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At least 181 records · Page 10Linked to original sources
PubMed2005

Proteomic profiling unravels insights into the molecular background underlying increased Aphanomyces euteiches-tolerance of Medicago truncatula.

To investigate the molecular mechanisms underlying susceptibility of legumes to the root pathogen Aphanomyces euteiches (oomycota), comparative proteomic studies have been carried out. In a first approach, we have analysed two Medicago truncatula lines of the French CORE collection (F83.005-5 (R2002) and F83.005-9 (R2002)), which showed either increased or decreased susceptibility to A. euteiches as compared to the widely adopted line A17. Several proteins were identified to be differentially induced after pathogen challenge in the two M. truncatula accessions with altered disease susceptibility, whereof proteins with increased abundances in the more resistant line F83.005-9 could be involved in mechanisms that lead to an improved disease resistance. Among these proteins, we identified two proteasome alpha subunits, which might be involved in defense response. To broaden our studies on A. euteiches-tolerance of M. truncatula, we investigated two other phenomena that lead to an either increased A. euteiches-resistance or to an enhanced susceptibility. The topic of an enhanced plant resistance to A. euteiches was studied in plants showing a bioprotective effect of a pre-established arbuscular mycorrhiza (AM) symbiosis. Evaluation of root fresh weights and pathogen spreading in the root system clearly indicate that mycorrhizal plants show increased A. euteiches-resistance as compared to non-mycorrhizal plants. Proteome analyses revealed the induction of similar protein patterns as in the M. truncatula accessions with comparatively high resistance level to A. euteiches. In a third approach, increased A. euteiches susceptibility was effected by exogenous abscisic acid (ABA) application prior to root infection. Evaluation of the abundance levels of a group of pathogenesis related class 10 (PR10)-like proteins, which were previously identified to be regulated after A. euteiches infection, revealed a correlation between the abundance levels of these proteins and the A. euteiches infection level or severity.

Abscisic Acid↗
PubMed2005

Proteome profiling of corneal epithelium and identification of marker proteins for keratoconus, a pilot study.

The purpose of this study is to identify corneal proteins differentially expressed between keratoconus and normal epithelial samples. Proteins from the corneal epithelium were isolated from 6 keratoconus and 6 myopia patients (controls) and separated by 2D-gel electrophoresis. Six % and 12% SDS-PAGE gels were used to separate low and high molecular weight proteins. Gels were silver stained and protein spots were defined by Melanie II software. The proteins that were most altered in expression comparing keratoconus and controls were extracted, trypsin-digested, and identified by mass spectroscopy. Approximately 200-500 protein spots were detected on each gel. Nineteen spots were identified as differentially expressed between keratoconus and reference epithelium including cytokeratin 3 (< 7.8 fold), gelsolin (1.6 fold), S100A4 (1.9 fold), and enolase 1 (0.72 fold). Another identified protein found at very high levels was cytokeratin 12. Gelsolin, cytokeratin 3, and cytokeratin 12 have previously been described to be involved in other corneal diseases. Three proteins, gelsolin, alpha enolase, and S100A4 were identified to be differentially expressed in keratoconus compared to reference epithelium and thus may be involved in the pathogenesis.

Biomarkers↗
PubMed2004

Proteomic profiling in microdissected hepatocellular carcinoma tissue using ProteinChip technology.

At present, the molecular mechanisms of hepatocellular carcinogenesis are not well understood. It is known, however, that cancer development and progression are accompanied by profound changes at the cellular and subcellular level, involving RNA/DNA and protein structure and function. Therefore, high-throughput, proteomic techniques targeting these biological molecules may provide novel insights into HCC genesis and prognosis. We characterized tissue protein profiles from 10 HCC patients using ProteinChip technology (SELDI) which is able to detect minute amounts of proteins and moreover to analyze complex protein pattern. Therefore, after histopathological examination, proteins from kryostat sections of non-tumorous hepatic tissue as well as from central and peripheral tumor areas were isolated from complete histological sections or from selected and microdissected tissue areas. Analysis on the SAX and WCX ProteinChip Arrays revealed 14-26, and 25-29 differentially expressed peaks respectively, which characterized non-tumorous and tumor tissue (p< or =0.05). One feature which allows differentiation between central tumor and peripheral tumor regions could only be detected in microdissected tissue. Using ProteinChip technology in combination with tissue microdissection it is possible to investigate complex changes at the protein level in hepatocellular cancer associated with tumor development and progression.

Biomarkers, Tumor↗
PubMed2006

Proteome profiles of mucosal immunoglobulin uptake in inflamed porcine gut.

Acquisition of passive immunity by endocytosis of intact immunoglobulins (Ig) from colostrum is critical for prevention of intestinal and systemic diseases in neonatal mammals. We compared proteome patterns of healthy and inflamed gut tissues from pre-term piglets to investigate the effect of inflammation on acquisition of passive immunity. A clear difference in the two-dimensional gel electrophoresis protein patterns between healthy and inflamed intestinal tissues was observed, suggesting that inflamed tissues failed to absorb and transfer Ig from colostrum to epithelial cells. We have mapped and identified the Ig proteins that are taken up by healthy intestinal tissues, and found that isoforms of the IgA and IgG heavy chain and Ig kappa and lambda light chains were internalized. Our results indicate that colostrum protein uptake in the porcine gut is a selective process that is obstructed in inflamed pre-term gut.

Animals↗
PubMed2006

Proteomic profiling of cold thyroid nodules.

Cold thyroid nodules (CTNs) represent a frequent endocrine disorder accounting for up to 85% of thyroid nodules in a population living in an iodine-deficient area. Benign CTNs need to be distinguished from thyroid cancer, which is relatively rare. The molecular etiology of benign CTNs is unresolved. To obtain novel insights into their pathogenesis, protein expression profiling was performed in a series of 27 solitary CTNs (10 follicular adenoma and 20 adenomatous nodules) and surrounding normal thyroid tissues using two-dimensional gel electrophoresis combined with mass spectrometry analysis, Western blotting, and immunohistochemistry. The proteome analysis revealed a specific fingerprint of CTNs with up-regulation of three functional systems: 1) thyroid cell proliferation, 2) turnover of thyroglobulin, and 3) H2O2 detoxification. Western blot analysis and immunohistochemistry confirmed the proteome data and showed that CTNs exhibit significant up-regulation of proteins involved in thyroid hormone synthesis yet are deficient in T4-containing thyroglobulin. This is consequential to intranodular iodide deficiency, mainly due to cytoplasmic sodium iodide symporter localization, and portrays the CTN as an activated proliferating lesion with an intranodular hypothyroid milieu. Furthermore, we provide preliminary evidence that up-regulation of H2O2 generation in CTNs could override the antioxidative system resulting in oxidative stress, which is suggested by the finding of raised 8-oxo-guanidine DNA adduct formation in CTNs.

Adult↗
PubMed2007

Proteome profile of cytosolic component of zebrafish liver generated by LC-ESI MS/MS combined with trypsin digestion and microwave-assisted acid hydrolysis.

The zebrafish genome has recently been sequenced and annotated allowing for high-throughput proteomic analysis. Here, we report for the first time a proteomic subset of zebrafish liver, an important organ for metabolizing toxins. Using a newly developed analytical procedure, we have identified 1204 proteins from the cytosolic component of a zebrafish liver tissue sample. Our methods involve cell-compartment fractionation of liver tissue samples, four levels of protein digestion, and off-line two-dimensional liquid chromatography (2-D LC) separations of resultant peptides. Proteins are identified using an electrospray ionization quadrupole time-of-flight tandem mass spectrometer (ESI-QTOF MS/MS), which provides high-resolution and high-accuracy mass measurement of peptide ions and their fragment ions. We demonstrate that greater proteome coverage can be achieved by combining the results obtained from four methods of protein digestion: three tryptic digests (one in buffer, one in methanol, and another in SDS), and a microwave-assisted acid hydrolysate of the protein extracts. Identified proteins--which included several groups of established protein biomarkers--were functionally classified. We discuss the functions and implications of these biomarkers within the context of zebrafish toxicology.

Animals↗
PubMed2004

Global investigation of p53-induced apoptosis through quantitative proteomic profiling using comparative amino acid-coded tagging.

p53-induced apoptosis plays a pivotal role in the suppression of tumorigenesis, and mutations in p53 have been found in more than 50% of human tumors. By comparing the proteome of a human colorectal cancer cell transfected with inducible p53 (DLD-1.p53) with that of the control DLD-1 cell line using amino acid-coded mass tagging (AACT)-assisted mass spectrometry, we have broadly identified proteins that are upregulated at the execution stage of the p53-mediated apoptosis. In cell culturing, the deuterium-labeled (heavy) amino acids were incorporated into the proteome of the induced DLD-1.p53 cells, whereas the DLD-1.vector cells were grown in the unlabeled medium. In high-throughput LC-ESI-MS/MS analyses, the AACT-containing peptides were paired with their unlabeled counterparts, and their relative spectral intensities, reflecting the differential protein expression, were quantified. In addition, our novel AACT-MS method utilized a number of different heavy amino acids as internal markers that significantly increased the peptide sequence coverage for both quantitation and identification purposes. As a result, we were able to identify differentially regulated protein isozymes that would be difficult to distinguish by ICAT-MS methods and to obtain a large dataset of the proteins with altered expression in the late stage of p53-induced apoptosis. The regulated proteins we identified are associated with several distinct functional categories: cell cycle arrest and p53 binding, protein chaperoning, plasma membrane dynamics, stress response, antioxidant enzymes, and anaerobic glycolysis. This result suggests that the p53-induced apoptosis involves the systematic activation of multiple pathways that are glycolysis-relevant, energy-dependent, oxidative stress-mediated, and possibly mediated through interorganelle crosstalks.

Amino Acid Sequence↗
PubMed2006

Proteomic profile changes in membranes of ethanol-tolerant Clostridium thermocellum.

Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, has potential for commercial exploitation in converting fibrous biomass to ethanol. However, ethanol concentrations above 1% (w/v) are inhibitory to growth and fermentation, and this limits industrial application of the organism. Recent work with ethanol-adapted strains suggested that protein changes occurred during ethanol adaptation, particularly in the membrane proteome. A two-stage Bicine-doubled sodium dodecyl sulfate-polyacrylamide gel electrophoresis protocol was designed to separate membrane proteins and circumvent problems associated with membrane protein analysis using traditional gel-based proteomics approaches. Wild-type and ethanol-adapted C. thermocellum membranes displayed similar spot diversity and approximately 60% of proteins identified from purified membrane fractions were observed to be differentially expressed in the two strains. A majority (73%) of differentially expressed proteins were down-regulated in the ethanol-adapted strain. Based on putative identifications, a significant proportion of these down-regulated proteins were involved with carbohydrate transport and metabolism. Approximately one-third of the up-regulated proteins in the ethanol-adapted species were associated with chemotaxis and signal transduction. Overall, the results suggested that membrane-associated proteins in the ethanol-adapted strain are either being synthesized in lower quantities or not properly incorporated into the cell membrane.

Bacterial Proteins↗
PubMedDate not supplied

Proteomic profiling of surface proteins on Th1 and Th2 cells.

We utilized mass spectrometry to profile cell surface protein differential expression on primary human T helper (Th1 and Th2) cells with the stable isotope labeling by amino acids in cell culture (SILAC) approach. Proteomic and microarray analyses were done concurrently and results were compared for 38 different genes. Although microarray studies displayed wide variability between donors for mRNA expression, these two approaches were shown to be corroborative for most gene products with the exception of a small subset of uncorrelated protein and message levels. The greatest differing Th1 to Th2 ratios were observed for BST2 (bone marrow stromal protein 2) and TRIM (T cell receptor interacting molecule). Both showed greater Th1 expression by proteomic methods, even though mRNA levels were approximately equal for both. To validate this method, we compared protein expression levels of a recently cloned molecule, B and T cell lymphocyte attenuator (BTLA), on Th1 and Th2 cell populations and showed greater protein expression on Th1 cells, which agrees with a previous analysis of higher BTLA mRNA expression in Th1 cells.(1).

Cell Differentiation↗
PubMed2005

Proteomic profiling of hepatic endoplasmic reticulum-associated proteins in an animal model of insulin resistance and metabolic dyslipidemia.

Hepatic insulin resistance and lipoprotein overproduction are common features of the metabolic syndrome and insulin-resistant states. A fructose-fed, insulin-resistant hamster model was recently developed to investigate mechanisms linking the development of hepatic insulin resistance and overproduction of atherogenic lipoproteins. Here we report a systematic analysis of protein expression profiles in the endoplasmic reticulum (ER) fractions isolated from livers of fructose-fed hamsters with the intention of identifying new candidate proteins involved in hepatic complications of insulin resistance and lipoprotein dysregulation. We have profiled hepatic ER-associated proteins from chow-fed (control) and fructose-fed (insulin-resistant) hamsters using two-dimensional gel electrophoresis and mass spectrometry. A total of 26 large scale two-dimensional gels of hepatic ER were used to identify 34 differentially expressed hepatic ER protein spots observed to be at least 2-fold differentially expressed with fructose feeding and the onset of insulin resistance. Differentially expressed proteins were identified by matrix-assisted laser desorption ionization-quadrupole time of flight (MALDI-Q-TOF), MALDI-TOF-postsource decay, and database mining using ProteinProspector MS-fit and MS-tag or the PROWL ProFound search engine using a focused rodent or mammalian search. Hepatic ER proteins ER60, ERp46, ERp29, glutamate dehydrogenase, and TAP1 were shown to be more than 2-fold down-regulated, whereas alpha-glucosidase, P-glycoprotein, fibrinogen, protein disulfide isomerase, GRP94, and apolipoprotein E were all found to be up-regulated in the hepatic ER of the fructose-fed hamster. Seven isoforms of ER60 in the hepatic ER were all shown to be down-regulated at least 2-fold in hepatocytes from fructosefed/insulin-resistant hamsters. Implications of the differential expression of positively identified protein factors in the development of hepatic insulin resistance and lipoprotein abnormalities are discussed.

Amino Acid Sequence↗
PubMed2005

[Effects of the Radix Ranuncoli Ternati extracts on Mycobacterium tuberculosis proteome profiling revealed by 2D electrophoresis].

Radix Ranuncoli Ternati is clinically effective traditional Chinese medicine for multidrug resistant tuberculosis. Its active components and mechanism of action remain unsolved. Two dimensional gel electrophoresis (2-DE) was employed to address this problem. Globlal proteome of Mycobacterium tuberculosis untreated and treated with Radix Ranuncoli Ternati were compared, and 22 protein spots were found to be expressed differentially. 3 protein spots which remarkably decreased in Mycobacterium tuberculosis treated with Radix Ranuncoli Ternati were subjected to matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis. The data obtained from peptide mass finger printing were used for database search. The 3 protein spots in gel were identified as cysA2 (thiosulfate sulfurtransferase), tsf (elongation factor EF-Ts) and hspX (heat shock protein X). These data provide insights into the changed global protein patterns of Mycobacterium tuberculosis treated with Radix Ranuncoli Ternati and may prove useful for further study in the mechanism in how Radix Ranuncoli Ternati influence the life of Mycobacterium tuberculosis. The differentially expressed proteins may be potential novel antituberculosis drug targets.

Antigens, Bacterial↗
PubMed2006

Functional proteomic profiling of glycan-processing enzymes.

Glycoconjugates play critical roles in regulating cellular and organismal functions. Consequently, defining the relative levels of these glycoconjugates under varied physiological conditions is important. Thus identifying and understanding the regulation of the enzymes that process these glycoconjugates are essential steps in understanding the role of this "glycocode" in development and disease. Activity-based affinity reagents are useful tools for probing these enzymes and should facilitate the unraveling of proteomes. One advantage of activity-based affinity probes is that they can simultaneously reveal multiple enzymes having similar activities. These probes can also be used to enrich proteomes of interest, thereby facilitating identification and cloning of new carbohydrate-processing enzymes. Here we review the current state of activity-based affinity probes for profiling carbohydrate-processing enzymes, focusing on successes and limitations, general design features, and a specific example describing profiling of exoglycosidases from cell lysates.

Affinity Labels↗
PubMed2003

A data review and re-assessment of ovarian cancer serum proteomic profiling.

BACKGROUND: The early detection of ovarian cancer has the potential to dramatically reduce mortality. Recently, the use of mass spectrometry to develop profiles of patient serum proteins, combined with advanced data mining algorithms has been reported as a promising method to achieve this goal. In this report, we analyze the Ovarian Dataset 8-7-02 downloaded from the Clinical Proteomics Program Databank website, using nonparametric statistics and stepwise discriminant analysis to develop rules to diagnose patients, as well as to understand general patterns in the data that may guide future research. RESULTS: The mass spectrometry serum profiles derived from cancer and controls exhibited numerous statistical differences. For example, use of the Wilcoxon test in comparing the intensity at each of the 15,154 mass to charge (M/Z) values between the cancer and controls, resulted in the detection of 3,591 M/Z values whose intensities differed by a p-value of 10-6 or less. The region containing the M/Z values of greatest statistical difference between cancer and controls occurred at M/Z values less than 500. For example the M/Z values of 2.7921478 and 245.53704 could be used to significantly separate the cancer from control groups. Three other sets of M/Z values were developed using a training set that could distinguish between cancer and control subjects in a test set with 100% sensitivity and specificity. CONCLUSION: The ability to discriminate between cancer and control subjects based on the M/Z values of 2.7921478 and 245.53704 reveals the existence of a significant non-biologic experimental bias between these two groups. This bias may invalidate attempts to use this dataset to find patterns of reproducible diagnostic value. To minimize false discovery, results using mass spectrometry and data mining algorithms should be carefully reviewed and benchmarked with routine statistical methods.

Artificial Intelligence↗
PubMed2005

Proteomic profiling identifies an UV-induced activation of cofilin-1 and destrin in human epidermis.

The human skin is the only line of defense against UV radiation. A series of responses to protect the skin are induced by UV radiation. In this study, a proteomic approach was used to study these responses. We have performed high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis of (solar simulated) UV-exposed reconstructed skin equivalents as well as native skin. Differentially expressed proteins were processed for mass spectrometric analysis, when consistent differences were observed in all individual human skin equivalents. In addition to proteins known to be involved in UV responses (HSP27, MnSOD, and PDX-2), we identified two novel proteins that were downregulated following UV exposure. Further analysis revealed that these proteins were the phosphorylated forms of the actin cytoskeleton modulators cofilin-1 and destrin. The de-phosphorylation of cofilin-1 was confirmed using western blotting of UV-exposed skin equivalents and ex vivo skin protein extracts. In conclusion, our study indicates the potency of a proteomic approach to study UV-induced changes in a tissue culture system mimicking human skin as well as excised human skin.

Actin Depolymerizing Factors↗
PubMed2005

Constructing support vector machine ensembles for cancer classification based on proteomic profiling.

In this study, we present a constructive algorithm for training cooperative support vector machine ensembles (CSVMEs). CSVME combines ensemble architecture design with cooperative training for individual SVMs in ensembles. Unlike most previous studies on training ensembles, CSVME puts emphasis on both accuracy and collaboration among individual SVMs in an ensemble. A group of SVMs selected on the basis of recursive classifier elimination is used in CSVME, and the number of the individual SVMs selected to construct CSVME is determined by 10-fold cross-validation. This kind of SVME has been tested on two ovarian cancer datasets previously obtained by proteomic mass spectrometry. By combining several individual SVMs, the proposed method achieves better performance than the SVME of all base SVMs.

Algorithms↗
PubMed2026

Large-Scale Proteomic Profiling of Incident Heart Failure and Its Subtypes in Older Adults.

BACKGROUND: Heart failure (HF) and its main subtypes, heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF), impose an enormous health burden on elders. Assessment of the circulating proteome to illuminate pathogenesis could open new opportunities for treatment. METHODS: We conducted a plasma proteomics screen of incident HF and its subtypes in 2 older population-based cohorts, the CHS (Cardiovascular Health Study) and the AGES-RS (Aging, Gene/Environment Susceptibility-Reykjavik Study). The 2 studies used SomaLogic platforms, with 4404 aptamers in common. Multivariable Cox models were fit to evaluate individual-protein associations with HF, HFpEF, and HFrEF separately in each cohort, and study-specific associations were combined by fixed-effects meta-analysis. Replication was performed in the ARIC (Atherosclerosis Risk in Communities) cohort. Two-sample Mendelian randomization of HF and its subtypes, along with colocalization analysis, was performed to support causal inference. RESULTS: Among 8599 participants, 1590 experienced incident HF (536 HFpEF, 471 HFrEF). There were 119 proteins associated with HF, 15 proteins with HFpEF, and 11 proteins with HFrEF, at Bonferroni-corrected significance. Among these, 9 have never previously been identified for cardiovascular diseases, and another 61 represent new associations with incident HF or its subtypes. Of these 70 proteins, 55 of the 66 available replicated externally. Mendelian randomization analysis revealed 7 proteins genetically associated with HF at nominal significance; 2 were separately associated with HFpEF, and another 2 with HFrEF. Seven of these 9 proteins (NPDC1 [neural proliferation differentiation and control protein 1], APOF [apolipoprotein F], LMAN2 [lectin, mannose-binding 2], ADIPOQ [adiponectin], CD14 [cluster of differentiation 14], ARHGAP1 [Rho GTPase-activating protein 1], C9 [complement 9]) showed new, possibly causal associations, although we did not detect evidence for colocalization. CONCLUSIONS: In this large-scale proteomic study involving 3 longitudinal cohorts of older adults, we identified and replicated 55 novel protein markers of HF or its subtypes, and 7 new, possibly causal proteins. These proteins may enhance risk prediction, improve understanding of pathobiology, and help prioritize targets for therapeutic development of these foremost disorders in elders.

Humans↗
PubMed2006

Proteomic profiling of primary breast cancer predicts axillary lymph node metastasis.

To determine if protein expression in primary breast cancers can predict axillary lymph node (ALN) metastasis, we assessed differences in protein expression between primary breast cancers with and without ALN metastasis using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Laser capture microdissection was performed on invasive breast cancer frozen sections from 65 patients undergoing resection with sentinel lymph node (SLN) or level I and II ALN dissection. Isolated proteins from these tumors were applied to immobilized metal affinity capture (IMAC-3) ProteinChip arrays and analyzed by SELDI-TOF-MS to generate unique protein profiles. Correlations between unique protein peaks and histologically confirmed ALN status and other known clinicopathologic factors were examined using ANOVA and multivariate logistic regression. Two metal-binding polypeptides at 4,871 and 8,596 Da were identified as significant risk factors for nodal metastasis (P = 0.034 and 0.015, respectively) in a multivariate analysis. Lymphovascular invasion (LVI) was the only clinicopathologic factor predictive of ALN metastasis (P = 0.0038). In a logistic regression model combining the 4,871 and 8,596 Da peaks with LVI, the area under the receiver operating characteristic curve was 0.87. Compared with patients with negative ALN, those with > or =2 positive ALN or non-SLN metastases were significantly more likely to have an increased peak at 4,871 Da (P = 0.016 and 0.0083, respectively). ProteinChip array analysis identified differential protein peaks in primary breast cancers that predict the presence and number of ALN metastases and non-SLN status.

Biopsy↗