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Results for “differential proteomics analysis”

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PubMed2005

Use of two-dimensional gel electrophoresis in predictive toxicology: identification of potential early protein biomarkers in chemically induced hepatocarcinogenesis.

Our current approach focused on the identification of potential early protein biomarker signatures which are indicative of the carcinogenic processes in rats exposed to 20 mg/kg of the liver carcinogen N-nitrosomorpholine (NNM). Treated liver was investigated at different timepoints. Therefore, proteins were separated by two-dimensional gel electrophoresis as a first step prior to identification of differentially expressed proteins by mass spectrometry. Proteomic analysis of liver samples after one day of exposure revealed significant upregulation of proteins involved in response to cellular stress induced by NNM (superoxide dismutase, heat shock protein 60, peroxiredoxin). Eighteen weeks after withdrawal of NNM, we were able to identify cancer-related proteins in rat liver bearing malignant, transformed cells (caspase-8 precursor, vimentin, Rho GDP dissociation inhibitor). Some of these proteins were already deregulated after three weeks of exposure indicating their potential usefulness as early predictive biomarkers for liver carcinogenicity (annexin A5, fructose-1,6-bisphosphatase). As regulatory toxicology approaches usually include the investigation of carcinogenicity in two-years studies in rodents, especially the detection of early protein biomarker signatures which precede the appearance of neoplasia, demonstrates the high potential of proteomics approaches to substantially reduce the time and costs of carcinogenicity testing.

Animals↗

PubMed2006

Differential steady-state tyrosine phosphorylation of two oligomeric forms of mitochondrial F0F1ATPsynthase: a structural proteomic analysis.

We investigated tyrosine phosphorylation of F(0)F(1)ATPsynthase using 3-D blue native (BN)-SDS-PAGE, a refinement of the electrophoretic analysis of mitochondrial complexes. Bovine heart mitochondria were detergent-solubilized and subjected to BN-PAGE. Bands of ATPsynthase monomer (Vmon) and dimer (Vdim) were excised and submitted to SDS-PAGE and immunoblotting. One protein corresponding to F(1)gamma subunit was detected by anti-phosphotyrosine antibody in monomer but not in dimer. This was confirmed by MS peptide mapping. LC-ESI/MS analysis after 3-D SDS-PAGE demonstrated phosphotyrosine in fragment 43-54. NetPhos scores predicted the phosphorylated residue to be Tyr52, in a solvent-accessible loop at the foot of the F(1) central stalk.

Animals↗

PubMed2006

Involvement of multitargets in paeoniflorin-induced preconditioning.

Paeoniflorin (PF) is the principal component of Paeoniae radix prescribed in traditional Chinese medicine. The delayed neuroprotection induced by PF preconditioning and its underlying mechanisms were investigated in rat middle cerebral artery occlusion (MCAO) and reperfusion model. At a dosage of 20 or 40 mg/kg, PF preconditioning 48 h before MCAO followed by 24-h reperfusion significantly reduced the mortality and infarct volume and reversed the neurological deficits caused by ischemia. Likewise, the ameliorative effects on mortality, infarct size, and neurological impairment induced by MCAO emerged as well when PF was administered 24 h, 48 h, or 5 days before MCAO at the dose of 20 mg/kg. Furthermore, comparative proteomics analysis was adopted to identify the differentially expressed proteins induced by PF preconditioning itself. The relative levels of 42 proteins were altered after PF preconditioning, among which 20 were elevated and 22 reduced. In summary, A(1) receptor-regulator of G protein signaling-K(ATP) signaling, arachidonic acid cascade, nitric oxide system, markers of neuronal damage, mitochondrial damage-related molecules, and the mitogen-activated protein kinase and nuclear factor-kappaB pathway are associated with the mechanisms of PF preconditioning.

Adenosine Triphosphate↗

PubMed2005

Identification of biomarkers for breast cancer in nipple aspiration and ductal lavage fluid.

PURPOSE: To establish a comprehensive proteomic approach for biomarker discovery and validation in breast fluid. EXPERIMENTAL DESIGN: A total of 95 specimens from three institutions were used including 10 nipple aspiration fluid (5 stage I/II cancerous breasts and 5 age-matched healthy controls), 42 ductal lavage fluid from 14 patients with unilateral stage I/II cancer (25 from 9 cancerous breasts and 17 from 7 contralateral breasts), and 42 ductal lavage fluid from 14 high-risk women (multiple ducts repeated lavage). Differentially expressed protein/peptides were discovered by proteomic analysis of training sample, using ProteinChip arrays and surface-enhanced laser desorption ionization (SELDI) time-of-flight mass spectrometry, and validated on independently collected testing samples. After protein identification, ELISA was done to confirm the SELDI findings. RESULTS: We were able to obtain reproducible protein profiles using minimal amount of protein (1 mug) by applying an optimized chip protocol and SELDI. We were able to select cancer-associated biomarkers despite large individual variability by applying both unsupervised and supervised cluster analysis. Furthermore, we were able to train and test candidate biomarkers on independently collected samples and identified one component of a multimarker panel as human neutrophil peptides 1 to 3. CONCLUSIONS: Breast fluid is a rich source of breast cancer biomarkers. In combination with high-throughput novel proteomic profiling technology and multicenter study design, markers that are highly specific to breast cancer can be discovered and validated. Our observations also suggest that persistent elevation of human neutrophil peptide in high-risk women may imply early onset of cancer not yet detectable by current detection method. Proof of this hypothesis requires follow-up on a larger study population.

Adenocarcinoma↗

PubMed2004

Proteomic analysis of brown planthopper: application to the study of carbamate toxicity.

Toxicity to o-sec-butylphenyl methylcarbamate compound (BPMC) was analyzed in the rice brown planthopper, Nilaparvata lugens, using a differential proteomics approach of identifying proteins on two dimensional-polyacrylamide gel electrophoresis (2D-PAGE). Proteome analysis from BPMC-treated brown planthopper resulted in the modulation of 22 proteins at the expression level as compared to control samples on coomassie brilliant blue (CBB) stained gels. Out of total 22 proteins, 10 proteins showed elevated expression, eight proteins showed decreased expression and four proteins showed specific expression after insecticide treatment. The N-terminal sequences of seven out of 22 proteins were determined by a gas-phase protein sequencer. The internal amino acid sequences of the 15 proteins were determined by the sequence analyses of peptides obtained by Cleveland peptide mapping method and were compared with those of the known proteins available in public databases and the EST database of the brown planthopper in our laboratory to understand the nature of the proteins. Sequence analyses revealed that the expression of putative serine/threonine protein kinase, paramyosin, HSP 90, beta-tubulin, calreticulin, ATP synthase, actin and tropomyosin was elevated, and that of beta-mitochondrial processing peptidase, dihydrolipoamide dehydrogenase, enolase and acyl-coA dehydrogenase was reduced due to the exposure of BPMC. The differential expression of these proteins reflects the overall change in cellular structure and metabolism after insecticide treatment.

Amino Acid Sequence↗

PubMed2005

Difference in mass analysis using labeled lysines (DIMAL-K): a new, efficient proteomic quantification method applied to the analysis of astrocytic secretomes.

Here we describe an original strategy for unbiased quantification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential predigestion labeling of lysine residues in complex protein mixtures. The method is relevant for proteomic analysis by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein labeling on lysine residues uses two closely related chemical reagents, S-methyl thioacetimidate and S-methyl thiopropionimidate. Using protein standards, we demonstrated that 1) the chemical labeling was quantitative, specific, and rapid; 2) the differentially labeled proteins co-migrated on two-dimensional gels; and 3) the identification by mass fingerprinting and the relative quantification of the proteins were possible from a single MALDI-TOF mass spectrum. The power of the method was tested by comparing and quantifying the secretion of proteins in normal and proinflammatory astrocytic secretomes (20 microg). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins.

Animals↗

PubMed2005

Proteomic analysis of nipple aspirate fluid using SELDI-TOF-MS.

Proteomic analysis of body fluids, including breast nipple aspirate fluid (NAF), holds promise to aid in early cancer detection. We conducted a prospective trial that collected NAF from women scheduled for diagnostic breast surgery to determine 1) the consistency of proteomic results, 2) protein masses associated with breast cancer, 3) subsets of women with a unique proteomic profile and 4) a breast cancer predictive model. NAF was collected preoperatively in 114 women and analyzed by SELDI-TOF mass spectrometry over a 3-50 kDa range using H4, NP and SAX ProteinChips. For all 3 chips, the same protein peaks were detected over 90% of the time in duplicate samples. The overall coefficient of variation was < or = 0.17% for each chip for the internal standard and < or = 0.29% for the unknown proteins. Seven candidate protein ion masses frequently expressed in NAF were identified. Three (5,200-H4, p=.04, 11,880-H4, p=.07 and 13,880 Da-SAX, p=.03) were differentially expressed in women with/without breast cancer. Protein expression differed between women with/without pathologic nipple discharge (PND), but the 5,200, 11,880 and 13,880 proteins remained associated with breast cancer even if PND samples were excluded. Subset analysis identified differences in expression between benign disease and DCIS and between DCIS and invasive cancer for the 5,200 and 33,400 Da proteins. The best cancer detection model included age, parity and the 11,880 Da protein, and excluded women with PND. 1) NAF proteomic analysis using SELDI-TOF is reproducible with the same sample set across different platforms, 2) differential proteomic expression exists between women/without breast cancer and 3) combining proteomic and clinical information that are available before surgery optimizes the prediction of which women have breast cancer.

Adult↗

PubMed2026

Sex-Specific Renal Proteomic Responses to Mycotoxins and Their Mitigation by Bioactive Food Ingredients.

This study aimed to evaluate sex-related differences in kidney toxicity induced by aflatoxin B1 (AFB1) and ochratoxin A (OTA), administered either individually or in combination, and to investigate the mitigating potential of fermented whey (FW) and pumpkin (P) using a proteomic approach in a sub-chronic rat model. Diets naturally contaminated with AFB1 and OTA fungal producers, or enriched with FW and P, were administered to 120 Wistar rats for 28 days. Afterwards, kidneys were collected and subjected to protein extraction and digestion to peptides. For proteomic analysis, peptides were separated using an LC-QTOF-MS system, and differentially expressed proteins between control and treated groups were statistically filtered (p < 0.05) to distinguish sex-related differences between males and females. Proteomic analysis revealed that non-supplemented diets induced significant protein alterations, with male kidneys showing greater sensitivity to mycotoxin exposure, particularly to OTA, and exhibiting a distinct expression pattern compared with females. Notably, FW and FW + P supplementation mitigated these adverse effects by reducing the number of differentially expressed proteins, stabilizing mitochondrial function, and enhancing xenobiotic metabolism pathways. These findings underscore the importance of sex-specific factors in evaluating the protective potential of bioactive ingredients against mycotoxin-induced renal injury.

Animals↗

PubMed2005

Proteomic analysis of human thyroid cell lines reveals reduced nuclear localization of Mn-SOD in poorly differentiated thyroid cancer cells.

Differential protein arrays between nuclear extracts of human thyroid cell lines obtained from tumors with different degree of differentiation were exploited to define molecular alterations occurring during thyroid tumor progression. Nuclear extracts from the well differentiated TPC-1 (from papillary carcinoma) and the poorly differentiated ARO (from anaplastic carcinoma) cells showed an overall similar pattern of protein expression as revealed by two-dimensional gel electrophoresis analysis. However, manganese-superoxide dismutase (Mn-SOD) was clearly identified by mass spectrometry procedures as significantly less expressed in ARO compared to TPC-1 cells. A reduced expression of Mn-SOD in the nuclear compartment was confirmed by Western blot and immunofluorescence analysis. A similar expression pattern of nuclear Mn-SOD was detected by immunohistochemistry in human thyroid tumors, with the lowest or absent detection in anaplastic carcinomas. Moreover, the levels of nuclear Mn-SOD in tumor cells were lower than in the normal thyrocytes. These data indicate that an altered nuclear expression of Mn-SOD parallels, together with changes in other elements of the antioxidant protective system, the loss of differentiation occurring during the progression of thyroid tumors.

Adenocarcinoma, Follicular↗

PubMed2026

Differential Proteomic Profiling of Responders and Non-responders to Direct-Acting Antivirals Treatment in Chronic Hepatitis C Virus Infection.

Hepatitis C Virus (HCV), particularly genotype 3 (GT-3), is highly prevalent in India and is associated with faster progression to cirrhosis, hepatocellular carcinoma, and higher treatment failure rates. Although Direct-Acting Antivirals (DAAs) have revolutionized HCV therapy, 5-10% of patients fail to achieve sustained virological response (SVR). This proteomic study aimed to identify changes in the proteomic profile before and after treatment of both responders and non-responders to HCV treatment. Paired plasma samples from HCV GT-3 infected patients were collected before and 12 weeks after initiating DAAs treatment, along with healthy controls. Quantitative proteomic analysis was performed on the paired samples. Differentially expressed proteins (DEPs) were identified and subjected to functional analysis including gene set enrichment analysis (GSEA) and protein-protein interaction (PPI) network analysis. GSEA revealed enrichment in extracellular matrix organization and innate immune pathways. Expression patterns of candidate proteins selected based on fold change and false discovery rate (FDR) criteria were further evaluated in an independent cohort. Western blot confirmed key expression trends of candidate proteins. Proteins linked to extracellular matrix remodeling and angiogenesis showed differential expression patterns. Successful validation of these candidate proteins in large independent cohorts holds potential to predict therapeutic outcomes.

Humans↗

PubMed2004

Presentation of differentially regulated proteins within a web-accessible proteome database system of microorganisms.

Web-accessible proteome databases represent indispensable tools for quantitative and comparative proteomics research. The majority of two-dimensional gel electrophoresis (2-DE) databases contains clickable 2-DE gel images and descriptive textual information such as protein name, Mr/pI values, methods of identification, cellular localization and other information on proteins. Although a great part of the work in comparative proteomics consists of the analysis of 2-DE gels using image analysis approaches, most proteome databases lack the ability to present protein abundance data and their alterations within experiments via the web. Now, differentially regulated proteins detected in microbial experiments by quantitative gel image analysis are presented in a web-accessible relational database DIFF (Differentially Regulated Proteins). The DIFF database is a part of the proteome database system for microbial research available at http://www.mpiib-berlin.mpg.de/2D-PAGE.

Bacterial Proteins↗

PubMed2006

Differential detection of S100A8 in transitional cell carcinoma of the bladder by pair wise tissue proteomic and immunohistochemical analysis.

The search for novel molecular markers of tumor invasion is vital if strategies are to become more effective in the diagnostic and prognostic management of transitional cell carcinoma of the bladder. Up to 50% of tumors detected at stage 1 (pT1) progress to a higher grade even after endoscopic surgical resection, and there are currently no protein markers of this aggressive, invasive phenotype. We have combined SELDI-TOF-MS, ClinProt magnetic bead enrichment, Nano-LC-ESI-ion trap tandem mass spectrometry and immunohistochemical analysis to the study of 12 invasive bladder cancer tissue biopsies paired with normal bladder tissue samples obtained from the same patients for the definition and identification of proteins up-regulated in the tumors. We report the inflammation-associated calcium binding protein S100A8 (MRP-8, calgranulin A) to be highly expressed in tumor cells in contrast to normal urothelium in 50% of the samples, as well as two unidentified protein markers at 5.75 and 6.89 kDa that were differentially detected in 9/12 and 10/12 tumor samples, respectively. These new markers, when fully characterized, may contribute to new target proteins for the prediction of aggressive, invasive bladder tumors.

Biomarkers, Tumor↗

PubMed2004

Differential proteomics: an overview of gel and non-gel based approaches.

The comprehensive analysis of gene expression in complex biological systems has demanded the development of new technologies to study the cell transcriptome and the cell proteome. Each approach has advantages and disadvantages from both the conceptual and the methodological viewpoints. Differential proteomics, the comparison of distinct proteomes (eg normal versus diseased cells, diseased versus treated cells etc) is of paramount importance. Several approaches can be used and these typically involve electrophoresis and/or chromatography combined with chemical or metabolic labelling and mass spectrometry. These approaches aim to identify molecular targets, namely proteins, involved in different physiopathological states. Incorporating this knowledge with knowledge from other technologies lays the foundations of active principles at the molecular level. Here, the various gel- and non-gel-based approaches that are used in a wide range of biological systems for the study of differentially expressed proteins will be reviewed.

Algorithms↗

PubMed2006

Analysis of neuron-like differentiation of human bone marrow mesenchymal stem cells.

The objective of the study was to evaluate differentiation of human bone marrow mesenchymal stem cells into true or pseudo neurons after treating with chemical induction medium in vitro. The morphological changes were assessed using interference contrast microscopy. Immunocytochemistry and Western blotting were performed using neuronal markers. Further evaluation was conducted with proteomic profiling, DNA microarray analysis and the whole-cell patch clamp test. After three hours of treatment with chemical induction medium, nearly three-fourths of the hMSCs changed to cells with a neuronal phenotype. The results of immunocytochemistry and Western blotting showed a high expression of neuronal markers in these cells at 3 h which decreased at 24 h. The proteomics analysis showed no change of proteins related to neuronal differentiation. DNA microarray showed downregulation of neuron related genes. The patch clamp test was unable to demonstrate any similarity to true neurons. Our findings suggest that neuron-like cells derived from chemical induction of hMSCs are not the genuine neurons as they resemble true neurons phenotypically but are different in genotypic and electrophysiological characteristics.

Biomarkers↗

PubMed2026

Editor's Choice - Aquaporin-4: A Predictor and Therapeutic Target for Permanent Paraplegia after Endovascular Thoraco-abdominal Aortic Aneurysm Repair.

OBJECTIVE: Endovascular thoraco-abdominal aortic aneurysm (TAAA) repair can impair spinal cord perfusion, leading to paraplegia. The mechanisms driving this devastating complication are poorly understood. This study aimed to interrogate the cerebrospinal fluid (CSF) proteome in patients after TAAA repair to identify biomarkers that herald permanent paraplegia. It also aimed to investigate a potential therapeutic target identified by proteomics using an in vivo model of ischaemic spinal cord injury (iSCI). METHODS: CSF was collected for proteomic analysis from patients before and following TAAA repair. A differentially expressed protein identified in human paraplegic subjects was subsequently interrogated in a rodent model of iSCI. The protein composition of CSF was analysed using tandem mass tag proteomics. Neurological examinations were carried out by a blinded neurologist and T2 weighted magnetic resonance imaging (MRI) was used to measure spinal cord volume and oedema. A rodent model of iSCI was used to investigate a clinically relevant therapeutic target informed by proteomic findings. RESULTS: CSF analysis was taken from 37 patients, all of whom had aneurysm repair using a custom branched and or fenestrated device (median age 73.5 years, range 67 - 78 years; 27 men, ten women; Crawford classification: six type I, 11 type II, 15 type III, three type IV, and two type V). Five patients remained permanently paraplegic and seven recovered from transient paraplegia. The CSF of patients who remained paraplegic contained approximately fourfold more aquaporin-4 (AQP4) (41.8 ± 19.2 ng/mL, n = 5) than those who recovered from paraplegia (10.8 ± 1.3 ng/mL, n = 7; p = .005) or did not develop paraplegia (10.8 ± 1.2 ng/mL, n = 25; p = .004). Permanently paraplegic patients had CSF AQP4 levels > 15 ng/mL and this was associated with greater cord oedema on T2 weighted MRI (1.77 ± 0.19 vs. 1.03 ± 0.36; p = .032). In a rodent model of iSCI, AQP4 inhibition preserved spinal neurons and glia in the dorsal horn and intermediate zones of white matter (p = .004) and protected against ischaemia induced paraplegia (p < .001). CONCLUSION: The AQP4 level in the CSF of a patient represents a prognostic marker of permanent paraplegia after TAAA repair and highlights a novel therapeutic target. These findings represent a conceptual advance in the management of iSCI.

Paraplegia↗

PubMed2006

Proteomic based investigation of rhamnolipid production by Pseudomonas chlororaphis strain NRRL B-30761.

We recently reported that a strain of the non-pathogenic bacterial species Pseudomonas chlororaphis was capable of producing the biosurfactant molecule, rhamnolipids. Previous to this report the organisms known to produce rhamnolipids were almost exclusively pathogens. The newly described P. chlororaphis strain produced rhamnolipids at room temperature in static minimal media, as opposed to previous reports of rhamnolipid production which occurred at elevated temperatures with mechanical agitation. The non-pathogenic nature and energy conserving production conditions make the P. chlororaphis strain an attractive candidate for commercial rhamnolipid production. However, little characterization of molecular/biochemical processes in P. chlororaphis have been reported. In order to achieve a greater understanding of the process by which P. chlororaphis produces rhamnolipids, a survey of proteins differentially expressed during rhamnolipid production was performed. Separation and measurement of the bacteria's proteome was achieved using Beckman Coulter's Proteome Lab PF2D packed column-based protein fractionation system. Statistical analysis of the data identified differentially expressed proteins and known orthologues of those proteins were identified using an AB 4700 Proteomics Analyzer mass spectrometer system. A list of proteins differentially expressed by P. chlororaphis strain NRRL B-30761 during rhamnolipid production was generated, and confirmed through a repetition of the entire separation process.

Bacterial Proteins↗

PubMed2002

Proteomic analysis of transcription factor interactions in myeloid stem cell development and leukaemia.

Recent results indicate that interactions of transcription factors with other nuclear proteins play an important role in stem cell development, lineage commitment and differentiation in the haematopoietic system, and the pathogenesis of myeloid leukaemias. High-throughput proteomics by mass spectrometric analysis of gel-separated proteins can identify multi-protein complexes and changes in the expression of multiple proteins simultaneously. This review describes an application of proteomic methods (2D gel electrophoresis (GE) and mass spectrometry (MS)), which can be used to identify regulated protein targets of transcription factors important in myeloid differentiation and leukaemia. This global high-throughput functional proteomics approach could lead to new insights into the network of protein-protein interactions and target proteins involved in myeloid stem cell development and leukaemia as well as provide new targets for rational pathogenesis-based therapies of leukaemia and cancer.

CCAAT-Enhancer-Binding Proteins↗

PubMed2004

Proteomic analysis of lymph.

This report provides the first proteomic analysis of normal ovine lymph. By establishing the fact that lymph is more than an ultrafiltrate of blood plasma, it documents that the lymph proteome contains an array of proteins that differentiates it from plasma. The protein chip technology, surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS), two-dimensional gel electrophoresis (2-D PAGE) and MS, were employed to examine the protein expression profiles of ovine lymph. Using a weak cation exchange chip surface to assay lymph and plasma samples by SELDI-TOF-MS showed that the analysis of peak maps from lymph contained three protein peaks that were found only in lymph, while analysis of peak maps from plasma samples showed that five protein peaks were found only in plasma. Lymph and plasma samples showed eight peaks that were common to both. There were also more ions present in plasma than in lymph, which is consistent with the 2-D PAGE analysis. MS analysis of a large number of protein spots from 2-D PAGE gels of lymph produced MS/MS sequences for 18 proteins that were identified by searching against a comprehensive protein sequence database. As in plasma, large protein spots of albumin dominated the protein pattern in lymph. Other major proteins identified in 2-D PAGE gels of lymph included, fibrinogen alpha- and beta-chains, immunoglobulin G (IgG) heavy chain, serotransferrin precursor, lactoferrin, and apolipoprotein A-1. Two proteins that were identified and were differentially expressed in lymph were glial fibrillary astrocyte acidic protein and neutrophil cytosol factor-1. By bringing the technologies of proteomics to bear on the analysis of lymph, it is possible to detect proteins in lymph that are quantitatively and qualitatively differentially expressed from those of plasma.

Animals↗