Proteomic analysis of nasal cells from cystic fibrosis patients and non-cystic fibrosis control individuals: search for novel biomarkers of cystic fibrosis lung disease

Potential biological markers for cystic fibrosis (CF) lung disease were identified by comparative proteomics profiling of nasal cells from deletion of phenylalanine residue 508 (F508del)-homozygous CF patients and non-CF controls. From the non-CF 2-DE gels, 65 spots were identified by MS, and a reference 2-DE map was thus established. The majority of those correspond to ubiquitously expressed proteins. Consistent with the epithelial origin of this tissue, some of the identified proteins are epithelial markers (e.g. cytokeratins, palate lung and nasal epithelium clone protein (PLUNC), and squamous cell carcinoma antigen 1). Comparison of this protein profile with the one similarly obtained for CF nasal cells revealed a set of differentially expressed proteins. These included proteins related to chronic inflammation and some others involved in oxidative stress injury. Alterations were also observed in the levels of cytoskeleton proteins, being probably implicated with cytoskeleton organization changes described to occur in CF-airways. Lower levels were found for some mitochondrial proteins suggesting an altered mitochondrial metabolism in CF. Differential expression was also found for two more enzymes that have not been previously associated to CF. Further studies will clarify the involvement of such proteins in CF pathophysiology and whether they are targets for CF therapy.     

Pharmacoproteomics of 4-phenylbutyrate-treated IB3-1 cystic fibrosis bronchial epithelial cells

4-Phenylbutyrate (4-PBA) is an oral butyrate derivative that has recently been approved for treatment of urea cycle disorders and is under investigation in clinical trials of cancer, hemoglobinopathies, and cystic fibrosis (CF). We hypothesized that proteome profiling of IB3-1 cystic fibrosis bronchial epithelial cells treated with 4-PBA would identify butyrate-responsive cellular chaperones, protein processing enzymes, and cell trafficking molecules associated with the amelioration of the chloride transport defect in these cells. Protein profiles were analyzed by two-dimensional gel electrophoresis and mass spectrometry. Over a pI range of 4-7 and molecular weight from 20 to 150 kDa a total of 85 differentially expressed proteins were detected. Most of the identified proteins were chaperones, catalytic enzymes, and proteins comprising structural elements, cellular defense, protein biosynthesis, trafficking activity, and ion transport. Subsets of these proteins were confirmed by immunoblot analysis. These data represent a first-draft of the pharmacoproteomics map of 4-PBA treated cystic fibrosis bronchial epithelial cells.     

The first two-dimensional reference map of the fission yeast, Schizosaccharomyces pombe proteins

Cytosolic proteins of Schizosaccharomyces pombe were separated by two-dimensional (2-D) gel electrophoresis, to construct the first 2-D reference map. In the pI range 4-7, more than 500 spots were detected by silver staining, and 70 different proteins corresponding to 111 spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and tandem mass spectrometry, where necessary. In the pI range 6-9, approximately 330 spots were detected, and 31 proteins corresponding to 38 spots were identified by mass spectrometry. More than 50% of the identified proteins were involved in amino acid, carbohydrate or nucleotide metabolism, and energy production. A second large group of identified proteins comprises heat shock and other stress related proteins and chaperones.     

Proteome analysis of Schizosaccharomyces pombe by two-dimensional gel electrophoresis and mass spectrometry

The fission yeast Schizosaccharomyces pombe (S. pombe) is a unicellular eukaryote and contains many genes and regulatory mechanisms that are close to those of mammals. In this study, we performed a global proteomic analysis of the fission yeast S. pombe wild type h(-S) L 972 proteome. More than 1,500 protein spots were visualized on silver stained 2-D gels in the 3-10 pI range with a high resolution and high reproducibility. Protein identification was carried out by MALDI-TOF-MS and/or nanoLC-MS/MS. Advantage of the complementarity of these two MS approaches was used to enhance the identification quality. So far, 364 proteins (representing 157 different proteins) have been identified. We report here the identification of 117 new proteins on our 2-D reference map of this yeast compared to the first reference map. Of these identified proteins, 40.1% were involved in metabolism. The present work provides a very useful tool for all studies relying on S. pombe as a model organism and is a considerable complement to the first reference map of S. pombe published recently by Sun and coworkers (Sun, N., Jang, J., Lee, S., Kim, S. et al.., Proteomics 2005, 5, 1574-1579).     

The 46,000-dalton tyrosine protein kinase substrate is widespread, whereas the 36,000-dalton substrate is only expressed at high levels in certain rodent tissues

Proteins of molecular mass 46,000 (p46) and 34,000-39,000 (p36) daltons are phosphorylated at tyrosine in Rous sarcoma virus-transformed chicken and mouse fibroblasts. p46 has recently been identified as an isozyme of enolase but the function of p36 is unknown. The expression of these proteins in various mouse and rat tissues has been examined. In most tissues, except muscle, p46 is found at relatively constant levels. In muscle, a more basic, related protein is present. In contrast, the abundance of p36 varies more widely from tissue to tissue, suggesting that it has a function in some but not all differentiated cells. By SDS gel electrophoresis and immunoblotting, high levels of p36 (60-120% of its relative abundance in fibroblasts) were found in small intestine, lung, and thymus, and intermediate levels (20-50%) were found in spleen, lymph nodes, and testes. No p36 was detectable in brain and muscle. Where studied, p36 mRNA expression paralleled protein levels. The cell types within each tissue expressing p36 were identified by immunofluorescence and immunoperoxidase staining. These cell types include all endothelial cells and fibroblastic cells examined, as well as various epithelial cells, cardiac muscle cells, macrophages, and testicular interstitial cells. We were unable to detect p36 in skeletal or smooth muscle cells, erythrocytes, nerve cells, or lymphocytes in any of the examined tissues. p36 appears to be concentrated in the terminal web region of intestinal columnar epithelial cells.     

Proteome analysis on an early transformed human bronchial epithelial cell line,BEP2D,after alpha-particle irradiation

To probe the mechanism of carcinogenesis of lung cancer at the molecular level and to find potential protein markers involved in the early phase of tumorgenesis, differential proteome analysis on primary passage cell line R15H, and early transformed cell line R15H20 derived from (238)Pu alpha-particle irradiation of human papillomavirus (HPV) 18-immortalized human bronchial epithelial cell line (BEP2D), was carried out using two-dimensional electrophoresis (2-DE) and peptide mass fingerprinting (PMF) with matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. Image analysis and Student's t-test (p < 0.05) showed that three protein spots were only expressed in R15H, intensities of 43 protein spots on the gels were altered between R15H and R15H20. Two of the three spots that were only expressed in R15H were identified as high mobility group protein 1. Two proteins decreased in abundance in R15H20 were identified as maspin precursor, a tumor suppressor and aminoacylase-1. Ornithine aminotransferase and peptidyl-prolyl cis-trans isomerase A that were increased in R15H20, were also identified. Relationships between these differentially expressed proteins and the carcinogenesis mechanism of lung cancer are discussed. The protein expression profile of the R15H cell line was also constructed during the study as a reference map for further comparative proteome analysis of the irradiation induced BEP2D cell line. Of the 90 spots analyzed with PMF in the 2-DE gel of R15H cell line, 50 proteins were identified by searching the nonredundant protein database SWISS-PROT/TrEMBL.     

Proteome analysis of Nelore bull (Bos taurus indicus) seminal plasma

The Nelore bull (Bos taurus indicus) seminal plasma proteome was analyzed by MALDI-TOF MS and two-dimensional gel electrophoresis. A total of 260 spots were visualized in the 2-DE gel (pI range 3-10) and 13 spots could be identified by peptide mass fingerprinting corresponding to 11 different polypeptides. The results allowed the creation of the first proteomic map of Bos taurus indicus seminal plasma. The roles of the identified proteins in the bull seminal plasma are discussed.     

Strategic shotgun proteomics approach for efficient construction of an expression map of targeted protein families in hepatoma cell lines

An expression map of the most abundant proteins in human hepatoma HepG2 cells was established by a combination of complementary shotgun proteomics approaches. Two-dimensional liquid chromatography (LC)-nano electrospray ionization (ESI) tandem mass spectrometry (MS/MS) as well as one-dimensional LC-matrix-assisted laser desorption/ionization MS/MS were evaluated and shown that additional separation introduced at the peptide level was not as efficient as simple prefractionation of protein extracts in extending the range and total number of proteins identified. Direct LC-nanoESI MS/MS analyses of peptides from total solubilized fraction and the excised gel bands from one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionated insolubilized fraction afforded the best combination in efficient construction of a nonredundant cell map. Compiling data from multiple variations of rapid shotgun proteomics analyses is nonetheless useful to increase sequence coverage and confidence of hits especially for those proteins identified primarily by a single or two peptide matches. While the returned hit score in general reflects the abundance of the respective proteins, it is not a reliable index for differential expression. Using another closely related hepatoma Hep3B as a comparative basis, 16 proteins with more than two-fold difference in expression level as defined by spot intensity in two-dimensional gel electrophoresis analysis were identified which notably include members of the heat shock protein (Hsp) and heterogeneous nuclear ribonucleoprotein (hnRPN) families. The observed higher expression level of hnRNP A2/B1 and Hsp90 in Hep3B led to a search for reported functional roles mediated in concert by both these multifunctional cellular chaperones. In agreement with the proposed model for telomerase and telomere bound proteins in promoting their interactions, data was obtained which demonstrated that the expression proteomics data could be correlated with longer telomeric length in tumorigenic Hep3B. This biological significance constitutes the basis for further delineation of the dynamic interactions and modifications of the two protein families and demonstrated how proteomic and biological investigation could be mutually substantiated in a productive cycle of hypothesis and pattern driven research.     

家蚕脂肪体蛋白质组学研究

通过高精度的双向电泳技术对家蚕末龄幼虫的脂肪体组织进行了研究,采用基质辅助质量飞行时间质谱对其中一些表达量较高的蛋白点进行了鉴定,并利用GPMAW软件结合家蚕基因组预测的蛋白质数据库构建了本地的肽质量指纹图谱数据库,对所得到的肽质量指纹图谱进行了分析。研究发现,经过双向凝胶电泳及其图象分析技术,银染可以分离出722个清晰蛋白点,这些蛋白质主要集中在分子量15～90kD区域,等电点pH4～8之间。MALDI-TOF-MS鉴定的41个蛋白点中都有较强的肽质量指纹信号峰,其中34个蛋白点得到了成功鉴定,其中包括了大量参与代谢的酶类、不同分子量的热激蛋白、重要的血液蛋白30K,Actin3等,这一结果对人们进一步认识家蚕脂肪体提供了有利的帮助。     

Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins

A proteomic approach was used for the identification of larval hemolymph proteins of Drosophila melanogaster. We report the initial establishment of a two-dimensional gel electrophoresis reference map for hemolymph proteins of third instar larvae of D. melanogaster. We used immobilized pH gradients of pH 4-7 (linear) and a 12-14% linear gradient polyacrylamide gel. The protein spots were silver-stained and analyzed by nanoLC-Q-Tof MS/MS (on-line nanoscale liquid chromatography quadrupole time of flight tandem mass spectrometry) or by Matrix assisted laser desorption time of flight MS (MALDI-TOF MS). Querying the SWISSPROT database with the mass spectrometric data yielded the identity of the proteins in the spots. The presented proteome map lists those protein spots identified to date. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level in different physiological conditions.     

A two-dimensional proteome map of maize endosperm

We have established a proteome reference map for maize (Zea mays L.) endosperm by means of two-dimensional gel electrophoresis and protein identification with LC-MS/MS analysis. This investigation focussed on proteins in major spots in a 4-7 pI range and 10-100 kDa M(r) range. Among the 632 protein spots processed, 496 were identified by matching against the NCBInr and ZMtuc-tus databases (using the SEQUEST software). Forty-two per cent of the proteins were identified against maize sequences, 23% against rice sequences and 21% against Arabidopsis sequences. Identified proteins were not only cytoplasmic but also nuclear, mitochondrial or amyloplastic. Metabolic processes, protein destination, protein synthesis, cell rescue, defense, cell death and ageing are the most abundant functional categories, comprising almost half of the 632 proteins analyzed in our study. This proteome map constitutes a powerful tool for physiological studies and is the first step for investigating the maize endosperm development.     

Proteomic analysis of proteins from bronchoalveolar lavage fluid reveals the action mechanism of ultrafine carbon black-induced lung injury in mice

Previous studies have shown that ultrafine carbon black (ufCB) could cause oxidative stress and lung injury, but the mechanisms have not been clearly demonstrated. In this study, 1-D gel electrophoresis coupled with LC/MS/MS (1-D geLC/MS/MS) was carried out with bronchoalveolar lavage fluid (BALF) to identify proteins associated with ufCB-induced lung injury. If required, Western blot was conducted additionally to validate proteins. Thirty-three proteins were identified, including leukemia inhibitory factor receptor (LIFR) and epidermal growth factor receptor (EGFR). Western blot analysis showed that ufCB exposure caused the increases of LIFR and EGFR in BALF and decreases of both receptors in lung tissues, suggesting the acceleration of epithelial shedding from the lung and increase of cell debris with membrane proteins EGFR and LIFR in BALF. There were strong correlations between vascular endothelial growth factor (VEGF) and albumin (p<0.01) or alpha2-macroglobulin (alpha2M) in BALF (p<0.05). Importantly, antioxidant ceruloplasmin (Cp) was shown to be produced from lung epithelial cells in response to ufCB exposure. This is the first study to apply 1-D ge LC/MS/MS and experimental studies to reveal the mechanisms involved in the pathogenesis of ufCB-induced lung injury.     

Proteome profiling of human epithelial ovarian cancer cell line TOV-112D

A proteome profiling of the epithelial ovarian cancer cell line TOV-112D was initiated as a protein expression reference in the study of ovarian cancer. Two complementary proteomic approaches were used in order to maximise protein identification: two-dimensional gel electrophoresis (2DE) protein separation coupled to matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and one-dimensional gel electrophoresis (1DE) coupled to liquid-chromatography tandem mass spectrometry (LC MS/MS). One hundred and seventy-two proteins have been identified among 288 spots selected on two-dimensional gels and a total of 579 proteins were identified with the 1DE LC MS/MS approach. This proteome profiling covers a wide range of protein expression and identifies several proteins known for their oncogenic properties. Bioinformatics tools were used to mine databases in order to determine whether the identified proteins have previously been implicated in pathways associated with carcinogenesis or cell proliferation. Indeed, several of the proteins have been reported to be specific ovarian cancer markers while others are common to many tumorigenic tissues or proliferating cells. The diversity of proteins found and their association with known oncogenic pathways validate this proteomic approach. The proteome 2D map of the TOV-112D cell line will provide a valuable resource in studies on differential protein expression of human ovarian carcinomas while the 1DE LC MS/MS approach gives a picture of the actual protein profile of the TOV-112D cell line. This work represents one of the most complete ovarian protein expression analysis reports to date and the first comparative study of gene expression profiling and proteomic patterns in ovarian cancer.     

Proteomic profiling of endothelial cells in human lung cancer

Genomic and proteomic analysis of normal and diseased tissues have yielded an abundance of molecular information for diagnostic and potential therapeutic targets. Changing the target of analysis from poorly accessible cells within tissues to easily accessible vascular endothelium has theoretical advantages in tissue-specific targeting. In this study, we sought to map a large-scale proteome of microvascular endothelium in human non-small cell lung cancer (NSCLC) and normal lung tissues, and identify lung cancer-related endothelial cell (EC)-selective proteins. Endothelial cells were isolated within NSCLC tissues and adjacent-normal lung tissue of lung cancer patients by using CD31-immunomagnetic beads. The complex proteins from the ECs were separated by one-dimensional gel electrophoresis, and the proteins in each gel band were digested by trypsin. Peptides were separated by online reverse-phase liquid-chromatography and analyzed by electrospray ionization (ESI) ion trap tandem mass spectrometry. Approximately 600-1000 proteins were identified in each individual sample. Five patient cases of paired individual data, extracted from the protein identification data sets of both normal- and cancer-derived ECs, were analyzed by subtractive proteomics. An average of 300 proteins was specifically identified from each lung cancer-derived EC isolate, compared to normal lung-derived ECs. With the use of several comparative analyses, we identified among those 300 proteins, 16 common candidate proteins that were detected in at least 3 of 5 cases specific to lung cancer-derived ECs. Proteins selectively identified in cancer-derived ECs, including coatomer protein complex, subunit gamma (COPG), and peroxiredoxin 4 (PRDX4), were validated by Western blot analysis. In an additional experiment in which 16 cancer samples were analyzed by immunohistochemistry, PRDX4, thymopoietin (TMPO), and COPG were confirmed to be abundantly expressed in lung cancer-derived ECs and in cancerous lung cells. Further ongoing analysis of these 16 candidate proteins will determine their potential applicability to NSCLC-specific diagnosis and therapeutics.     

Proteomic Analysis of Cd-Responsive Proteins in Solanum torvum

Proteomic changes induced by Cd have been described in plants in different scenarios. However, there has been no proteomic study on Cd toxicity, including any low Cd-accumulating species. Here, we investigate the response of a low Cd-accumulating species, Solanum torvum, to Cd toxicity at the root proteomic level using two-dimensional gel electrophoresis (2-DGE). The root 2-DGE map consisted of at least 927 reproducible protein spots, of which 45 were classified as differentially expressed proteins based on three replicated separations. MALDI-TOF MS analysis identified 19 of these spots, and MALDI-TOF/TOF MS analysis identified 8 of the spots. The eight proteins identified were two S-adenosylmethionine (SAM) synthetases, actin, an ATP synthase subunit, two tubulin proteins, alcohol dehydrogenase (ADH), and 14-3-3 protein 4. These proteins are involved in phytohormone synthesis, defense responses, energy metabolism, and cytoskeleton construction. Thus, our proteomic analysis revealed that Cd stress promotes an increase in the abundance of proteins involved in antioxidant defenses and anti-stress protection.