial hosts during fermentation processes. This chapter gives an overview of current RNA analysis techniques. The bacterial stress and starvation responses, which are of potential importance for industrial microbial bioprocesses are summarised.

Proteomic Profiling Identifies an UV-Induced Activation of Cofilin-1 and Destrin in Human Epidermis

The human skin is the only line of defense against UV radiation. A series of responses to protect the skin are induced by UV radiation. In this study, a proteomic approach was used to study these responses. We have performed high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis of (solar simulated) UV-exposed reconstructed skin equivalents as well as native skin. Differentially expressed proteins were processed for mass spectrometric analysis, when consistent differences were observed in all individual human skin equivalents. In addition to proteins known to be involved in UV responses (HSP27, MnSOD, and PDX-2), we identified two novel proteins that were downregulated following UV exposure. Further analysis revealed that these proteins were the phosphorylated forms of the actin cytoskeleton modulators cofilin-1 and destrin. The de-phosphorylation of cofilin-1 was confirmed using western blotting of UV-exposed skin equivalents and ex vivo skin protein extracts. In conclusion, our study indicates the potency of a proteomic approach to study UV-induced changes in a tissue culture system mimicking human skin as well as excised human skin.

Proteome analysis of the silkworm (Bombyx mori. L) colleterial gland during different development stages

The silkworm, Bombyx mori, colleterial gland developed very slowly until 2 days before emergence, then markedly enlarged due to the accumulation of a glue-like substances (mainly including 85% water and 11% proteins). However, the No glue (Ng) mutant female moth secreted only very little glue-like substance and laid loose eggs naturally. High-resolution two-dimensional polyacrylamide gel electrophoresis, followed by computer-assisted analysis, was used to screen the secretory region of colleterial gland protein patterns during different development stages to find quantitative and qualitative difference in protein expression during the pupae and moth stages. More than 700 protein spots were resolved in different developmental stages from the secretory region of the glands and most of the proteins were distributed in the mass range from 30 to 70 kD with pH 4-8. Through comparison and analysis, it was found that 3 proteins were only expressed in the later pupae stage (one or two days before emergence) and moth stage. Furthermore, these proteins were not expressed in the Ng mutant especially actin. There was a great variation of some protein expression volume during the development. Protein spots that changed more than 1.5-fold in expression level (relative to day 9), including 6 spots that were down-regulated and 2 spots that were up-regulated in expression were excised for identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Results indicated that actins that participated or regulated the exocytosis of colleterial gland and other differentially expressed proteins might be related to colleterial gland development or the secretion of a glue-like substance.

The protein profile of acetazolamide-treated sera in mice bearing Lewis neoplasm

The aim of the present research is to analyze the proteome of neoplasm serum before and after treated with acetazolamide (20, 40, 80 mg kg −1 d −1 for 3 days p.o.). The Lewis lung carcinoma mice were used and carried out a comprehensive proteomic analysis by using the technologies of high-resolution two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and mass spectrometry (MS). The results showed that the acetazolamide could dramatically reduce the lung metastasis and primary tumor growth. Its most potent inhibition rate on lung metastases was reach to 77.7% at the dose of 80 mg kg −1 d −1. The two dimension electrophoresis and software analysis reveal 393 protein spots in control gel, 385 protein spots were detected in treated gel and matched 209 protein spots with control gel, indicating that intensive changes had occurred during the process of treatment. Two obviously different spots were cut off from gel and for the peptide mass fingerprinting. Data base searching showed the two proteins' peptide much more mach with Histone H2B fragment and Ubc-like protein CROC1 fragment. The results suggest that acetazolamide has a strong anti-tumor and anti-metastasis effect on Lewis-lung-carcinoma. The mechanism may be related to its regulation on plenty of proteins, in particular, on upregulation of H2B and CROC-1 expression of postreplicational DNA repair related protein in serum.

Studies on middle silkgland proteins of cocoon colour sex-limited silkworm (Bombyx mori L.) using two-dimensional polyacrylamide gel electrophoresis.

Qualitative and quantitative differences in proteins expressed in the middle silkglands of male and female silkworm larvae that differ in silk colour were investigated by high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by computer assisted image analysis. About 1000 protein spots were resolved in both the sexes and most proteins were shown to be distributed in the area from 15 kDa to 70 kDa and pH 4-8. It was found that some proteins displayed higher expression in yellow cocoon, while two proteins were only expressed in female silkworm silkgland tissue through the comparison and analysis by two-D software. These proteins especially existed in female silkworm middle silkgland tissue of yellow cocoon. Furthermore, these proteins might be involved in the expression of cocoon colour phenotype

Proteome analysis of rice tissues by two-dimensional electrophoresis: an approach to the investigation of gibberellin regulated proteins

Protein databases constructed using high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) were used to explore the proteome expressed in various rice tissues. Proteins from leaf sheath, root, and cultured suspension cells were systematically analyzed using 2D-PAGE, mass spectrometry and Edman sequencing, followed by database searching. In all, 79 of the 431 spots detected by 2D-PAGE in the leaf sheath, 73 of the 508 spots in the root and 140 of the 962 spots in the cultured suspension cells could be identified. Protein lists were constructed for each tissue and used to investigate the effects of gibberellin (GA) treatment. In the leaf sheath, root and cultured suspension cells, 8, 21, and 14 of the identified proteins, respectively, were regulated by GA. These proteins included polypeptides involved in general metabolism, energy production, transcriptional regulation and signal transduction in the leaf sheath; in metabolism and defense in the root; and in metabolism, energy production, cell growth, defense and signal transduction in the cultured suspension cells. These results indicate that the proteome databases assembled in these studies will be useful for the rapid assessment of changes in protein content in specific tissues, and that proteins regulated by GA may play a significant role in tissue growth.

Analysis of Protein Expression along the Normal to Tension Wood Gradient in Eucalyptus gunnii

AbstractThis paper examines the contribution of various xylem proteins to wood formation inEucalyptus gunnii. Proteins were extracted from differentiating xylem harvested from a crooked tree, separated by high-resolution two-dimensional polyacrylamide gel electrophoresis, visualised by silver nitrate staining and analysed with a computer-assisted system for single protein spot quantification. Growth strain measurements allowed xylem samples to be classified quantitatively from tension wood to normal wood. Regression of lignin content on growth strain showed that a decrease in lignin content corresponded to decreasing growth strain values,i.e., presence of tension wood. Out of the 140 studied protein spots, 12 were significantly associated with growth strain: 7 being less abundant in tension wood and 5 being more abundant in tension wood. A clustered-correlation analysis was performed to study protein expression simultaneously along the gradient of gravistimulated stressed xylem tissue. Proteins were found to form “expression clusters”.

Proteome analysis in the study of the bacterial heat‐shock response

In recent years, it has become clear that, in addition to the regulation of the expression of specific genes, there are global regulatory systems that control the simultaneous expression of a large number of genes in response to a variety of environmental stresses. The first of these global control systems, and of substantial importance, is the heat-shock response. The heat-shock response is characterized by the induction of a large set of proteins (heat-shock proteins-HSPs) upon shifts to higher temperature and upon exposure to conditions in which proteins are denatured (i.e., alcohols, heavy metals). The heat-shock response is universal and many of the heat-shock proteins are highly conserved among species. In bacteria, the heat-shock response has been studied extensively in several Gram-positive bacteria (Bacillus subtilis) and in the Gram-negative bacteria (i.e., Escherichia coli, Agrobacterium tumefaciens). The first recognition of the molecular abundance of the bacterial heat-shock proteins took place with the introduction of high-resolution two-dimensional polyacrylamide gels (2D gels) to analyze complex mixtures of cellular proteins. Two-dimensional gels, followed by mass spectrometry, were used to define the heat-shock stimulons in several bacteria, and to study the regulatory elements that control the heat-shock response. Here, we review the heat-shock response and its regulation in bacteria. The review will emphasize the use of proteome analysis in the study of this response, and will point out those open questions that can be investigated with proteomics, including mass spectrometry techniques.

Electrophoretic characteristics of monoclonal immunoglobulin G of different subclasses

Monoclonal IgG are commonly observed in various B cell disorders, of which multiple myeloma is the most clinically relevant. In a series of serum samples, we identified by immunofixation 73 monoclonal IgG, including 63 IgG 1, 4 IgG 2, 5 IgG 3, and 1 IgG 4. The light chains were of κ type in 45 cases, and of λ type in 28 cases. These monoclonal IgG were further characterized by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) in various isoelectric focusing conditions, as well as by 3-DE (2-DE of the proteins extracted from agarose after serum protein agarose electrophoresis). After 2-DE, 38 out of 73 monoclonal γ chains (52%) were visualized using immobilized pH 3–10 gradients for isoelectric focusing. In 6 cases (8%), γ chains were only detected using alkaline immobilized pH 6–11 gradients. In 3 cases (4%), 3-DE revealed monoclonal γ chains hidden by polyclonal γ chains. Finally, in 26 cases (36%), no monoclonal γ chains were clearly visualized. Sixty-one monoclonal light chains (84%) were detected using immobilized pH 3–10 gradients, whereas 12 (16%) were not. Monoclonal γ chains and light chains were highly heterogeneous in terms of p I and M r. However, a statistically significant correlation ( P<0.05) was observed between the position of the monoclonal IgG in agarose gel and the p I of their heavy and light chains ( R=0.733, multiple linear regression). Because of the extreme diversity of their heavy and light chains, it appears that a classification of monoclonal IgG based only on their electrophoretic properties is not possible.

Identification of new proteins in follicular fluid of mature human follicles.

Proteins present in human follicular fluid (HFF) have been poorly characterized to date. The purpose of our study was to analyse the protein content and identify new proteins originating from fluid of mature human follicles. A total of six females from infertile couples referred for in vitro fertilization (IVF) were stimulated and 44 follicular fluid samples from mature follicles yielding an oocyte were collected 34-36 h after human chorionic gonadotropin administration. HFF samples were processed for high-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Comparative analysis of the 2-D gels revealed up to 600 spots, of which four were selected because of variations in their expression level. Using direct sequencing procedures (Edman degradation) or matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS), these four spots were identified as three new proteins: thioredoxin peroxydase 1 (TDPX1), transthyretin (TTR) and retinol-binding protein (RBP). The proteins identified here may emerge as potential candidates for specific functions during folliculogenesis and may prove useful as biomedical markers for follicle and/or oocyte maturation.

Identification of specific proteins in different lymphocyte populations by proteomic tools

The solubilized proteins of purified CD19(+) (B), CD8(+) (T) as well as CD4(+) (T) lymphocytes were separated by high resolution two-dimensional polyacrylamide gel electrophoresis, and the gels were analyzed using Melanie 3.0. Nine gels were studied, three for each lymphocyte population. After image analysis, 1411 +/- 73 spots (mean + SD) were detected. The protein pattern of B lymphocytes segregated from the one of T lymphocytes by ascendant heuristic clustering analysis. In addition, computer analysis separated CD8(+) from CD4(+) lymphocytes. When a search was performed in order to detect subsets of specific spots (presence vs. absence), a group of three spots, detected in the area of the protein maps corresponding to isoelectric point (pI) of 5.2 to 5.4 and molecular weight (M(r)) of 50 to 51 kDa, were found in both CD8(+) and CD4(+) cells, but not in CD19(+) cells. Mass spectrometry analysis revealed that these spots were associated with several proteins such as vimentin, tubulin, desmin and cytokeratin. Two spots, located in the area of the gel corresponding to pI of about 5.0 and a M(r) of 30 kDa, appeared as CD8(+) cell associated. Mass spectrometry analysis showed that the two spots were related to the same non-identified protein. Moreover internal peptides sequences matched with two human expressed sequence tags: gi/9759776, gi/12798420. No spots were found as only B cell associated.

Identification of specific proteins in different lymphocyte populations by proteomic tools

The solubilized proteins of purified CD19+ (B), CD8+ (T) as well as CD4+ (T) lymphocytes were separated by high resolution two-dimensional polyacrylamide gel electrophoresis, and the gels were analyzed using Melanie 3.0. Nine gels were studied, three for each lymphocyte population. After image analysis, 1411 ± 73 spots (mean + SD) were detected. The protein pattern of B lymphocytes segregated from the one of T lymphocytes by ascendant heuristic clustering analysis. In addition, computer analysis separated CD8+ from CD4+ lymphocytes. When a search was performed in order to detect subsets of specific spots (presence vs. absence), a group of three spots, detected in the area of the protein maps corresponding to isoelectric point (pI) of 5.2 to 5.4 and molecular weight (Mr) of 50 to 51 kDa, were found in both CD8+ and CD4+ cells, but not in CD19+ cells. Mass spectrometry analysis revealed that these spots were associated with several proteins such as vimentin, tubulin, desmin and cytokeratin. Two spots, located in the area of the gel corresponding to pI of about 5.0 and a Mr of 30 kDa, appeared as CD8+ cell associated. Mass spectrometry analysis showed that the two spots were related to the same non-identified protein. Moreover internal peptides sequences matched with two human expressed sequence tags: gi|9759776, gi|12798420. No spots were found as only B cell associated.