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PubMed2006

Meta-analysis of differentiating mouse embryonic stem cell gene expression kinetics reveals early change of a small gene set.

Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC) lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined, undifferentiated ESC in culture. In each dataset, we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets, despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest, we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728_at, 8430410A17Rik, Klf2, Nr0b1, Sox2, Tcl1, and Zfp42) showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis, this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types.

Adaptation, Physiological↗
PubMed2005

Rapid detection of fetal aneuploidy using proteomics approaches on amniotic fluid supernatant.

OBJECTIVE: Conventional chromosomal studies or fluorescent in situ hybridization takes days to diagnose fetal aneuploidies during amniocentesis. Here, we evaluated the value of mass spectrometry-based clinical proteomics analysis on amniotic fluid supernatant (AFS) as a rapid detection of fetal aneuploidies. METHODS: Proteomics profiles generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after fractionating samples with functionalized magnetic beads were used for differentiating 60 normal karyotypic from 20 aneuploid AFS. After the discriminating models were generated using genetic algorithm, we evaluated the clinical efficacy of the models in detecting aneuploidies in two batches (each n=30) of AFS prior to the release of chromosomal diagnoses. RESULTS: Within hours, the two-step proteomics analysis of AFS with the C18 model, followed by the weak cation exchange model, was able to detect aneuploid AFS at 3.3% disease prevalence rate with 100% sensitivity, 72 to 96% specificity, 11 to 50% positive predictive value, and 100% negative predictive value. CONCLUSION: Clinical proteomics analysis of AFS using magnetic beads-based sample preparation and MALDI-TOF-MS can be used as a rapid detection for fetal aneuploidies. With perfect sensitivity and negative predictive value of the two-step proteomics method, it may be used for rapid detection of aneuploid AFS immediately after amniocentesis. Further large-scale examinations are apparently needed to verify the clinical value of this rapid detection.

Amniotic Fluid↗
PubMed2003

Early drug safety evaluation: biomarkers, signatures, and fingerprints.

When target organ toxicity arises in animal models during routine drug safety evaluation, it raises several key questions: Is this target organ toxicity related to the pharmacology? What is the mode of action (MOA)? Is the target organ toxicity relevant to humans? Pathology or prior knowledge of the compound class may provide clues on a possible MOA for toxicity. However, if this deductive approach yields no results, the inductive approach offered by new technologies can generate novel research leads. For example, toxicogenomics can generate a gene expression profile of the toxicity that can be compared with reference compounds or with other candidate drugs. Similarly, proteomic analysis of the protein profile at the toxic vs. the efficacious dose can provide clues on MOA for the toxicity and may allow differentiation of the pathways of the toxic response from those required for pharmacological activity.

Animals↗
PubMed2006

Comparative proteomic analysis between the invasive and commensal strains of Staphylococcus epidermidis.

Staphylococcus epidermidis is one of the major causative agents for nosocomial infections. To reveal the pathogenesis factors, we performed the comparative proteomic analysis of invasive ATCC35984 and commensal ATCC12228 strains by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. The differentially expressed proteins were involved in carbohydrate metabolism, sugar binding, lipid degradation and amino acid binding. In addition, we demonstrated that the trap gene was transcribed by 3.657+/-0.156 (P<0.01) -fold higher in ATCC35984 than in ATCC12228. Levels of accumulation-associated protein (AAP) were found to be low by the immuno-dot blotting assay in ATCC12228, which is unable to form biofilm. Our results suggest that the target of RNAIII activating protein and AAP may contribute to S. epidermidis virulence and biofilm formation.

Amino Acids↗
PubMed2004

Proteome analysis of adipogenesis.

Adipose tissue plays a crucial endocrine role in controlling whole body glucose homeostasis and insulin sensitivity. Given the substantial rise in obesity and obesity-related diseases such as diabetes, it is important to understand the molecular basis of adipocyte differentiation and its control. Many studies have successfully exploited gene array technology to monitor changes in the profile of expressed genes during adipocyte differentiation, although this method only measures changes at the level of individual mRNA species. Using two-dimensional polyacrylamide gel electrophoresis, high-throughput image analysis, and candidate picking coupled with sequencing mass spectrometry, we have followed the changes in protein expression profile that occur during the differentiation of 3T3-L1 fibroblasts into adipocytes in response to dexamethasone, isobutyl methyl xanthine and insulin, or to the PPARgamma agonist, ciglitazone. Using this technique we have found alterations in the profile of over 2000 protein species during adipogenesis. Our studies reveal previously unknown alterations during adipogenesis in the expression or mobility (on sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of coactosin, which promotes actin filament destabilization, several signalling molecules, including RhoGDI-1, RhoGDI-2 and EHD1, and NEDD5 a protein involved in cytokinesis.

3T3-L1 Cells↗
PubMed2026

Discovery and validation of novel plasma protein biomarkers for severe tuberculosis patients.

OBJECTIVE: Severe tuberculosis (STB) imposes a substantial disease burden, yet reliable biomarkers for distinguishing STB from mild/moderate tuberculosis (MTB) remain scarce. This study aimed to identify and independently validate plasma protein biomarkers associated with tuberculosis severity. METHODS: In this multicenter prospective study, 298 adults with confirmed pulmonary tuberculosis were enrolled into screening (n&#x2009;=&#x2009;128) and independent validation (n&#x2009;=&#x2009;170) cohorts. Plasma samples were analysed using data-independent acquisition proteomics. Differentially expressed proteins were screened via Limma and four machine-learning algorithms, with candidate proteins measured by enzyme-linked immunosorbent assays. Receiver operating characteristic analysis assessed individual and combined diagnostic performance. RESULTS: STB patients were older and presented with lymphopenia, hypoalbuminemia, neutrophilia, and elevated lactate dehydrogenase. Among 166 differentially expressed proteins, HSPA5, HSP90B1, EEF1D, and SULT1A1 were selected for validation. In STB patients, HSPA5, HSP90B1, and EEF1D were upregulated, whereas SULT1A1 was downregulated. The four-protein panel achieved an AUC of 0.908 (95% CI 0.864-0.952), with 87.5% sensitivity and 83.8% specificity, modestly outperforming HSPA5 alone (AUC&#x2009;=&#x2009;0.894). Functional enrichment implicated cholesterol metabolism, immune-inflammatory pathways, and endoplasmic reticulum stress. CONCLUSIONS: The four-protein panel effectively discriminated STB from MTB; however, its marginal improvement over HSPA5 alone suggests that an HSPA5-based assay may offer a simpler, more practical, and potentially cost-effective strategy for severity stratification.

Humans↗
PubMed2003

Analysis of the quorum-sensing regulon of the opportunistic pathogen Burkholderia cepacia H111 by proteomics.

Burkholderia cepacia H111, an important pathogen for persons suffering from cystic fibrosis, employs a quorum-sensing (QS) system, cep, to control expression of virulence factors as well as the formation of biofilms. The QS system is thought to ensure that pathogenic traits are only expressed when the bacterial population density is high enough to overwhelm the host before it is able to mount an efficient response. In this study, we compared the protein pattern of the intracellular, extracellular, and surface protein fractions of an AHL-deficient cepI mutant with the one of the parent strain H111 by means of two-dimensional gel electrophoresis (2-DE). Our analysis showed that 55 proteins out of 985 detected spots were differentially expressed; these are expected to represent QS-controlled gene products. Addition of the respective signal molecules to the growth medium of the cep mutant fully restored the wild-type protein expression profile. In total about 5% of the B. cepacia proteome was downregulated and 1% upregulated in the cepI mutant, indicating that quorum sensing represents a global regulatory system. Nineteen proteins were identified with high confidence by N-terminal sequence analysis.

Burkholderia cepacia↗
PubMed2006

Comparative proteomic analysis of nucleic acid-binding proteins in ten human tumor cell lines.

Failure in regulation of genes involved in growth and division of cells may result in pathological conditions, particularly cancer. Regulation is exerted at various levels of the transcriptional and post-transcriptional processes involving mainly nucleic acid-binding proteins. Here, we systematically explored the proteome of ten different cell lines in search for proteins potentially serving as molecular markers and/or targets for monitoring prognostic outcome and clinical therapies. High-throughput analysis, two-dimensional electrophoresis coupled to matrix-assisted laser desorption/ionization mass spectrometry, identified 72 nucleotide-binding proteins and their interacting partners, which were differentially expressed in the cell lines investigated. Out of the 72 identified proteins, 33 of them were specifically expressed in a single cell line (for e.g., replication protein A 32 kDa, transcription intermediary factor 1, heterogeneous ribonucleoproteins). Moreover, tumor-related proteins including breast carcinoma amplified sequence 2, zinc finger proteins, chromobox protein homologs were identified in individual cell lines. The present findings demonstrate that rich protein information can be obtained by means of proteomic analysis for better understanding of oncogenesis and pathogenesis in a global way, which in turn represents the basis for the rational designs of diagnostic and therapeutic methods.

Biomarkers, Tumor↗
PubMed2003

An approach to quantitative proteome analysis by labeling tryptophan residues.

This report describes a method for quantification and sequence identification of individual proteins in complex mixtures. The method is based on labeling with the chemical reagent 2-nitrobenzenesulfenyl chloride (NBSCl) in conjunction with tandem mass spectrometry. In this method, selective introduction of the 2-nitrobenzenesulfenyl (NBS) moiety onto tryptophan residues is achieved, and a 6 Da mass differential is generated using (13)C(6)-labeled NBSCl (NBSCl-(13)C(6)) and (12)C(6)-labeled NBSCl (NBSCl-(12)C(6)). The 6 Da mass differential between the NBS-(12)C(6)-labeled and the NBS-(13)C(6)-labeled peptides assigns a mass signature to all tryptophan-containing peptides in any pool of proteolytic digests for protein identification through peptide mass mapping. Using this strategy, we compared the protein expression in rat sera using a normal (control) rat (Crj:Wistar) and a hyperglycemic rat (GK/Crj). The stable isotope dilution techniques used in this method provide highly accurate relative quantification. The NBS approach offers a widely applicable means of analyzing protein mixtures derived from biological samples, and the method described here presents an effective and simplified approach to proteome analysis.

Animals↗
PubMed2006

[Determination of differentially expressed proteins and it's significance among chronic sinusitis, nasal polyps and normal nasal mucosa].

OBJECTIVE: To investigate the differentially expressed proteins among chronic sinusitis, nasal polyps and normal nasal mucosa by means of proteomic technology, and select the candidate biomarkers of chronic sinusitis and nasal polyps. METHODS: Proteins extracted from chronic sinusitis, nasal polyps and normal nasal mucosa were separated and the differentially expressed proteins were identified by series of proteomic tools, including immobilized pH4-7 gradient two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, modified coomassie brilliant blue staining, images scanning by the Image Scanner apparatus, PDQuest analysis software, peptide mass fingerprinting based on matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) by in-gel digestion extract, and Mascot searching in NCBInr and SWISS-PROT databases. RESULTS: The 2-DE patterns with high resolution and reproducibility were obtained. The protein spots separated and visualized in chronic sinusitis, nasal polyps and normal nasal mucosa gel were 1020 +/- 40, 1112 +/- 10 and 1008 +/- 25, respectively. And the match rates were (93 +/- 2)%, (95 +/- 1)% [see text] (90 +/- 3)% respectively. Thirteen differentially expressed spots were found from chronic sinusitis, nasal polyps and normal nasal mucosa gel. We selected and recommend Keratin 8 and APOA1 proteins as candidate biomarkers of nasal polyps, and PLUNC protein, PACAP protein, NKEF-B and SOD as candidate biomarkers of chronic sinusitis. CONCLUSIONS: The differentially expressed proteins among chronic sinusitis, nasal polyps and normal nasal mucosa can be efficiently and relatively reliably identified via the techniques of proteomics. These techniques will play a very important role in the researches for new objective indicators possibly employed in the future classifying, staging and prognosis.

Adolescent↗
PubMed2005

Altered calmodulin response to light in the suprachiasmatic nucleus of PAC1 receptor knockout mice revealed by proteomic analysis.

In mammals circadian rhythms are generated by a light-entrainable oscillator located in the hypothalamic suprachiasmatic nucleus (SCN). Light signals reach the SCN via a monosynaptic neuronal pathway, the retinohypothalamic tract, originating in a subset of retinal ganglion cells. The nerve terminals of these cells contain the classical neurotransmitter glutamate and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), and there is evidence that these two transmitters interact to mediate photoentrainment of the oscillator in the SCN. To elucidate light-provoked PACAP receptor signaling we used proteomic analysis. Wild-type mice and mice lacking the PAC1 receptor (PAC1-/-) were light stimulated at early night, and the SCN was examined for proteins that were differentially expressed using two-dimensional gel electrophoresis and identification by tandem mass spectrometry. The most striking finding, which was subsequently confirmed by Western blotting, was a significant reduction of calmodulin (CaM) in wild-type mice as compared with PAC1-/- mice. Analysis at the mRNA level by quantitative in situ hybridization histochemistry was inconclusive, indicating that a translational mechanism might be involved. The findings indicate that PAC1 receptor signaling in the SCN in response to light stimulation induces a down-regulation of CaM expression and that CaM is involved in the photic-entrainment mechanism.

Animals↗
PubMed2006

Proteins differentially expressed in response to nicotine in five rat brain regions: identification using a 2-DE/MS-based proteomics approach.

To determine protein expression patterns within the central nervous system (CNS) in response to nicotine, 2-DE/MS was performed on samples from five brain regions of rats that had received nicotine bitartrate by osmotic minipump infusion at a dose of 3.15 mg/kg/day for 7 days. After spot matching and statistical analysis, 41 spots in the amygdala, 49 in the nucleus accumbens (NA), 46 in the prefrontal cortex (PFC), 36 in the striatum, and 28 in the ventral tegmental area (VTA) showed significant differences in the nicotine-treated compared with control samples. Using MALDI-TOF MS peptide fingerprinting, 14 proteins in the amygdala, 11 in the NA, 19 in the PFC, 13 in the striatum, and 19 in the VTA were identified. Several proteins (e.g. dynamin 1, laminin receptors, aldolase A, enolase 1 alpha, Hsc70-ps1, and N-ethylmaleimide-sensitive fusion protein) were differentially expressed in multiple brain regions. By gene ontology analysis, these differentially expressed proteins were grouped into biological process categories, such as energy metabolism, synaptic function, and oxidative stress metabolism. These data, in combination with microarray analysis of mRNA transcripts, have the potential to identify the CNS gene products that show coordinated changes in expression at both the RNA and protein levels in response to nicotine.

Animals↗
PubMedDate not supplied

Proteomics analysis of phosphotyrosyl-proteins in human lumbar cerebrospinal fluid.

Cerebrospinal fluid (CSF) is a secretion product of several different central nervous system (CNS) structures, including the choroid plexus in the ventricles. Pathological CNS processes are reflected in the protein composition of CSF. To elucidate the molecular events that occur in the homeostatic and pathological processes of the CNS, the high-throughput characterization of differentially expressed proteins, and of post-translationally modified proteins, is needed for proteomics studies of CSF. Among the post-translational modifications of proteins, phosphorylation is the most common and important mechanism for the reversible regulation of protein function. In this study, CSF phosphotyrosyl (p-Tyr)-proteins were detected with antibodies and were analyzed with proteomics methods. Three different combination methods--1D gel electrophoresis and Western blotting, immunoprecipitation and 2D gel electrophoresis, and 2D gel electrophoresis and Western blotting--were used to detect p-Tyr-proteins in human lumbar CSF samples. Six protein spots, representing four proteins on a 2D Western blot, were identified as p-Tyr-proteins with the 2D gel electrophoresis and Western blotting method. Those four p-Tyr-proteins are kallikrein-6 precursor, complement C4 gamma-chain, gelsolin, and ceruloplasmin precursor. Additionally, four other nonphosphorylated CSF proteins--beta-2-glycoprotein I precursor, fibulin-1 precursor, EGF-containing fibulin-like extracellur matrix protein 1 precursor, and angiotensinogen precursor--were characterized for the first time.

Blotting, Western↗
PubMed2006

Identification of breast cancer metastasis-associated proteins in an isogenic tumor metastasis model using two-dimensional gel electrophoresis and liquid chromatography-ion trap-mass spectrometry.

To better understand the molecular mechanisms underlying breast cancer metastasis and search for potential markers for metastatic progression, we have developed a highly metastatic variant of human MDA-MB-435 breast cancer cell line through in vivo stepwise selection of pulmonary metastatic cells caused by parental MDA-MB-435 cells in the athymic mice. Comparative proteomic analysis using 2-DE and LC-IT-MS revealed that 102 protein spots were reproducibly altered more than three-fold between the selected variant and its parental counterpart. Eleven differentially expressed protein spots were identified with high confidence using SEQUEST with uninterpreted tandem mass raw data. Cathepsin D precursor, peroxiredoxin 6 (PDX6), heat shock protein 27 (HSP27), HSP60, tropomyosin 1 (TPM1), TPM2, TPM3, TPM4, 14-3-3 protein epsilon, and tumor protein D54 were up-regulated in the highly metastatic variant, whereas alpha B-crystalline (CRAB) was only detected in its parental counterpart. Differential expression was confirmed for four proteins including PDX6, CRAB, TPM4, and HSP60 by real-time quantitative PCR and Western blotting analysis in our model. Immunohistochemical analysis in 80 breast cancer donors demonstrated a significant association of TPM4 (p = 0.002), HSP60 (p = 0.001), PDX6 (p = 0.002) but not CRAB (p = 0.113) staining with the presence of lymph node metastasis. In addition, TPM4 staining was also associated with clinical stage (p = 0.000), but no significant association was found between TPM4, PDX6, CRAB, and HSP60 expression and tumor size, hormone receptor, and HER-2 status (p > 0.05). The functional implication of these identified proteins was also discussed. These proteomic data are valuable and informative for understanding breast cancer metastasis and searching for potential markers for metastatic progression.

Animals↗
PubMed2006

ProteinChip technology is a useful method in the pathogenesis and diagnosis of endometriosis: a preliminary study.

OBJECTIVE: To test the feasibility of ProteinChip (Ciphergen Biosystems, Inc., Fremont, CA) technology as a proteomic tool in discovering and identifying proteins that are differentially expressed in endometrium, endometriotic tissue, and normal peritoneum from women with and without endometriosis. DESIGN: Differential analysis of protein expression in women with and without endometriosis. SETTING: University hospital. PATIENT(S): A total of nine patients during their secretory phase (days 20-22) were selected for this study on the basis of cycle phase and presence/or absence of endometriosis. INTERVENTION(S): Twelve tissues used in the study included six endometrial biopsies from women with mild endometriosis (n = 3) and a normal pelvis (n = 3) as well as paired samples of peritoneal endometriotic lesions (n = 3) and macroscopically normal peritoneum biopsies (n = 3) from three women with endometriosis. MAIN OUTCOME MEASURE(S): Numerous expression differences were observed in the above comparisons, representing both up-regulation and down-regulation in protein and peptide expression levels. RESULT(S): Endometrial expression for a number of proteins and peptides in the range of 2.8-12.3 kDa was 3-24 times lower in women with endometriosis than in those without endometriosis. When compared with normal peritoneum, endometriotic lesions showed an increased expression for a set of proteins and peptides in the range of 3-96 kDa, and especially an up-regulated cluster of proteins between 22 and 23 kDa, identified to be transgelin, a smooth muscle actin-binding protein. CONCLUSION(S): This preliminary study demonstrated that differential protein profiling by using ProteinChip array technology is feasible, reproducible, and may be developed into a powerful tool for endometriosis research.

Biomarkers↗
PubMed2003

Functional and structural proteomics: a critical appraisal.

The notion of functional proteomics (description of changes in protein expression during differentiation of cells) and structural proteomics (elucidation of the primary structure of the components in the proteomic pattern) is reviewed. Quantitative and qualitative aspects of the proteome analysis are discussed and evaluated. A list of projects is given that are of high priority for the elucidation of the proteome. This includes, e.g., the study of the repertoire of the proteome and the description of molecular pathways during the clonal cell replication. The paper re-evaluates a number of methodological issues, such as the comparison of the use of Ampholines and Immobilines, various staining procedures and labeling procedures.

Autoradiography↗
PubMed2006

Comparative analysis of two-dimensional electrophoresis maps (2-DE) of Helicobacter pylori from Brazilian patients with chronic gastritis and duodenal ulcer: a preliminary report.

Helicobacter pylori is a bacterium recognized as the major cause of peptic ulcer and chronic gastritis. Recently, a proteome-based approach was developed to investigate pathogenic factors related to H. pylori. In this preliminary study, H. pylori strains were isolated from gastric biopsies of patients with chronic gastritis and duodenal ulcers. A partial proteomic analysis of H. pylori strains was performed by bacterial lyses and proteins were separated by two-dimensional gel electrophoresis (2-DE). A comparative analysis was performed to verify a differential protein expression between these two 2-DE maps. These data should be useful to clarify the role of different proteins related to bacterial pathogenesis. This study will be completed using a larger number of samples and protein identification of H. pylori by MALDI-TOF mass spectrometry.

Bacterial Proteins↗