Identification of proteins differentially expressed during chondrogenesis of mesenchymal cells

We performed comparative proteome analysis of mesenchymal cells and chondrocytes to identify proteins differentially expressed during chondrogenesis. Nine such proteins were identified. Type II collagen, matrilin-1, carbonic anhydrase-II (CA-II), 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase-2, and aldo-keto reductase were increased during chondrogenesis, whereas cellular retinoic acid binding protein-I (CRABP-I), CRABP-II, cytoplasmic type 5 actin, and fatty acid binding protein were decreased or almost disappeared. Expression of type II collagen, matrilin-1, PAPS synthetase-2, and CA-II was regulated by extracellular signal-regulated protein kinase, protein kinase C, and p38 kinase, signaling molecules known to regulate chondrogenesis.     

Identification of differentially expressed proteins in Ficedula flycatchers

This is the first report on the large-scale identification and comparison of proteins in non-model organisms, Ficedula flycatchers. It highlights the potential of proteomics approaches in both non-sequenced and non-model organisms for identification of differentially expressed proteins. Not surprisingly, more than 55% of the proteins failed to be identified even though the MS spectra were of high quality. Nevertheless, the protein information obtained in this study will serve as a valuable resource for continued research.     

Identification of differentially expressed proteins in curcumin-treated prostate cancer cell lines

Due to high prevalence and slow progression of prostate cancer, primary prevention appears to be attractive strategy for its eradication. During the last decade, curcumin (diferuloylmethane), a natural compound from the root of turmeric (Curcuma longa), was described as a potent chemopreventive agent. Curcumin exhibits anti-inflammatory, anticarcinogenic, antiproliferative, antiangiogenic, and antioxidant properties in various cancer cell models. This study was designed to identify proteins involved in the anticancer activity of curcumin in androgen-dependent (22Rv1) and -independent (PC-3) human prostate cancer cell lines using two-dimensional difference in gel electrophoresis (2D-DIGE). Out of 425 differentially expressed spots, we describe here the MALDI-TOF-MS analysis of 192 spots of interest, selected by their expression profile. This approach allowed the identification of 60 differentially expressed proteins (32 in 22Rv1 cells and 47 in PC-3 cells). Nineteen proteins are regulated in both cell lines. Further bioinformatic analysis shows that proteins modulated by curcumin are implicated in protein folding (such as heat-shock protein PPP2R1A; RNA splicing proteins RBM17, DDX39; cell death proteins HMGB1 and NPM1; proteins involved in androgen receptor signaling, NPM1 and FKBP4/FKBP52), and that this compound could have an impact on miR-141, miR-152, and miR-183 expression. Taken together, these data support the hypothesis that curcumin is an interesting chemopreventive agent as it modulates the expression of proteins that potentially contribute to prostate carcinogenesis.     

Identification of differentially expressed proteins during larval molting of Helicoverpa armigera

Insect molting involves many molecular processes, such as protein degradation and protein synthesis in the epidermis. Various proteins have been implicated in these processes. The differentially expressed proteins during larval molting of Helicoverpa armigera were investigated using two-dimensional electrophoresis (2-D-PAGE) and matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALTI-TOF-MS). Four larval tissues sampled during molting and feeding were examined. Seventy-seven differentially expressed proteins were identified in these tissues, including 20 proteins from the fifth-molting epidermis (fifth instar molting to sixth instar), 36 proteins from the fifth-molting hemolymph, and 21 from the fifth-molting fat bodies. No obviously different spots were identified from the fifth-molting midgut under these experimental conditions. After application of MALTI-TOF-MS and similarity analysis comparing results to a Drosophila protein database, 30 proteins were identified: 10 proteins from the fifth-molting epidermis, 11 proteins from the hemolymph, and 9 proteins from fat bodies. These proteins were separated into 5 groups according to their probable functions, such as enzymes, regulators, protein hydrolases, receptors, and proteins with unknown functions. These differentially expressed proteins were proposed to be involved in the Helicoverpa molting cascade.     

Identification of differentially expressed proteins using automated meta-analysis of proteomic articles

An automated method for searching for differentially expressed proteins (DEP) in proteomic articles has been developed and tested. Full-text proteomics-related articles were selected using an electronic version of the journal Proteomics and PubMedCentral. The list of proteins most frequently mentioned in the articles shares 86% identity with the list of human frequently identified DEPs published recently (Petrak et al., Proteomics (2008) 8, 1744). Regardless of the goal or design or methods identification of DEP delivers annexins and peroxiredoxins, as well as alpha-enolase, triosephosphate isomerase, and heat-shock protein HSP60. Among the most often mentioned proteins were also serum albumin, cathepsin D and vimentin. In regard to protein function the most often mentioned proteins were involved in inflammation and the immune response. According to GenRIF and UniProtKB annotations, most of these proteins are linked to the pathogenesis of tumor diseases, or diseases of the cardiovascular and nervous systems.     

Identification and characterisation of differentially expressed leaf proteins among Vitis species

Grapes are commercially grown worldwide for fresh fruit and wine. They are mainly classified into bunch and muscadine grapes. These species differ in their sugar content and composition, photosynthetic efficiency and tolerance to abiotic and biotic stresses. Grape berry relies on carbohydrates produced during photosynthesis to support its development and composition. In view of the unique physiology and genetic make‐up of muscadine grape, a proteomics study was performed to increase our knowledge of Vitis leaf proteome in order to improve enological and disease tolerance characteristics of grape species. A high throughput two‐dimensional gel electrophoresis (2‐DE) was conducted on muscadine, bunch and hybrid bunch leaf proteins. The differentially expressed proteins were excised from 2‐DE gels, subjected to in‐gel trypsin digestion, and analysed in MALDI/TOF mass spectrometer. The mass spectra were collected and protein identification was performed by searching against Viridiplantae database using Matrix Science algorithm. Proteins were mapped to universal protein resource to study gene ontology. We have discovered >255 proteins with pIs between 3.5 and 8.0 and molecular weight between 12 and 100 kDa among the Vitis species. Comparative analysis of leaf proteome showed that 54 polypeptides varied qualitatively and quantitatively among the three Vitis species studied. Of these, seven proteins were unique to muscadine, two proteins were present in both muscadine and bunch, while 28 proteins were common to all the three species. Bioinformatic analysis of these proteins showed that they are involved in signal transduction pathway, transport of metabolites, energy metabolism, protein trafficking, photosynthesis and defence. We have also identified proteins unique to muscadine grape that are involved in defence and stress tolerance. In addition, photosynthesis‐related proteins were found to be more abundant in Vitis vinifera grape compared to other Vitis species.     

Identification of proteins differentially expressed in gastric cancer cells with high metastatic potential for invasion to lymph nodes

In a search for proteins involved in cancer metastasis, we analyzed proteomes of the human gastric cancer cell OCUM-2M and its metastatic subline OCUM-2MLN. We observed that aspartate aminotransferase (AAT), D-site binding protein (DBP), and anterior gradient protein 2 (AGR2) are differentially expressed in metastatic OCUM-2MLN cells. Measurement of protein expression in clinical samples indicated that DBP and AAT are also down-regulated in metastatic adenocarcinoma. Additionally, urokinase-type tissue plasminogen activator is up-regulated in OCUM-2MLN cells and also in metastatic gastric cancer samples. Collectively, these results raise a possibility that AAT, DBP and AGR2 are functionally implicated in the invasiveness of gastric cancer cells.     

Identification of differentially expressed proteins in ruminal epithelium in response to a concentrate-supplemented diet

Ruminal epithelium adapts to dietary change with well-coordinated alterations in metabolism, proliferation, and permeability. To further understand the molecular events controlling diet effects, the aim of this study was to evaluate protein expression patterns of ruminal epithelium in response to various feeding regimes. Sheep were fed with a concentrate-supplemented diet for up to 6 wk. The control group received hay only. Proteome analysis with differential in gel electrophoresis technology revealed that, after 2 days, 60 proteins were significantly modulated in ruminal epithelium in a comparison between hay-fed and concentrate-fed sheep (P < 0.05). Forty proteins were upregulated and 20 proteins were downregulated in response to concentrate diet. After 6 wk of this diet, only 14 proteins were differentially expressed. Among these, 11 proteins were upregulated and 3 downregulated. To identify proteins that were modulated by dietary change, two-dimensional electrophoresis was coupled with liquid chromatography electrospray ionization mass spectrometry. The differential expression of selected proteins, such as esterase D, annexin 5, peroxiredoxin 6, carbonic anhydrase I, and actin-related protein 3, was verified by immunoblotting and/or mRNA analysis. The identified proteins were mainly associated with functions related to cellular stress, metabolism, and differentiation. These results suggest new candidate proteins that may contribute to a better understanding of the signaling pathways and mechanisms that mediate rumen epithelial adaptation to high-concentrate diet.     

Identification of differentially expressed proteins in colorectal cancer by proteomics: down-regulation of secretagogin

To identify proteins with colorectal cancer-specific regulation, comparative 2-DE of individual-matched normal and neoplastic colorectal tissue specimens was performed. We found 15 protein spots with concordantly increased and 20 protein spots with concordantly decreased intensity in tumor tissue (expression regulation more than fivefold). Nine of these proteins were identified by MS/MS. Interestingly, one of the proteins, which exhibited a marked down-regulation in colorectal cancer tissues, was the recently identified endocrine cell-expressed protein secretagogin. The reduction of the secretagogin content in colorectal cancer tissues was confirmed by comparative immunoblotting (n = 17) and RT-PCR (n = 22) as well as by immunohistochemistry (n = 45) of individual-matched neoplastic and normal colorectal tissue specimens. Immunohistochemistry revealed absence of secretagogin-expressing cells in most of the colorectal cancer tissue specimens. However, some colorectal cancers were characterized by secretagogin-expressing cells. In normal mucosa, positively stained cells exhibited a neuroendocrine cell-characteristic morphology and mucosal location. In colorectal cancer tissues, secretagogin-expressing cells were characterized by a malignant morphology. Our findings might represent the basis for the clinical application of secretagogin as a biomarker for a distinct subgroup of colorectal cancers.     

Properties of purified folate-binding proteins from chronic myelogenous leukemia cells

Folate-binding protein(s) from chronic myelogenous leukemia cells have been purified using acid dialysis, ammonium sulfate fractionation and affinity chromatography. The purified preparation which migrates as a single band on disc electrophoresis could be separated by DEAE agarose chromatography into two folate-binding proteins (binders I and II) which bind molar equivalents of folic acid. One binder (I) eluted from DEAE at 1 mM sodium phosphate, pH 6.0, and the other (II) at 100 mM sodium phosphate, pH 7.4. Analysis of the purified mixture, which contained more than 90% binder II, by sedimentation equilibrium centrifugation indicated a homogeneous protein with a calculated molecular weight of 44000. Antiserum raised against the purified mixture gave a single precipitin line by immunodiffusion against a preparation of partially purified cell lysate.Hydrolysis of the more acidic binder (II) with neuraminidase converted it to a weakly acidic protein similar to binder I suggesting that these binders are glycoproteins which differ in sialic acid content. With isoelectric focusing, the binding of folic acid would be demonstrated at pH 6.7, 7.3, 7.8 and 8.2 for binder I, and at pH 5.1, 5.8 and 6.5 for binder II. Binders I and II had equally high affinity for folic acid and dihydroflate, lower affinity of N⁵-methyl-tetrahydrofolate, and no apparent affinity for N⁵-formytetrahydrofolate or methotrexate.     

Comparative proteomic analysis of chronic myelogenous leukemia cells: inside the mechanism of imatinib resistance

Imatinib is the first molecular targeted therapy that has shown clinical success, but imatinib acquired resistance, although a rare event, is critical during the therapy of chronic myelogenous leukaemia (CML). With the aim of better understanding the molecular mechanisms accompanying acquisition of resistance to this drug, a comparative proteomic approach was undertaken on CML cell lines LAMA 84 S (imatinib sensitive) and LAMA 84 R (imatinib resistant). Forty-four differentially expressed proteins were identified and categorized into five main functional classes: (I) heat shock proteins and chaperones; (II) nucleic acid interacting proteins (binding/synthesis/stability); (III) structural proteins, (IV) cell signaling, and (V) metabolic enzymes. Several heat shock proteins known to complex Bcr-Abl were overexpressed in imatinib resistant cells, showing a possible involvement of these proteins in the mechanism of resistance. HnRNPs also resulted in being up-regulated in imatinib resistant cells. These proteins have been shown to be strongly and directly related to Bcr-Abl activity. To our knowledge, this is the first direct proteomic comparison of imatinib sensitive/resistant CML cell lines.     

Identification of Streptomyces coelicolor proteins that are differentially expressed in the presence of plant material

Streptomyces coelicolor and Lemna minor were used as a model to study the modulation of bacterial gene expression during plant-streptomycete interactions. S. coelicolor was grown in minimal medium with and without L. minor fronds. Bacterial proteomes were analyzed by two-dimensional gel electrophoresis, and a comparison of the two culture conditions resulted in identification of 31 proteins that were induced or repressed by the presence of plant material. One-half of these proteins were identified by peptide mass fingerprinting by using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The induced proteins were involved in energetic metabolism (glycolysis, pentose phosphate pathway, oxidative phosphorylation), protein synthesis, degradation of amino acids, alkenes, or cellulose, tellurite resistance, and growth under general physiological or oxidative stress conditions. The repressed proteins were proteins synthesized under starvation stress conditions. These results suggest that root exudates provide additional carbon sources to the bacteria and that physiological adaptations are required for efficient bacterial growth in the presence of plants.     

Identification of differentially expressed proteins in the cervical mucosa of HIV-1-resistant sex workers

Novel tools are necessary to understand mechanisms of altered susceptibility to HIV-1 infection in women of the Pumwani Sex Worker cohort, Kenya. In this cohort, more than 140 of the 2000 participants have been characterized to be relatively resistant to HIV-1 infection. Given that sexual transmission of HIV-1 occurs through mucosal surfaces such as that in the cervicovaginal environment, our hypothesis is that innate immune factors in the genital tract may play a role in HIV-1 infection resistance. Understanding this mechanism may help develop microbicides and/or vaccines against HIV-1. A quantitative proteomics technique (2D-DIGE: two-dimensional difference in-gel electrophoresis) was used to examine cervical mucosa of HIV-1 resistant women ( n = 10) for biomarkers of HIV-1 resistance. Over 15 proteins were found to be differentially expressed between HIV-1-resistant women and control groups ( n = 29), some which show a greater than 8-fold change. HIV-1-resistant women overexpressed several antiproteases, including those from the serpin B family, and also cystatin A, a known anti-HIV-1 factor. Immunoblotting for a selection of the identified proteins confirmed the DIGE volume differences. Validation of these results on a larger sample of individuals will provide further evidence these biomarkers are associated with HIV-1 resistance and could help aid in the development of effective microbicides against HIV-1.     

Identification of differentially expressed proteins from Burkholderia pseudomallei isolated during primary and relapsing melioidosis

Burkholderia pseudomallei causes septicemic melioidosis with a high rate of relapse, however microbial determinants of relapse are unknown. Proteins were analyzed from sequential B. pseudomallei isolates from primary and relapsing melioidosis. Analysis by isotope tagging for relative and absolute quantitation revealed that factors required for nitric oxide detoxification (HmpA) and necessary for anaerobic growth (ArcA, ArcC and ArcB) were highly expressed in the relapse isolate. Two-dimensional gel electrophoresis revealed up-regulation of a putative hemolysin-coregulated protein in the primary isolate, and flagellin and HSP20/alpha crystalline in the relapse isolate. These observations provide targets for further analysis of latency and virulence of melioidosis.     

Properties of purified folate-binding proteins from chronic myelogenous leukemia cells.

Folate-binding protein(s) from chronic myelogenous leukemia cells have been purified using acid dialysis, ammonium sulfate fractionation and affinity chromatography. The purified preparation which migrates as a single band on disc electrophoresis could be separated by DEAE agarose chromatography into two folate-binding proteins (binders I and II) which bind molar equivalents of folic acid. One binder (I) eluted from DEAE at 1 mM sodium phosphate, pH 6.0, and the other (II) at 100 mM sodium phosphate, pH 7.4. Analysis of the purified mixture, which contained more than 90% binder II, by sedimentation equilibrium centrifugation indicated a homogeneous protein with a calculated molecular weight of 44000. Antiserum raised against the purified mixture gave a single precipitin line by immunodiffusion against a preparation of partially purified cell lysate. Hydrolysis of the more acidic binder (II) with neuraminidase converted it to a weakly acidic protein similar to binder I, suggesting that these binders are glycoproteins which differ in sialic acid content. With isoelectric focusing, the binding of folic acid could be demonstrated at pH 6.7, 7.3, 7.8 and 8.2 for binder I, and at pH 5.1, 5.8, and 6.5 for binder II. Binders I and II had equally high affinity for folic acid and dihydrofolate, lower affinity for N5-methyl-tetrahydrofolate, and no apparent affinity for N5-formyltetrahydrofolate or methotrexate.     

Inhibition of polysome assembly enhances imatinib activity against chronic myelogenous leukemia and overcomes imatinib resistance

Dysregulated mRNA translation is implicated in the pathogenesis of many human cancers including chronic myelogenous leukemia (CML). Because our prior work has specifically implicated translation initiation in CML, we tested compounds that could modulate translation initiation and polysomal mRNA assembly. Here, we evaluated the activity of one such compound, CGP57380, against CML cells and explored its mechanisms of action. First, using polysomal mRNA profiles, we found that imatinib and CGP57380 could independently, and cooperatively, impair polysomal mRNA loading. Imatinib and CGP57380 also synergistically inhibited the growth of Ba/F3-Bcr-Abl and K562 cells via impaired cell cycle entry and increased apoptosis. Mechanistically, CGP57380 inhibited efficient polysomal assembly via two processes. First, it enhanced imatinib-mediated inhibition of eukaryotic initiation factor 4F induction, and second, it independently impaired phosphorylation of ribosomal protein S6 on the preinitiation complex. We also identified multiple substrates of the mTOR, Rsk, and Mnk kinases as targets of CGP57380. Finally, we found a novel negative-feedback loop to the mitogen-activated protein kinase/Mnk pathway that is triggered by CGP57380 and demonstrated that an interruption of the loop further increased the activity of the combination against imatinib-sensitive and -resistant CML cells. Together, this work supports the inhibition of translation initiation as a therapeutic strategy for treating cancers fueled by dysregulated translation.     

Accentuation of differentially expressed proteins using phage technology

Protein profiling is frequently used to elucidate disease-specific or differentially expressed proteins. While recent developments have resulted in improved differential profiling, alternative expression platforms that complement existing techniques are continually being explored. We developed a novel method utilizing the amplification and selection capabilities of random peptide-expressing M13 bacteriophage to accentuate differentially expressed proteins in biologic specimens. While the current study used this method to demonstrate differentially expressed proteins in lung cancer tissue in comparison to normal lung tissue, this approach is applicable to a wide range of sample types.