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Results for “differential proteomics analysis”

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PubMed2006

Differential label-free quantitative proteomic analysis of Shewanella oneidensis cultured under aerobic and suboxic conditions by accurate mass and time tag approach.

We describe the application of LC-MS without the use of stable isotope labeling for differential quantitative proteomic analysis of whole cell lysates of Shewanella oneidensis MR-1 cultured under aerobic and suboxic conditions. LC-MS/MS was used to initially identify peptide sequences, and LC-FTICR was used to confirm these identifications as well as measure relative peptide abundances. 2343 peptides covering 668 proteins were identified with high confidence and quantified. Among these proteins, a subset of 56 changed significantly using statistical approaches such as statistical analysis of microarrays, whereas another subset of 56 that were annotated as performing housekeeping functions remained essentially unchanged in relative abundance. Numerous proteins involved in anaerobic energy metabolism exhibited up to a 10-fold increase in relative abundance when S. oneidensis was transitioned from aerobic to suboxic conditions.

Amino Acid Sequence↗

PubMed2003

Multiplex proteomic analysis by two-dimensional differential in-gel electrophoresis.

This paper describes the use of fluorescence two-dimensional differential in-gel electrophoresis in a multiplex analysis of two distinct proteomes. As a model system, cerebral cortex tissues were analyzed from neurokinin1 receptor knockout (NK(1)R-/-) and wild type (NK(1)R+/+) mice in an attempt to identify molecular pathways involved in the function of this protein. Paired NK(1)R-/- and NK(1)R+/+ samples were labeled with fluorescent Cy3 and Cy5 dyes and electrophoresed on the same two-dimensional gels. Scanning the gels at wavelengths specific for each dye revealed the two different proteomes which were overlaid and the differences in abundance of specific protein spots were determined by the Amersham Biosciences DeCyder Differential In-gel Analysis software. A Cy2-labeled sample pool was co-electrophoresed with all Cy3- and Cy5-labeled sample pairs as an internal standard providing a link for inter-gel comparisons and for more robust statistical analysis of the data. Eight spots were found to be upregulated and two downregulated in the NK(1)R-/- mice compared to NK(1)R+/+ controls. Matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass fingerprinting was used to identify the proteins. The results illustrate the power of this multiplex proteomics technology and illustrate how proteomics can be used to understand gene function.

Animals↗

PubMed2001

Genomic and proteomic analysis of the myeloid differentiation program.

Although the mature neutrophil is one of the better characterized mammalian cell types, the mechanisms of myeloid differentiation are incompletely understood at the molecular level. A mouse promyelocytic cell line (MPRO), derived from murine bone marrow cells and arrested developmentally by a dominant-negative retinoic acid receptor, morphologically differentiates to mature neutrophils in the presence of 10 microM retinoic acid. An extensive catalog was prepared of the gene expression changes that occur during morphologic maturation. To do this, 3'-end differential display, oligonucleotide chip array hybridization, and 2-dimensional protein electrophoresis were used. A large number of genes whose mRNA levels are modulated during differentiation of MPRO cells were identified. The results suggest the involvement of several transcription regulatory factors not previously implicated in this process, but they also emphasize the importance of events other than the production of new transcription factors. Furthermore, gene expression patterns were compared at the level of mRNA and protein, and the correlation between 2 parameters was studied. (Blood. 2001;98:513-524)

Animals↗

PubMed2002

Genomic and proteomic analysis of the myeloid differentiation program: global analysis of gene expression during induced differentiation in the MPRO cell line.

We have used an approach using 2-dimensional gel electrophoresis with mass spectrometry analysis combined with oligonucleotide chip hybridization for a comprehensive and quantitative study of the temporal patterns of protein and mRNA expression during myeloid development in the MPRO murine cell line. This global analysis detected 123 known proteins and 29 "new" proteins out of 220 protein spots identified by tandem mass spectroscopy, including proteins in 12 functional categories such as transcription factors and cytokines. Bioinformatic analysis of these proteins revealed clusters with functional importance to myeloid differentiation. Previous analyses have found that for a substantial number of genes the absolute amount of protein in the cell is not strongly correlated to the amount of mRNA. These conclusions were based on simultaneous measurement of mRNA and protein at just a single time point. Here, however, we are able to investigate the relationship between mRNA and protein in terms of simultaneous changes in their levels over multiple time points. This is the first time such a relationship has been studied, and we find that it gives a much stronger correlation, consistent with the hypothesis that a substantial proportion of protein change is a consequence of changed mRNA levels, rather than posttranscriptional effects. Cycloheximide inhibition also showed that most of the proteins detected by gel electrophoresis were relatively stable. Specific investigation of transcription factor mRNA representation showed considerable similarity to those of mature human neutrophils and highlighted several transcription factors and other functional nuclear proteins whose mRNA levels change prominently during MPRO differentiation but which have not been investigated previously in the context of myeloid development. Data are available online at http://bioinfo.mbb.yale.edu/expression/myelopoiesis.

Animals↗

PubMedDate not supplied

Proteomic analysis of erythrocyte membranes by soft Immobiline gels combined with differential protein extraction.

Improvements in proteome analysis of erythrocyte membrane proteins by two-dimensional electrophoresis are here reported. In particular, a differential extraction procedure was set up allowing separation of integral membrane proteins from peripheral species. Moreover, the use of dilute Immobiline gels (down to as low as 3% T matrix) permitted a better penetration and transfer inside the gel of proteins with large M(r). These protocol modifications, combined with sample delipidation and alkylation prior to electrophoresis, which prevented generation of homo- and hetero-oligomers following disulfide scrambling phenomena, allowed the display of more than 500 spots in the pI/M(r) plane. Among those, noteworthy was the presence of high levels of filamentous proteins, such as alpha-spectrin and ankyrins, or integral membrane proteins, such as band 3, band 4.1 and 4.2, not displayed or barely present in other maps exploiting immobilized pH gradients in the first dimension. Accordingly, our results show that this 2D mapping technique is a valuable tool in exploring pathologies related to genetic defects associated to membrane proteins.

Detergents↗

PubMed2007

A comparative proteome and phosphoproteome analysis of differentially regulated proteins during fertilization in the self-incompatible species Solanum chacoense Bitt.

We have used 2-DE for a time-course study of the changes in protein and phosphoprotein expression that occur immediately after fertilization in Solanum chacoense. The phosphorylation status of the detected proteins was determined with three methods: in vivo labeling, immunodetection, and phosphoprotein-specific staining. Using a pI range of 4-7, 262 phosphorylated proteins could be mapped to the 619 proteins detected by Sypro Ruby staining, representing 42% of the total proteins. Among these phosphoproteins, antibodies detected 184 proteins from which 78 were also detected with either of the other two methods (42%). Pro-Q Diamond phosphoprotein stain detected 111 proteins, of which 76 were also detected with either of the other two methods (68%). The 32P in vivo labeling method detected 90 spots from which 78 were also detected with either of other two methods (87%). On comparing before and after fertilization profiles, 38 proteins and phosphoproteins presented a reproducible change in their accumulation profiles. Among these, 24 spots were selected and analyzed by LC-MS/MS using a hybrid quadrupole-TOF (Q-TOF) instrument. Peptide data were searched against publicly available protein and EST databases, and the putative roles of the identified proteins in early fertilization events are discussed.

Fertilization↗

PubMed2006

The proteomic analysis of an adipocyte differentiated from human mesenchymal stem cells using two-dimensional gel electrophoresis.

Adipose tissues play a crucial endocrine role in the control of whole body glucose homeostasis and insulin sensitivity. Considering the current substantial rise in obesity and obesity-related diseases, including diabetes, it is important to understand the molecular basis of adipocyte differentiation and its control. In this study, we have analyzed the protein expression inherent to adipogenic differentiation, by 2-DE, MALDI-TOF, and RT-PCR. This study focused on proteins that were differentially expressed by the differentiation of human mesenchymal stem cells (hMSCs) to adipocytes. We conducted 2-DE for each set of proteins in the cytosol of adipocytes that had differentiated from hMSC, in a pH range from 3-10. Thirty-two protein spots were shown to have different expression levels. Among these, eight up-regulated proteins were identified by MALDI-TOF/MS, as the following: syntaxin binding protein 3, OSBP-related protein 3, phosphodiesterase, glycophorin, immunoglobulin kappa chain variable region, peroxisome proliferative activated receptor gamma (PPAR-gamma), bA528A10.3.1 (novel protein similar to KIAA01616, isoform 1), and T cell receptor V-beta 4. Four proteins: syntaxin-3, OSBP-related protein 3, PPAR-gamma and glycophorin were associated with adipogenesis.

Adipocytes↗

PubMed2003

Proteomic changes during disturbance of cholesterol metabolism by azacoprostane treatment in Caenorhabditis elegans.

Although nematodes like Caenorhabditis elegans are incapable of de novo cholesterol biosynthesis, they can utilize nonfunctional sterols by converting them into cholesterol and other sterols for cellular function. The results reported previously and presented here suggest that blocking of sterol conversion to cholesterol in C. elegans by 25-azacoprostane-HCl (azacoprostane) treatment causes a serious defect in germ cell development, growth, cuticle development, and motility behavior. To establish a biochemical basis for these physiological abnormalities, we performed proteomic analysis of mixed stage worms that had been treated with the drug. Our results from a differential display proteomic analysis revealed significant decreases in the levels of proteins involved in collagen and cytoskeleton organization such as protein disulfide isomerase (6.7-fold), beta-tubulin (5.41-fold), and NEX-1 protein (>30-fold). Also reduced were enzymes involved in energy production such as phosphoglycerate kinase (4.8-fold) and phosphoenolpyruvate carboxykinase (8.5-fold), a target for antifilarial drugs such as azacoprostane. In particular, reductions in the expression of lipoprotein families such as vitellogenin-2 (7.7-fold) and vitellogenin-6 (5.4-fold) were prominent in the drug-treated worms, indicating that sterol metabolism disturbance caused by azacoprostane treatment is tightly coupled with suppression of the lipid transfer-related proteins at the protein level. However, competitive quantitative reverse transcriptase polymerase chain reaction showed that the transcriptional levels of vit-2, vit-6, and their receptors (e.g. rme-2 and lrp-1) in drug-treated worms were 3- to 5-fold higher than those in the untreated group, suggesting a presence of a sterol regulatory element-binding protein (SREBP)-like pathway in these genes. In fact, multiple predicted sterol regulatory elements or related regulatory sequences responding to sterols were found to be located at the 5'-flanking regions in vit-2 and lrp-1 genes, and their transcriptional activities fluctuated highly in response to changes in sterol concentration. Thus, many physiological abnormalities caused by azacoprostane-mediated sterol metabolism disturbance appear to be exerted at least in part through SREBP pathway in C. elegans.

Animals↗

PubMedDate not supplied

Comparison of statistical approaches for the analysis of proteome expression data of differentiating neural stem cells.

Comparative proteomic studies often use statistical tests included in the software for the analysis of digitized images of two-dimensional electrophoresis gels. As these programs include only limited capabilities for statistical analysis, many studies do not further describe their statistical approach. To find potential differences produced by different data processing, we compared the results of (1) Student's t-test using a spreadsheet program, (2) the intrinsic algorithms implemented in the Phoretix 2D gel analysis software, and (3) the SAM algorithm originally developed for microarray analysis. We applied the algorithms to proteome data of undifferentiated neural stem cells versus in vitro differentiated neural stem cells. We found (1) 367 spots differentially expressed using Student's t-test, (2) 203 spots using the algorithms in Phoretix 2D, and (3) 119 spots using the algorithms in SAM, respectively, with an overlap of 42 spots detected by all three algorithms. Applying different statistical approaches on the same dataset resulted in divergent set of protein spots labeled as statistically "significant". Currently, there is no agreement on statistical data processing of 2DE datasets, but the statistical tests applied in 2DE studies should be documented. Tools for the statistical analysis of proteome data should be implemented and documented in the existing 2DE software.

Animals↗

PubMed2006

[Introduction of proteomic approach to environmental medicine].

Recent progress in life science technology and the availability of much information on genes obtained by genome analysis has enabled us to analyze the changes of proteins on a large scale. Sets of proteins are called proteomes, and proteomics is the scientific field of proteome analysis including differential, post translational modification and interaction analyses. Various proteomic techniques, particularly two-dimensional gel electrophoresis (2-DE), mass spectrometry, protein chip methods, and surface plasmon resonance (SPR), are very useful for acquiring proteomes in cells, tissues and body fluid, and for analyzing interactions between a protein and other biofactors including proteins. A proteomic approach is also useful for determining biomarkers of diseases and key proteins involved in various stages of metabolism such as differentiation, cell cycle and apoptosis. Environmental pollutants including endocrine disruptors inhibit activities of various organs in wild animals and humans. Proteomic approaches could be very useful tools for elucidating the mechanisms of damage caused by environmental pollutants. In this review, we describe the application of a proteomic approach to the field of environmental medicine.

Animals↗

PubMed2005

Protein profiling of 3T3-L1 adipocyte differentiation and (tumor necrosis factor alpha-mediated) starvation.

The increased incidence of obesity and related disorders in Western societies requires a thorough understanding of the adipogenic process. Data at the protein level of this process are scarce. Therefore we performed a proteome analysis of differentiating and starving 3T3-L1 cells using two-dimensional gel electrophoresis combined with mass spectrometry. Effects of different starvation conditions were examined by subjecting 3T3-L1 adipocytes to caloric restriction, either in the absence or the presence of the lipolysis inducer tumor necrosis factor-alpha. Ninety-three differentially expressed proteins were found during differentiation and starvation of 3T3-L1 cells, 50 of which were identified. GenMAPP/MAPP-finder software revealed a non-reciprocal regulation of the glycolytic pathway during 3T3-L1 differentiation followed by starvation. Furthermore, proteins involved in growth regulation, cytoskeletal rearrangements and protein modification, 16 of which have not been described before in 3T3-L1 cells, were identified. In conclusion, our data provide valuable information for further understanding of the adipogenic process.

3T3-L1 Cells↗

PubMed2006

Proteomic profiling of differentiating osteoblasts.

The major event that triggers osteogenesis is the transition of mesenchymal stem cells into bone-forming, differentiating osteoblast cells. Osteoblast differentiation is the primary event of bone formation, exemplified by the synthesis, deposition and mineralization of extracellular matrix. Osteoblast differentiation is controlled tightly by sequential activation of diverse transcription factors that regulate the expression of specific genes. The spatial and temporal regulation of the differentiation process is not completely understood at the cellular or molecular level. Recent advances in mass spectrometry-based proteomics have allowed for the systematic qualitative and quantitative profiling of differentiating osteoblasts, enabling a better understanding of the multiple factors and signaling events that control the differentiation process at a molecular level. This review focuses on recent developments in the proteomic analysis of differentiating osteoblasts, including advances, challenges and future prospects of using mass spectrometry to investigate the local and systemic factors regulating bone formation and its homeostasis.

Animals↗

PubMed2002

Evaluation of two-dimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system.

The technique of fluorescent two-dimensional (2D) difference gel electrophoresis for differential protein expression analysis has been evaluated using a model breast cancer cell system of ErbB-2 overexpression. Labeling of paired cell lysate samples with N-hydroxy succinimidyl ester-derivatives of fluorescent Cy3 and Cy5 dyes for separation on the same 2D gel enabled quantitative, sensitive, and reproducible differential expression analysis of the cell lines. SyproRuby staining was shown to be a highly sensitive and 2D difference gel electrophoresis-compatible method for post-electrophoretic visualization of proteins, which could then be picked and identified by matrix-assisted laser-desorption ionization mass spectroscopy. Indeed, from these experiments, we have identified multiple proteins that are likely to be involved in ErbB-2-mediated transformation. A triple dye labeling methodology was used to identify proteins differentially expressed in the cell system over a time course of growth factor stimulation. A Cy2-labeled pool of samples was used as a standard with all Cy3- and Cy5-labeled sample pairs to facilitate cross-gel quantitative analysis. DeCyder (Amersham Biosciences, Inc.) software was used to distinguish clear statistical differences in protein expression over time and between the cell lines.

Breast Neoplasms↗

PubMed2004

Proteomic analysis of interleukin 6-induced differentiation in mouse myeloid leukemia cells.

Cytokine-induced differentiation of myeloid leukemia cells has important therapeutic implications, but the mechanism remains to be clarified. M1 cell, a mouse acute myeloid leukemia cell line, which underwent growth inhibition, terminal differentiation and apoptosis in response to IL-6, was selected as an experimental model to study on the molecular mechanisms of myeloid cell differentiation on a proteome-wide scale. Cell differentiation was evaluated by cell morphology and CD11b expression. With two-dimensional (2D) gel analyses, 17 protein spots showed obvious changes in quantity during the process of differentiation were found. With matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) or/and nano-electrospray ionization MS/MS (ESI-MS/MS) analysis, 15 protein spots were identified. The mRNA levels of these 15 proteins during differentiation were also examined using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. Except two proteins, the mRNA levels demonstrated similar expression patterns to what the proteomic analysis revealed. The identified proteins were known to be involved in different cellular functions, including protein synthesis, transcription, signal transduction, cell cycle control, cell rescue and defense, cellular organization, and metabolism. Notably, seven proteins were not described before to be involved in differentiation. Our data provide novel information for a better understanding of the mechanisms by which terminal differentiation of acute myeloid leukemia cells induced by IL-6.

Animals↗

PubMed2006

A proteomic analysis of protein variations during differentiation of v-myb-transformed monoblasts.

v-myb oncogene of avian myeloblastosis virus (AMV) transforms myelomonocytic cells in vitro and induces acute monoblastic leukemia in vivo. The transforming effect of the v-myb can be suppressed using phorbol ester (TPA) or histone deacetylase inhibitor trichostatin A (TSA), the inducers of cell differentiation that are in clinical trials. In this study, we used proteomics-based approach to identify proteins with variable expression in differentiated BM2 cells. Proteome variations induced by TPA and TSA were compared to examine the mechanism of differentiation-promoting effects of these drugs. We found that expression of several proteins participating in cell cytoskeleton rearrangement, heat shock response, proteosynthesis and cell signaling was altered in TPA- or TSA-treated cells. We present here the first comparative proteome analysis of v-myb-transformed monoblasts BM2 focused on identification of proteins involved in their terminal differentiation.

Animals↗

PubMed2004

Proteomic analysis of diet-induced hypercholesterolemic mice.

We report here a proteomic analysis of differentially expressed liver proteins of both C57BL/6J (B6, atherosclerosis-susceptible strain) and C3H/HeJ mice (C3H, atherosclerosis-resistant strain), which were fed either control or a high-fat enriched atherogenic diet for eight weeks. We observed differential patterns of plasma lipids between the two strains when both were fed atherogenic diets. That is, although low density lipoprotein cholesterol level was highly elevated in both, the levels of total cholesterol and triglyceride in B6 mice were much lower than those in C3H mice when they were fed atherogenic diets. However, the high density lipoprotein cholesterol level was increased in the latter but decreased in the former. Histopathological observation revealed that more prominent lipid droplets were present in B6 mice than in C3H mice, when they were maintained on the atherogenic diets. Proteomic analysis of liver tissues of these two strains showed that a total of 30 proteins were significantly changed in the livers obtained from both strains after being fed the atherogenic diet. Of these, 14 protein spots including carbonic anhydrase III, senescence marker protein 30 and selenium binding protein 2 were differentially changed only in B6 mice, which was also confirmed in part by Western blotting. An additional 16 protein spots including glutathione S-transferase subclass, apolipoprotein E and chaperonin proteins were changed in both strains. We also identified 28 proteins that were differentially expressed in the livers of both B6 and C3H mice, regardless of diet feeding condition. Of these, 4 protein spots in B6 mice and 11 protein spots in C3H mice were up-regulated. Thirteen strain specific protein spots including antioxidant protein 2, apolipoprotein E and apolipoprotein A-I were also detected in different positions in two-dimensional electrophoresis. These results suggest a clear distinction in differential expression of oxidative stress proteins and lipid metabolism related proteins between the two strains in response to atherogenic diet feeding, which might account for their difference in susceptibility to atherogenesis.

Animals↗

PubMed2003

Proteome analysis of secreted proteins during osteoclast differentiation using two different methods: two-dimensional electrophoresis and isotope-coded affinity tags analysis with two-dimensional chromatography.

Bone is maintained by two cell types, bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts express two factors, osteoprotegerin and receptor activator of NF-kappaB ligand (RANKL), inhibiting and promoting osteoclast differentiation, respectively. In contrast, modulators of bone resorption expressed by osteoclasts have not been so well studied enough. In the present study, we demonstrate proteome analysis of secreted proteins during osteoclast differentiation to elucidate the molecular mechanism of bone resorption and bone remodeling. To achieve this objective, we chose RAW264.7 cells with RANKL as a homogeneous osteoclast differentiation model and used two methods, two-dimensional gel electrophoresis (2-DE) and isotope-coded affinity tags (ICAT) analysis with two-dimensional liquid chromatography. We found 23 spots in 2-DE and 19 proteins in ICAT analysis which were expressed differently during osteoclast differentiation. These two methods gave us closely related but different information about proteins, suggesting they are complementary or at least supplementary methods at present. Cathepsins, osteopontin, legumain, macrophage inflammatory protein-1alpha, and other proteins were observed as up- or down-regulated proteins and are discussed in the context of osteoclast differentiation and bone resorption. In addition to confirming previous observations, this study indicates novel proteins related to osteoclast differentiation which are potential therapeutic targets for the treatment of bone diseases, such as osteoporosis.

Animals↗

PubMed2006

Proteome analysis of salt stress response in the cyanobacterium Synechocystis sp. strain PCC 6803.

In the present study, changes in protein synthesis patterns after salt shock visualized by 35S-methionine labeling and the changed protein composition in salt-acclimated cells of the cyanobacterium Synechocystis sp. strain PCC 6803 were analyzed by a combination of 2-DE for protein separation and PMF for protein identification. As a basis for the differential analysis, a proteome map with 500 identified protein spots comprising 337 different protein species was established. Fifty-five proteins were found, which are induced by salt shock or accumulated after long-term salt acclimation. Some of the proteins are salt stress-specific, such as enzymes involved in the synthesis of the compatible solute glucosylglycerol, while most of them are involved in general stress acclimation. Particularly, heat-shock proteins and proteins acting against lesions by reactive oxygen species were found. Moreover, changes in enzymes involved in basic carbohydrate metabolism were detected. The dynamic of the proteome of salt-stressed Synechocystis cells was compared to previous data concerning transcriptome analysis revealing that 89% of the proteins induced shortly after salt shock were also found to be induced at the RNA level. However, 42% of the stably up-regulated proteins in salt-acclimated cells were not detected previously using DNA microarrays. The comparison of transcriptomic and proteomic analyses shows the significance of post-transcriptional regulatory mechanisms in acclimation of Synechocystis to high salt concentrations.

Bacterial Proteins↗