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Results for “differential proteomics analysis”

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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↗

PubMed2004

Proteomic analysis of nuclear proteins from proliferative and differentiated human colonic intestinal epithelial cells.

Self-renewing tissues such as the intestine contain progenitor proliferating cells which subsequently differentiate. Cell proliferation and differentiation involve gene regulation processes which take place in the nucleus. A human intestinal epithelial cell line model (Caco2/TC7) which reproduces these dynamic processes has been used to perform proteomic studies on nuclear proteins. Nuclei from Caco2/TC7 cells at proliferative and differentiated stages were purified by subcellular fractionation. After two-dimensional gel electrophoresis separation and ruthenium staining, 400 protein spots were detected by image analysis. Eighty-five spots corresponding to 60 different proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry in nuclei from proliferative cells. Comparison of nuclear proteomes from proliferative or differentiated cells by differential display resulted in the identification of differentially expressed proteins such as nucleolin, hnRNP A2/B1 and hnRNP A1. By using Western blot analysis, we found that the expression and number of specific isoforms of these nuclear proteins decreased in differentiated cells. Immunocytochemistry experiments also showed that in proliferative cells nucleolin was distributed in nucleoli-like bodies. In contrast, hnRNPs A2/B1 and A1 were dispersed throughout the nucleus. This study of the nuclear proteome from intestinal epithelial cells represents the first step towards the establishment of a protein database which will be a valuable resource in future studies on the differential expression of nuclear proteins in response to physiological, pharmacological and pathological modulations.

Caco-2 Cells↗

PubMed2005

Proteomics analysis by two-dimensional differential gel electrophoresis reveals the lack of a broad response of Neisseria meningitidis to in vitro-produced AI-2.

To investigate the effect of the autoinducer AI-2 on protein expression in Neisseria meningitidis, a luxS mutant of strain MC58 was grown in the presence and absence of in vitro-produced AI-2, and differential protein expression was assessed by two-dimensional differential gel electrophoresis. N. meningitidis did not show a global response to AI-2 signaling activity.

Bacterial Proteins↗

PubMed2003

Comparative proteomic analysis of proliferating and functionally differentiated mammary epithelial cells.

Proliferation and differentiation of mammary epithelial cells are governed by hormonal stimuli, cell-cell, and cell-matrix interactions. Terminal differentiation of mammary epithelial cells depends upon the action of the lactogenic hormones, insulin, glucocorticoids, and prolactin that enable them to synthesize and secrete milk proteins. These differentiated cells are polarized and carry out vectorial transport of milk constituents across the apical plasma membrane. To gain additional insights into the mechanisms governing differentiation of mammary epithelial cells, we identified proteins whose expression distinguishes proliferating from differentiated mammary epithelial cells. For this purpose we made use of the HC11 mammary epithelial line, which is capable of differentiation in response to lactogenic hormones. Using two-dimensional gel electrophoresis and mass spectrometry, we found about 60 proteins whose expression levels changed in between these two differentiation states. Bioinformatic analysis revealed differential expression of cytoskeletal components, molecular chaperones and regulators of protein folding and stability, calcium-binding proteins, and components of RNA-processing pathways. The actin cytoskeleton is asymmetrically distributed in differentiated epithelial cells, and the identification of proteins involved in mRNA binding and localization suggests that asymmetry might in part be achieved by controlling cellular localization of mRNAs. The proteins identified provide insights into the differentiation of mammary epithelial cells and the regulation of this process.

Animals↗

PubMed2006

Statistical analysis of the experimental variation in the proteomic characterization of human plasma by two-dimensional difference gel electrophoresis.

The complexity of human plasma presents a number of challenges to the efficient and reproducible proteomic analysis of differential expression in response to disease. Before individual variation and disease-specific protein biomarkers can be identified from human plasma, the experimental variability inherent in the protein separation and detection techniques must be quantified. We report on the variation found in two-dimensional difference gel electrophoresis (2-D DIGE) analysis of human plasma. Eight aliquots of a human plasma sample were subjected to top-6 highest abundant protein depletion and were subsequently analyzed in triplicate for a total of 24 DIGE samples on 12 gels. Spot-wise standard deviation estimates indicated that fold changes greater than 2 can be detected with a manageable number of replicates in simple ANOVA experiments with human plasma. Mixed-effects statistical modeling quantified the effect of the dyes, and segregated the spot-wise variance into components of sample preparation, gel-to-gel differences, and random error. The gel-to-gel component was found to be the largest source of variation, followed by the sample preparation step. An improved protocol for the depletion of the top-6 high-abundance proteins is suggested, which, along with the use of statistical modeling and future improvements in gel quality and image processing, can further reduce the variation and increase the efficiency of 2-D DIGE proteomic analysis of human plasma.

Analysis of Variance↗

PubMed2006

Proteomic analysis of Candida albicans yeast and hyphal cell wall and associated proteins.

Candida albicans is an important human pathogen that causes systemic infections, predominantly among populations with weakened immune systems. The morphological transition from the yeast to the hyphal state is one of the key factors in C. albicans pathogenesis. Owing to their location at the host-pathogen interface, the cell wall and associated proteins are of interest, especially with respect to the yeast to hyphal transition. This study entailed the proteomic analysis of differentially regulated proteins involved in this transition. The protein profiles of C. albicans DTT/SDS-extractible proteins and the cyanogen bromide (CNBr)/trypsin-extractable proteins of a cell wall-enriched fraction from yeast and hyphae were compared. In total, 107 spots were identified from the DTT/SDS-extractible cell wall-enriched fraction, corresponding to 82 unique proteins. Of these DTT/SDS-extractible proteins, 14 proteins were upregulated and 10 were downregulated in response to hyphal induction. Approximately 6-9% of total cell wall-protein-enriched fraction was found to be resistant to DTT/SDS extraction. Analysis of the DTT/SDS-resistant fraction using a CNBr/trypsin extraction resulted in the identification of 29 proteins. Of these, 17 were identified only in the hyphae, four were identified only in the yeast, and eight were identified in both the yeast and hyphae.

Amino Acid Sequence↗

PubMed2006

Increased expression and phosphorylation of liver glutamine synthetase in well-differentiated hepatocellular carcinoma tissues from patients infected with hepatitis C virus.

Hepatocellular carcinoma (HCC) is one of the most common fatal cancers, and chronic infection with hepatitis C virus (HCV) is thought to be one of the main causes in Japan. To identify diagnostic or therapeutic biomarkers for HCC associated with HCV (HCV-HCC), we tried to elucidate the factors related to the products from cancerous tissues of HCV-infected patients. From proteomic differential display analysis of liver tissue samples from HCV-HCC cancerous tissues and corresponding non-cancerous tissues from patients, three protein spots of the same molecular mass (42 kDa), whose expression increased in well-differentiated cancerous tissues, were detected. Although their pI were different, they were identified as glutamine synthetase (GS) by PMF with MALDI-TOF MS and by Western blotting using anti-GS specific mAb. Immunohistochemical analysis showed that tumor tissue consists of two parts, GS-positive cell and GS-negative cell regions, suggesting that GS-producing cells grew in the tumor tissue as a nodule in nodules. The tryptic peptides of the most acidic GS isoform lost the signal of 899.5 Da, corresponding a peptide of SASIRIPR, and gained a signal of 1059.5 Da, which was submitted to PSD analysis. PSD analysis showed the neutral loss by elimination of two phosphate groups, supposed to be on serine residues of the 899.5-Da peptide, from serine 320 to arginine 327 in GS. PMF followed by PSD analysis is thought to be useful for the determination of phosphorylation sites of proteins showing molecular heterogeneity.

Amino Acid Sequence↗

PubMed2005

MSight: an image analysis software for liquid chromatography-mass spectrometry.

Images obtained from high-throughput mass spectrometry (MS) contain information that remains hidden when looking at a single spectrum at a time. Image processing of liquid chromatography-MS datasets can be extremely useful for quality control, experimental monitoring and knowledge extraction. The importance of imaging in differential analysis of proteomic experiments has already been established through two-dimensional gels and can now be foreseen with MS images. We present MSight, a new software designed to construct and manipulate MS images, as well as to facilitate their analysis and comparison.

Chromatography, Liquid↗

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↗

PubMed2000

Measurement of bacterial gene expression in vivo.

The complexities of bacterial gene expression during mammalian infection cannot be addressed by in vitro experiments. We know that the infected host represents a complex and dynamic environment, which is modified during the infection process, presenting a variety of stimuli to which the pathogen must respond if it is to be successful. This response involves hundreds of ivi (in vivo-induced) genes which have recently been identified in animal and cell culture models using a variety of technologies including in vivo expression technology, differential fluorescence induction, subtractive hybridization and differential display. Proteomic analysis is beginning to be used to identify IVI proteins, and has benefited from the availability of genome sequences for increasing numbers of bacterial pathogens. The patterns of bacterial gene expression during infection remain to be investigated. Are ivi genes expressed in an organ-specific or cell-type-specific fashion? New approaches are required to answer these questions. The uses of the immunologically based in vivo antigen technology system, in situ PCR and DNA microarray analysis are considered. This review considers existing methods for examining bacterial gene expression in vivo, and describes emerging approaches that should further our understanding in the future.

Animals↗

PubMed2003

Proteome analysis reveals novel proteins associated with proliferation and differentiation of the colorectal cancer cell line Caco-2.

Here, we describe a proteomics approach to study protein expression changes in differentiating Caco-2 cells. Caco-2 is a colorectal carcinoma cell line, which upon differentiation loses its tumorigenic phenotype and displays characteristics of mature enterocytes, including brush borders with microvilli. Cells were grown in culture flasks and harvested at different stages of differentiation (days post-confluence: -3, 0, 3, 7, 10, 14, and 18). Two-dimensional gel electrophoresis was used to analyse proteome changes. Approximately 1400 protein spots were detected within the Caco-2 proteome, within the pH 4-7 range. Two-dimensional gel electrophoresis allowed for the detection of 18 proteins from which the levels of expression were found to be associated with differentiation. Of these proteins, 11 were identified by means of MALDI-TOF or NANO-ESI-MS/MS mass spectrometry and include liver fatty acid binding protein (FABL), three forms of alpha-enolase (ENOA), nucleoside diphosphate kinase A (NDKA), cofilin-1 (COF1), translationally controlled tumour protein (TCTP), mitochondrial 60-kDa heat shock protein (CH60), probable protein disulfide isomerase (ER60), creatine kinase B (KCRB), and glutathione S-transferase alpha (GTA1). Thus, proteomics revealed that the differentiation-related change in phenotype of Caco-2 involves changes in a variety of distinct biochemical pathways. Some of these proteins have not been shown before to be associated with Caco-2 differentiation (ER60; COF1; CH60; NDKA; TCTP and ENOA). Therefore, processes related to protein folding and disulfide bridge formation, cytoskeleton formation and maintenance, nucleotide metabolism, glycolysis as well as tumorigenesis-associated proteins may be involved in Caco-2 differentiation. Changes in the expression of CH60, TCTP, GTA1, NDKA, and FABL have also been reported to be associated with in vivo colon carcinogenesis. These findings illustrate that a combination of proteomics and cell culture is a useful approach to find markers for Caco-2 differentiation, which could contribute to the comprehension of the process of colon carcinogenesis.

Alkaline Phosphatase↗

PubMed2003

HysTag--a novel proteomic quantification tool applied to differential display analysis of membrane proteins from distinct areas of mouse brain.

A novel isotopically labeled cysteine-tagging and complexity-reducing reagent, called HysTag, has been synthesized and used for quantitative proteomics of proteins from enriched plasma membrane preparations from mouse fore- and hindbrain. The reagent is a 10-mer derivatized peptide, H(2)N-(His)(6)-Ala-Arg-Ala-Cys(2-thiopyridyl disulfide)-CO(2)H, which consists of four functional elements: i) an affinity ligand (His(6)-tag), ii) a tryptic cleavage site (-Arg-Ala-), iii) Ala-9 residue that contains four (d(4)) or no (d(0)) deuterium atoms, and iv) a thiol-reactive group (2-thiopyridyl disulfide). For differential analysis cysteine residues in the compared samples are modified using either (d(4)) or (d(0)) reagent. The HysTag peptide is preserved in Lys-C digestion of proteins and allows charge-based selection of cysteine-containing peptides, whereas subsequent tryptic digestion reduces the labeling group to a di-peptide, which does not hinder effective fragmentation. Furthermore, we found that tagged peptides containing Ala-d(4) co-elute with their d(0)-labeled counterparts. To demonstrate effectiveness of the reagent, a differential analysis of mouse forebrain versus hindbrain plasma membranes was performed. Enriched plasma membrane fractions were partially denatured, reduced, and reacted with the reagent. Digestion with endoproteinase Lys-C was carried out on nonsolubilized membranes. The membranes were sedimented by ultra centrifugation, and the tagged peptides were isolated by Ni(2+) affinity or cation-exchange chromatography. Finally, the tagged peptides were cleaved with trypsin to release the histidine tag (residues 1-8 of the reagent) followed by liquid chromatography tandem mass spectroscopy for relative protein quantification and identification. A total of 355 unique proteins were identified, among which 281 could be quantified. Among a large majority of proteins with ratios close to one, a few proteins with significant quantitative changes were retrieved. The HysTag offers advantages compared with the isotope-coded affinity tag reagent, because the HysTag reagent is easy to synthesize, economical due to use of deuterium instead of (13)C isotope label, and allows robust purification and flexibility through the affinity tag, which can be extended to different peptide functionalities.

Amino Acid Sequence↗

PubMedDate not supplied

Proteome and transcriptome analysis of retinoic acid-induced differentiation of human acute promyelocytic leukemia cells, NB4.

Acute promyelocytic leukemia (APL) is characterized by a translocation t(15:17) that fuses the retinoic acid receptor gene with the promyelocytic gene, which blocks differentiation to normal granulocytes. NB4 cells, derived from human acute promyelocytic leukemia, display this genotype and phenotype. All trans-retinoic acid (ATRA) induces differentiation of NB4 cell cultures in vitro and APL in vivo, although resistance to differentiation therapy frequently develops. To identify genes involved in differentiation, we compared gene expression at the mRNA and protein levels using microarray analyses and two-dimensional (2D) difference gel electrophoresis (DIGE), plus MALDI-TOF-TOF mass spectrometry. Differentially expressed transcripts were identified using oligonucleotide-based microarrays with targets representing almost 14 000 genes. Real time PCR was performed on a subset of genes whose products were shown to be differentially expressed using proteomic and/or genomic approaches. Our analyses identified 46 genes that were differentially expressed in NB4 +/- ATRA; 22 were identified using 2D-DIGE and 24 using microarray analysis. All but four of these genes were expressed at higher levels in differentiated cells, and several controlled cell structure (internal and cytoskelatal) or signal transduction. We observed that proteome analysis with DIGE and silver-stained 2D gel electrophoresis analyses revealed significant differences between the two measurement approaches. Furthermore, our data showed significant discordance between gene expression at the protein and transcript levels.

Cell Differentiation↗

PubMed2002

Integrated genomic and proteomic analysis of signaling pathways in dendritic cell differentiation and maturation.

Dendritic cells (DCs) are antigen-presenting cells that play a major role in initiating primary immune responses. Their phenotypic and functional characteristics are intimately linked to their stage of maturation. The specific biochemical pathways and genes whose expression mediates differentiation of progenitors to DCs and their maturation are largely undefined. We recently utilized two approaches, DNA microarrays and proteomics, to analyze the expression profile of human CD14(+) blood monocytes and their derived DCs. Approximately 4% of the genes or proteins expressed were found to be regulated during DC differentiation. Most of these genes were not previously associated with DCs and included genes highly relevant to DC functions (genes involved in antigen presentation, cell adhesion and motility, lipid metabolism). Genes involved in specific signaling pathways, including IkappaBalpha, PPAR-gamma and C/EBPalpha as well as two members of the family of transcription factors, interferon regulatory factors (IRFs), were also modified. Modulation of IRF gene expression is of particular interest because of their functional roles in innate and adaptive immune responses. IRF-family members control the expression of proteins that include type-1 interferons, interleukin-12, interleukin-15, MHC molecules and adhesion molecules. They have also been found to play an important role in lymphocyte development. In contrast to DC differentiation, very few genes were modified at the transcript level during DC maturation as determined by microarray experiments. Further analysis suggested that DC maturation is largely controlled by posttranscriptional and posttranslational modifications. The use of proteomics is therefore necessary for a full comprehension of DC maturation process.

Animals↗

PubMed2026

Single-Cell Proteomics Reveals Proteome Remodeling and Cellular Heterogeneity During NGF-Induced PC12 Neuronal Differentiation.

Single-cell proteomics enables direct measurement of cellular heterogeneity during dynamic biological processes, but its application to fragile and highly adherent neuronal models remains challenging. Here, we developed and applied an optimized single-cell proteomics workflow to characterize proteome remodeling during nerve growth factor (NGF)-induced differentiation of PC12 cells. To enable reliable single-cell analysis, we implemented gentle dissociation, antiaggregation strategies, and thermal inkjet-based cell dispensing, achieving high accuracy in single-cell isolation. Inclusion of n-dodecyl-β-d-maltoside (DDM) improved recovery of membrane-associated and low-solubility proteins. Coupled with LC-ion mobility-mass spectrometry, this workflow enabled quantification of 2,000-3,000 proteins per cell across the differentiation time course. Single-cell proteomic analysis revealed progressive and heterogeneous proteome remodeling during differentiation. While undifferentiated cells formed a relatively homogeneous population, later stages (Days 4-6) exhibited increased variability, including multimodal protein abundance distributions and separation into distinct subpopulations. Dimensionality reduction, clustering, and non-negative matrix factorization identified multiple coexisting proteomic states within the same time points, reflecting asynchronous differentiation trajectories. These subpopulations were characterized by coordinated differences in pathways related to intracellular trafficking, protein translation, cytoskeletal organization, and neuronal maturation. Comparison with bulk proteomics demonstrated that proteins associated with differentiated neuronal states, including those involved in neurite formation and structural remodeling, are underrepresented in population-averaged measurements but are enriched within specific single-cell subpopulations. Temporal and cluster-resolved analyses further revealed distinct protein expression trajectories, including early decreases in cell cycle and metabolic pathways and later increases in neuronal structural and regulatory proteins. Together, this study establishes an optimized workflow for single-cell proteomics of neuronal systems and demonstrates that NGF-induced PC12 differentiation proceeds through heterogeneous and divergent proteomic states that are not resolved by bulk analysis.

Animals↗

PubMed2006

[Proteomics of breast cancer: From differential to functional analysis].

From differential analysis to identify biomarkers, to functional analysis for finding new therapeutic targets, proteomics bring new comprehensive information for a better understanding of the molecular basis of oncology and new perspectives for the clinic. However the major limitation of proteomic investigations, more generally of post-genomic approaches, remains the molecular and cellular complexity of the mammary gland that is still a major challenge.

Biomarkers, Tumor↗

PubMed2026

Beyond ion channel dysfunction: Integration of the transcriptome and proteome from patient-specific re-engineered cardiac cells, and population-level QT genome-wide association study reveals broad cellular dysfunction.

BACKGROUND: Congenital long QT syndrome (LQTS) is a cardiac channelopathy with increased risk of cardiac-triggered syncope/seizures, sudden cardiac arrest, and sudden cardiac death. OBJECTIVE: This study aimed to describe the transcriptomic and proteomic profiles in patient-derived inducible pluripotent stem cell-derived cardiomyocyte (iPSC-CM) models of the 3 canonical genotypes of congenital LQTS: LQT1, LQT2, and LQT3 and integrate these omics-level findings with each other and with population/clinical level QT-genome-wide association study (GWAS) data. METHODS: LQT1, LQT2, LQT3 and respective isogenic control iPSC-CMs were cultured, and RNA and protein samples were collected. RNA sequencing and mass spectrometry-enabled proteomic analysis was performed. PrediXcan analysis was performed using QT GWAS summary statistics and transcriptome expression data. Differential gene and protein expression and ingenuity pathway analysis (IPA) was performed comparing each LQT genotype with its respective isogenic control. RESULTS: 1645 differentially expressed genes (DEGs) were identified; 13 were altered in all 3 LQTS genotypes. IPA analysis of DEGs revealed 301 altered pathways; 47 were altered in all LQTS genotypes. Proteomic analysis identified 2561 differentially expressed proteins (DEPs); 30 were altered in all 3 genotypes. IPA analysis of DEPs identified 646 altered pathways. 306 genes/proteins were identified as significantly altered in both the transcriptome and proteome; pathway analysis of these 301 genes identified 201 altered pathways. 7 pathways were altered in all 3 LQTS genotypes in both the transcriptome and proteome. Integration of the population-level PrediXcan results and the cardiomyocyte-derived omics results identified multiple shared pathways. CONCLUSION: Multi-omics analysis of LQTS and integration of omics results with QT GWAS data reveals that primary LQTS-causative ion channel defects precipitate secondary alterations in a wide range of cellular pathways. Our findings suggest more broad molecular level changes throughout the cell. This study lays the foundation for further exploration of broad cellular changes resulting from ion channel disturbances and how they contribute to disease mechanism.

Humans↗

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↗