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PubMed2006

Differential display proteomic analysis of Picea meyeri pollen germination and pollen-tube growth after inhibition of actin polymerization by latrunculin B.

To investigate roles of the actin cytoskeleton in growth of the pollen tube of Picea meyeri, we used the actin polymerization inhibitor latrunculin B (LATB) under quantitatively controlled conditions. At low concentrations, LATB inhibited polymerization of the actin cytoskeleton in the growing pollen tube, which rapidly inhibited tip growth. The proteomic approach was used to analyse protein expression-profile changes during pollen germination and subsequent pollen-tube development with disturbed organization of the actin cytoskeleton. Two-dimensional electrophoresis and staining with Coomassie Brilliant Blue revealed nearly 600 protein spots. A total of 84 of these were differentially displayed at different hours with varying doses of LATB, and 53 upregulated or downregulated proteins were identified by mass spectrometry. These proteins were grouped into distinct functional categories including signalling, actin cytoskeleton organization, cell expansion and carbohydrate metabolism. Moreover, actin disruption affected the morphology of Golgi stacks, mitochondria and amyloplasts, along with a differential expression of proteins involved in their functions. These findings provide new insights into the multifaceted mechanism of actin cytoskeleton functions and its interaction with signalling, cell-expansion machinery and energy-providing pathways.

Actin Cytoskeleton↗
PubMed2006

2-D differential membrane proteome analysis of scarce protein samples.

Proteome studies with small sample amounts are difficult to perform, especially when membrane proteins are the focus of interest. In our study a new method for the analysis of scarce membrane protein samples combining large gel 2-D-CTAB/SDS-PAGE with fluorescence dye saturation labelling (satDIGE) was developed, allowing a highly sensitive differential analysis of different cell states. After Triton X-114 phase partitioning, enriched membrane protein samples of T cells were labelled at cysteine residues using fluorescence dyes and separated by large gel 2D-CTAB/SDS-PAGE. For a differential analysis 3 mug protein was found to be sufficient to detect proteins in a widespread well-separated diagonal spot pattern.

Amino Acid Sequence↗
PubMed2006

2D gel blood serum biomarkers reveal differential clinical proteomics of the neurodegenerative diseases.

This review addresses the challenges of neuroproteomics and recent progress in biomarkers and tests for neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. The review will discuss how the application of quantitative 2D gel electrophoresis, combined with appropriate single-variable and multivariate biostatistics, allows for selection of disease-specific serum biomarkers. It will also address how the use of large cohorts of specifically targeted patient blood serum samples and complimentary age-matched controls, in parallel with the use of selected panels of these biomarkers, are being applied to the development of blood tests to specifically address unmet pressing needs in the differential diagnosis of these diseases, and to provide potential avenues for mechanism-based drug targeting and treatment monitoring. While exploring recent findings in this area, the review discusses differences in critical pathways of immune/inflammation and amyloid formation between Parkinson's disease and amyotrophic lateral sclerosis, as well as discernable synergistic relationships between these pathways that are revealed by this approach. The potential for pathway measurement in blood tests for differential diagnosis, disease burden and therapeutic monitoring is also outlined.

Biomarkers↗
PubMedDate not supplied

Differential radioactive proteomic analysis of microdissected renal cell carcinoma tissue by 54 cm isoelectric focusing in serial immobilized pH gradient gels.

We present a proof of principle study, using laser microdissection and pressure catapulting (LMPC) of two clinical tissue samples, each containing approximately 3.8 microg renal cell carcinoma protein and 3.8 microg normal kidney protein respectively from one patient. The study involved separate radio-iodination of each sample with both (125)I and (131)I, dual inverse replicate sample loading to high resolution 54 cm "daisy chain" serial immobilized pH gradient isoelectric focusing (IPG-IEF) 2D-PAGE gels, co-electrophoretic separation of cross-labeled proteins from different samples, and precision multiplex differential radioactive imaging to obtain signals specific for each sample coelectrophoresed within single gels but labeled with different isotopes of iodine, providing extremely precise intra-gel estimates of the abundance ratio for protein spots from both samples. Twelve multiplexed analytical radioactive SDS-gels from 4 serial IPG-IEF gels provided 24 individual radioactive images for a comprehensive analytical protein multiplex quantification study. A further 12 SDS gels containing (125)I-labeled sample were coelectrophoresed with preparative protein amounts obtained from whole tissue sections for the mass spectrometric identification of comigrating proteins. This consumed <40% of the (125)I-labeled sample, and <20% of the (131)I-labeled sample from the respective original 3.8 microg samples. Twenty-nine proteins were identified by mass spectrometry with PMF scores >70 that were >2-fold differentially abundant between the samples and t-test probabilities <0.05. We conclude that this combination of technologies provides excellent quality protein multiplex data for the differential abundance analysis of large numbers of proteins from extremely small samples, and is applicable to a broad range of clinical and related applications.

Carcinoma, Renal Cell↗
PubMedDate not supplied

Proteome analysis of NIH3T3 cells transformed by activated Galpha12: regulation of leukemia-associated protein SET.

Galpha(12), the alpha-subunit of the G12 family of heterotrimeric G proteins is involved in the regulation of cell proliferation and neoplastic transformation. GTPase-deficient, constitutively activated mutant of Galpha(12) (Galpha(12)Q229L or Galpha(12)QL) has been previously shown to induce oncogenic transformation of NIH3T3 cells promoting serum- and anchorage-independent growth. Reduced growth-factor dependent, autonomous cell growth forms a critical defining point at which a normal cell turns into an oncogenic one. To identify the underlying mechanism involved in such growth-factor/serum independent growth of Galpha(12)QL-transformed NIH3T3, we carried out a two-dimensional differential proteome analysis of Galpha(12)QL-transformed NIH3T3 cells and cells expressing vector control. This analysis revealed a total of 22 protein-spots whose expression was altered by more than 3-folds. Two of these spots were identified by MALDI-MS analysis as proliferating cell nuclear antigen (PCNA) and myeloid-leukemia-associated SET protein. The increased expressions of these proteins in Galpha(12)QL cells were validated by immunoblot analysis. Furthermore, transient transfection studies with NIH3T3 cells indicated that the expression of activated Galpha(12) readily increased the expression of SET protein by 24 h. As SET has been previously reported to be an inhibitor of phosphatase PP2A, the nuclear phosphatase activity was monitored in cells expressing activated Galpha(12). Our results indicate that the nuclear phosphatase activity is inhibited by greater than 50% in Galpha(12)QL cells compared to vector control cells. Thus, our results from differential proteome analysis presented here report for the first time a role for SET in Galpha(12)-mediated signaling pathways and a role for Galpha(12) in the regulation of the leukemia-associated SET-protein expression.

Animals↗
PubMed2006

Proteome and differential expression analysis of membrane and cytosolic proteins from Mycobacterium avium subsp. paratuberculosis strains K-10 and 187.

Little is known of protein expression in Mycobacterium avium subsp. paratuberculosis and how this contributes to pathogenesis. In the present study, proteins from both membranes and cytosol were prepared from two strains of M. avium subsp. paratuberculosis, i.e., laboratory-adapted strain K-10 and a recent isolate, strain 187, obtained from a cow exhibiting clinical signs of Johne's disease. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosol and membrane proteins from K-10 and 187 showed marked differences in protein expression. Relative levels of protein expression from both M. avium subsp. paratuberculosis strains were measured by using amine-reactive isobaric tagging reagents (iTRAQ) and tandem mass spectroscopy. Protein identification and relative expression data were obtained for 874 membrane and cytosolic proteins from the M. avium subsp. paratuberculosis proteome. These data showed a number of significant differences in protein expression between strain K-10 and clinical isolate 187. Examples of proteins expressed at higher levels in clinical isolate 187 compared to strain K-10 are AtpC, RpoA, and several proteins involved in fatty acid biosynthesis. In contrast, proteins such as AhpC and several proteins involved in nitrogen metabolism were expressed at higher levels in strain K-10 compared to strain 187. These data may provide insights into the proteins whose expression is important in natural infection but are modified once M. avium subsp. paratuberculosis is adapted to laboratory cultivation. Results from these studies will provide tools for developing a better understanding of M. avium subsp. paratuberculosis infection in the host and offer potential as diagnostic reagents and vaccine candidates.

Bacterial Proteins↗
PubMed2006

Analysis of chicken serum proteome and differential protein expression during development in single-comb White Leghorn hens.

Serum is believed to harbor thousands of distinct proteins that are either actively secreted or leak from various blood cells or tissues. Exploring protein composition in serum may accelerate the discovery of novel protein biomarkers for specific economic traits in livestock species. This study analyzed serum protein composition to establish a 2-DE reference map, and monitored protein dynamics of single-comb White Leghorn hens at 8, 19 and 23 weeks after hatching. A total of 119 CBB-stained and 315 silver-stained serum protein spots were analyzed by MALDI-TOF MS. Of these, 98 CBB-stained and 94 silver-stained protein spots were significantly matched to existing chicken proteins. The identified spots represented 30 distinctive proteins in the serum of laying hens. To compare protein expression during development, expression levels of 47 protein spots were quantified by relative spot volume with Melanie 3 software. Ten protein spots increased and 3 protein spots decreased as hen age increased. Previous research has suggested that some of these proteins play critical roles in egg production. The differentially expressed proteins with unknown identities will be valuable candidates for further explorations of their roles in egg production of laying hens.

Age Factors↗
PubMed2005

Identification of thioredoxin h-reducible disulphides in proteomes by differential labelling of cysteines: insight into recognition and regulation of proteins in barley seeds by thioredoxin h.

Using thiol-specific fluorescence labelling, over 30 putative target proteins of thioredoxin h with diverse structures and functions have been identified in seeds of barley and other plants. To gain insight at the structural level into the specificity of target protein reduction by thioredoxin h, thioredoxin h-reducible disulphide bonds in individual target proteins are identified using a novel strategy based on differential alkylation of cysteine thiol groups by iodoacetamide and 4-vinylpyridine. This method enables the accessible cysteine side chains in the thiol form (carbamidomethylated) to be distinguished from those inaccessible or disulphide bound form (pyridylethylated) according to the mass difference in the peptide mass maps obtained by matrix-assistend laser desorption/ionisation-time of flight mass spectrometry. Using this approach, in vitro reduction of disulphides in recombinant barley alpha-amylase/subtilisin inhibitor (BASI) by barley thioredoxin h isoform 1 was analysed. Furthermore, the method was coupled with two-dimensional electrophoresis for convenient thioredoxin h-reducible disulphide identification in barley seed extracts without the need for protein purification or production of recombinant proteins. Mass shifts of 15 peptides, induced by treatment with thioredoxin h and differential alkylation, identified specific reduction of nine disulphides in BASI, four alpha-amylase/trypsin inhibitors and a protein of unknown function. Two specific disulphides, located structurally close to the alpha-amylase binding surfaces of BASI and alpha-amylase inhibitor BMAI-1 were demonstrated to be reduced to a particularly high extent. For the first time, specificity of thioredoxin h for particular disulphide bonds is demonstrated, providing a basis to study structural aspects of the recognition mechanism and regulation of target proteins.

Allergens↗
PubMed2002

Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients.

A reproducible high-resolution protein separation method is the basis for a successful differential proteome analysis. Of the techniques currently available, two-dimensional gel electrophoresis is most widely used, because of its robustness under various experimental conditions. With the introduction of narrow range immobilized pH gradient (IPG) strips (also referred to as ultra-zoom gels) in the first dimension, the depth of analysis, i.e. the number of proteins that can be resolved, has increased substantially. However, for poorly understood reasons isoelectric focusing on ultra-zoom gels in the alkaline region above pH 7 has suffered from problems with resolution and reproducibility. To tackle these difficulties we have optimized the separation of semipreparative amounts of proteins on alkaline IPG strips by focusing on two important phenomena: counteracting water transport during isoelectric focusing and migration of dithiothreitol (DTT) in alkaline pH gradients. The first problem was alleviated by the addition of glycerol and isopropanol to the focusing medium, leading to a significant improvement in the resolution above pH 7. Even better results were obtained by the introduction of excess of the reducing agent DTT at the cathode. With these adaptations together with an optimized composition of the IPG strip, separation efficiency in the pH 6.2-8.2 range is now comparable to the widely used acidic ultra-zoom gels. We further demonstrated the usefulness of these modifications up to pH 9.5, although further improvements are still needed in that range. Thus, by extending the range covered by conventional ultra-zoom gels, the depth of analysis of two-dimensional gel electrophoresis can be significantly increased, underlining the importance of this method in differential proteomics.

2-Propanol↗
PubMed2004

Differential analysis of two-dimension gel electrophoresis profiles from the normal-metaplasia-dysplasia-carcinoma tissue of human bronchial epithelium.

Processes involved in malignant transformation of the lung from preneoplasia are poorly understood. To better understand this process, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) profiles of proteins from the normal, metaplasia, dysplasia and carcinoma tissues of human bronchial epithelia were examined by differential proteomic analysis. The selected differential protein-spots were identified by peptide mass fingerprint based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database searching. The average spots for normal epithelium, metaplasia, dysplasia and invasive carcinoma were 1189.50 +/- 39.89, 1227.00 +/- 37.90, 1273.00 +/- 43.31 and 1326.00 +/- 66.63, respectively. Well-resolved, reproducible 2-D PAGE patterns of the normal-metaplasia-dysplasia-carcinoma tissues of bronchial epithelia were obtained. After matching, the number of spots of differential proteins between normal tissue and metaplasia, metaplasia and dysplasia, and dysplasia and invasive cancer tissues were 31.50 +/- 7.67, 41.00 +/- 9.07 and 56.00 +/- 8.96, respectively. In total, 35 differential proteins, expressed only at the later stage of a two-stage comparison, were identified, some of which are known to be involved in regulating the processes of proliferation, differentiation and signal transduction. Current data in this study, for the first time, provide the basis for identification of potential tumor markers of human lung squamous carcinoma and their involvement in the progression of malignant transformation of bronchial epithelium.

Bronchi↗
PubMed2002

Familial combined hyperlipidemia plasma stimulates protein secretion by HepG2 cells: identification of fibronectin in the differential secretion proteome.

The aim of this study was to evaluate whether soluble factors in plasma of familial combined hyperlipidemia (FCHL) patients affect hepatic protein secretion. Cultured human hepatocytes, i.e., HepG2 cells, were incubated with fasting plasma (20%, v/v, in DMEM) from untreated FCHL patients or normolipidemic controls. Overall protein secretion was 10-15% higher after incubation with FCHL plasma. This was specifically caused by an increase in four secreted proteins, with estimated sizes of 240, 180, 120, and <40 kD (P < 0.001, P < 0.006, P < 0.002, P < 0.02, respectively). The 240 kD protein in the secretion proteome was identified as fibronectin by mass spectrometry. Plasma fibronectin concentrations were elevated in FCHL patients, confirming biological relevance of these data. Overall protein secretion by HepG2 cells correlated with concentrations of triglycerides (r = 0.61, P < 0.001) in the applied plasma samples. VLDL+IDL isolated from FCHL patients, induced a higher protein secretion than lipoproteins isolated from controls (P < 0.001). Remarkably, secretion of apoB, the structural protein of VLDL, was stimulated to a similar extent by FCHL and control plasma. FCHL plasma did not induce excess secretion of apoB by HepG2 cells compared with control plasma. FCHL plasma did stimulate secretion of several distinct hepatic proteins, among which fibronectin was identified.

Apolipoproteins B↗
PubMed2004

Applying proteomic methodologies to analyze the effect of hexamethylene bisacetamide (HMBA) on proliferation and differentiation of human gastric carcinoma BGC-823 cells.

Human gastric carcinoma BGC-823 cells underwent morphological differentiation and cell cycle arrest in vitro when treated with 5mM hexamethylene bisacetamide (HMBA) for 48h. To further understand the mechanism of HMBA-induced differentiation, proteomic methodologies were applied to screen and identify altered proteins involved in the commitment of BGC-823 cells to differentiate. Five distinct altered proteins were acquired by two-dimensional (2-D) PAGE and were consequently identified as ras-related protein rab-35 (Rab-35), splice truncated isoform of transmembrane protease, serine 3 (serine TADG-12), regulator of G-protein signaling 1 (RGS1), ret finger protein-like 1 (RFPL1) and F-actin capping protein alpha-3 subunit (GSG3) by analysis of mass spectrograph. Of the five proteins, serine TADG-12 down-regulated under the detectable level after HMBA treatment, Rab-35, RGS1 and RFPL1 sharply up-regulated within the HMBA-induced BGC-823 cells, and GSG3, appearing in both treated and untreated cells, remarkably increased within BGC-823 cells after HMBA stimulation. Our results implicate that the molecular mechanism of BGC-823 cell differentiation in response to HMBA may involved in complex processes including a signaling network linking vesicle transport, actin cytoskeleton remodeling except for morphology differentiation, cell cycle G1 arrest.

Acetamides↗
PubMed2002

The application of 2D gel-based proteomics methods to the study of breast cancer.

The protein complement of breast cells consists of many thousands of proteins. Recent developments in 2D gel electrophoresis technology have made studies requiring the quantitative analysis of a differential proteome, such as comparison between normal and malignant cells or investigation of drug effects on cells, truly feasible. Computer software plays a central part in the comparisons between multiple gels required for such experiments. In addition, software tools allow patterns of coexpression of proteins to be studied, offering potential insights into protein regulation, interactions, and functions, especially when combined with complementary data on gene expression. In this paper, the technology and limitations of 2D gel-based proteomics are reviewed. Techniques for comparing sets of gels at a global level as well as identifying specific protein features that differentiate gels are discussed. Our own experience of studying the breast cell proteome is used to illustrate the difficulties and achievements of differential proteomics.

Breast Neoplasms↗
PubMed2006

Proteomic analysis of the transition from quiescent to proliferating stages in rat liver hepatectomy model.

The 70% (or 2/3) partial hepatectomy (PHx) rat liver model provides an effective medium for study of the transition and regulation of hepatocytes from quiescent to proliferating phase. Although the gene expression pattern has come under intense scrutiny, a differential proteomic study could help to reveal the mechanism of how the process is initiated and regulated. The proteomic changes were analyzed in two groups, 7 h after 70% PHx test group and sham-operation control group, by two-dimensional gel electrophoresis with 907 +/- 33 and 910 +/- 64 spots on gels, respectively. Twelve down-regulated spots and twenty-six up-regulated spots were recognized using ImageMaster software and were identified by matrix-assisted laser desorption/ionization-mass spectrometry-quadrupole time of flight mass spectrometry and/or tandem mass spectrometry reconfirmation. Some of the differential proteins were associated with stress defense, lipid metabolism, and macromolecular biosynthesis while the others were shown to be involved in regulating transcript factors associated with liver regeneration. A "proteomic model" for liver regeneration was suggested based on our data and related scientific literature to interpret the differential proteome pattern that reflected the transition of cells from quiescent to proliferating state, including but not limited to the rat liver after 70% PHx.

Animals↗
PubMedDate not supplied

Enabling proteomics discovery through visual analysis. The peptide permutation and protein prediction tool.

Proteins play a key role in cellular processes, making proteomics central to understanding systems biology. MS techniques provide a means to observe entire proteomes at a global level. Yet, high-throughput MS proteomics techniques generate data faster than it can currently be analyzed. The success of proteomics depends on high-throughput experimental techniques coupled with sophisticated visual analysis and data-mining methods. Visual analysis has been applied successfully in a number of fields plagued with huge, complex data sets and will likely be an important tool in proteomics discovery. PQuad, a novel visualization of MS proteomics data, provides powerful analysis capabilities that support a number of proteomic data applications. In particular, PQuad supports differential proteomics by simplifying the comparison of peptide sets from different experimental conditions as well as different protein identification or confidence scoring techniques. Finally, PQuad supports data validation and quality control by providing a variety of resolutions for huge amounts of data to reveal errors undetected by other methods.

Algorithms↗
PubMed2006

Statistics for proteomics: experimental design and 2-DE differential analysis.

Proteomics relies on the separation of complex protein mixtures using bidimensional electrophoresis. This approach is largely used to detect the expression variations of proteins prepared from two or more samples. Recently, attention was drawn on the reliability of the results published in literature. Among the critical points identified were experimental design, differential analysis and the problem of missing data, all problems where statistics can be of help. Using examples and terms understandable by biologists, we describe how a collaboration between biologists and statisticians can improve reliability of results and confidence in conclusions.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2004

Troponin T as a marker of differentiation revealed by proteomic analysis in renal arterioles.

Renovascular hypertension is characterized by stenosis of the renal artery and high plasma renin levels. The renal phenotype is characterized by high levels of renin in the hypoperfused kidney due to the recruitment of renin-producing cells along the afferent arterioles. This increase in myoepithelioïd cells is due mainly to the differentiation of existing smooth muscle cells with acquisition of a secretory phenotype. To understand the molecular mechanisms involved in this recruitment, we used the established rat model of renovascular hypertension known as the two-kidney, one-clip model in the Lewis rat. Renal arterioles were isolated using magnetized iron suspension. Differential proteomic analysis was performed using 2-D polyacrylamide gel electrophoresis followed by mass spectrometry. Comparative analysis of soluble proteins extracted from afferent arterioles of clipped and contralateral kidneys showed 14 proteins significantly differentially expressed by at least a factor of 2. These proteins were identified by mass spectrometry. The most striking protein revealed by proteomics is troponin T, which is down-regulated in the afferent arterioles of the clipped kidney. Confocal microscopy showed that troponin T is specific of the smooth muscle phenotype and absent in the myoepithelioïd phenotype. Our data suggest that troponin T is only present in renal smooth muscle cells.

Animals↗
PubMed2005

[Comparative proteomics analysis of differentially expressed metastasis-associated proteins in human ovarian cancer cell lines].

OBJECTIVE: To find the key proteins associated with metastasis of ovarian cancer, and find potential diagnostic markers and therapeutic targets of this malignancy. METHODS: A comparative proteomic strategy, in a combination of two-dimensional electrophoresis separation and mass spectrometry identification, was adopted to search for proteome alternations in an ovarian cancer mother cell line HO-8910 and its highly metastatic cell subline HO-8910PM. RESULTS: Twenty-one significantly different spots (two-fold increase or decrease) were detected between the two cell lines, of which 17 candidate proteins were successfully identified and characterized. Compared with those in HO-8910 mother cell line, 16 proteins were significantly up-regulated, while 5 proteins down-regulated in the highly metastatic cell subline HO-8910PM. The seventeen identified proteins could be functionally classified into 7 groups as following: zinc finger protein, calcium-binding protein, DNA repair and synthesis protein, cell regulatory protein, metabolism-related protein, cell surface antigen, cell signals and transducing protein. CONCLUSIONS: The results suggest that an obviously differential proteomic expression exists between the human ovarian cancer mother cell line HO-8910 and highly metastatic cell subline HO-8910PM. It provides a clue for further identification of metastasis-related proteins, novel diagnostic markers as well as therapeutic targets of this malignancy.

Cell Line, Tumor↗