bluuebunny/crossref_metadata_2025_split huggingface dataset:
Strategies for the enrichment and identification of basic proteins in proteome projects
Authors: Bae Soo‐Han , Harris Andrew G. , Hains Peter G. , Chen Hong , Garfin David E. , Hazell Stuart L. , Paik Young‐Ki , Walsh Bradley J. , Cordwell Stuart J. 
Published: May 2003

AbstractTwo‐dimensional gel electrophoresis (2‐DE) is currently the method of choice for separating complex mixtures of proteins for visual comparison in proteome analysis. This technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins. Proteins with extremely alkaline isoelectric points (pI) are often very poorly represented using 2‐DE technology, even when complex mixtures are separated using commercially available pH 6–11 or pH 7–10 immobilized pH gradients. The genome of the human gut pathogen, Helicobacter pylori, is dominated by genes encoding basic proteins, and is therefore a useful model for examining methodology suitable for separating such proteins. H. pylori proteins were separated on pH 6–11 and novel pH 9–12 immobilized pH gradients and 65 protein spots were subjected to matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry, leading to the identification of 49 unique proteins. No proteins were characterized with a theoretical pI of greater than 10.23. A second approach to examine extremely alkaline proteins (pI > 9.0) utilized a prefractionation isoelectric focusing. Proteins were separated into two fractions using Gradiflow technology, and the extremely basic fraction subjected to both sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and liquid chromatography (LC) – tandem mass spectrometry post‐tryptic digest, allowing the identification of 17 and 13 proteins, respectively. Gradiflow separations were highly specific for proteins with pI > 9.0, however, a single LC separation only allowed the identification of peptides from highly abundant proteins. These methods and those encompassing multiple LC ‘dimensions’ may be a useful complement to 2‐DE for ‘near‐to‐total’ proteome coverage in the alkaline pH range.
Serum biomarkers of hepatitis B virus infected liver inflammation: A proteomic study
Authors: He Qing‐Yu , Lau George K. K. , Zhou Yuan , Yuen Siu‐Tsan , Lin Marie C. , Kung Hsiang‐Fu , Chiu Jen‐Fu 
Published: May 2003

AbstractHepatitis B virus (HBV), a serious infectious and widespread human pathogen, represents a major health problem worldwide. Chronic HBV infection has a very high risk of evolving into hepatocellular carcinoma. Although considerable progress was made during the recent past, the pathogenesis of HBV infection is still elusive and a definite diagnosis of HBV infected liver information still relies on biopsy histological test. In this report, we used proteomics technology to globally examine HBV infected serum samples aiming at searching for disease‐associated proteins that can be used as serological biomarkers for diagnosis and/or target proteins for pathogenetic study. By comparing with normal and HBV negative serum samples, we found that at least seven proteins were significantly changed in HBV infected sera. These greatly altered proteins were identified to be haptoglobin β and α2 chain, apolipoprotein A‐I and A‐IV, α1‐antitrypsin, transthyretin and DNA topoisomerase IIβ. The alteration of these proteins is displayed not only in quantity but also in patterns (or specificity), which can be correlated with necroinflammatory scores. In particular, apolipoprotein A‐I presents heterogeneous change in expression level with different isoforms and α1‐antitrypsin produces evidently different fragments implying diverse cleavage pathways. These unique phenomena appear specific to HBV infection. A combination simultaneously considering the quantities and isoforms of these proteins could be a useful serum biomarker (or index) for HBV diagnosis and therapy.
ProteomeWeb: A web‐based interface for the display and interrogation of proteomes
Authors: Babnigg György , Giometti Carol S. 
Published: May 2003

AbstractThe analysis of proteomes, i.e., the proteins expressed by biological organisms under a given set of conditions at a given time, requires separating complex protein mixtures into discrete protein components, measuring their relative abundances, and identifying the individual protein components. Many types of data are generated during the course of proteome analysis, including graphic images of the protein profiles, flat files containing numeric data, spreadsheets for assimilating numeric data, and relational database tables for integrating data from multiple experiments. As part of a project to describe the proteomes of microbes of interest to the U.S. Department of Energy, a World‐Wide Web‐based interface has been developed for the display of protein profiles generated by two‐dimensional gel electrophoresis. The web interface is capable of obtaining protein identifications on the fly, interrogating the quantitative data in the context of available genome sequence information, and relating the proteome data to existing metabolic pathway databases. Analysis of protein expression profiles is expedited, providing the capability to efficiently determine the gene locations for proteins modulated in abundance in response to different growth conditions and to locate the positions of the proteins within specific metabolic pathways. The proteome of the archaeon Methanococcus jannaschii, a microbe for which the complete genome sequence is available, is used to demonstrate the capabilities of this evolving web interface (http://proteomeweb.anl.gov).
Enrichment of Escherichia coli proteins by column chromatography on reactive dye columns
Authors: Birch Rosslyn M. , O'Byrne Conor , Booth Ian R. , Cash Phillip 
Published: May 2003

AbstractThe reliable identification and analysis of the low abundance proteins expressed by a cell remains a key challenge in the study of cellular proteomes. The analysis of low abundance proteins is a particular problem when using two‐dimensional gel electrophoresis (2‐DE) to resolve the cellular proteins since the technology is unable to display the wide dynamic range of protein levels typically synthesized by cells. We have investigated the use of reactive dye compounds for the enrichment of low abundance cellular proteins prior to analysis by 2‐DE. The capacity of reactive dye compounds to bind specific protein species was used as the basis for a general chromatographic tool for protein enrichment. Six reactive dye compounds were investigated in detail for the analysis of Escherichia coli proteins. Whole bacterial cell lysates were passed down columns prepared with the reactive dye compounds. The bound proteins were eluted with 1.5 M NaCl and analyzed by 2‐DE. Distinctive protein profiles were observed for the bound proteins recovered from the different reactive dye compounds. Selected proteins enriched by these methods were identified by peptide mass mapping. The enrichment procedure developed using reactive dye compounds were used to investigate acid‐induced changes in the proteome of E. coli grown at either pH 7.0 or pH 5.8. Increased levels of expression were observed for a number of proteins (for example, GdhA, PanC, ProC, TkrA, EF‐TS and YodA) were observed for E. coli grown at pH 5.8. Five identified proteins (AroG, FabI, GlyA, PurA and EF‐Tu) showed reduced levels of synthesis for bacteria grown at pH 5.8 compared to pH 7.0. In the case of PanC and FabI the altered expression profiles were only reliably demonstrated using the enrichment protocols. One theme emerging from these data was that the expression of proteins concerned with one‐carbon metabolism was perturbed at pH 5.8, which may point to a previously unrecognized affect of low pH stress on the physiology of E. coli cells. We conclude that the prefractionation of cell lysates on reactive dye columns will serve as a valuable generic tool for the analysis of low abundance proteins expressed by both prokaryotic and eukaryotic cells.
Navigated laser capture microdissection as an alternative to direct histological staining for proteomic analysis of brain samples
Authors: Moulédous Lionel , Hunt Sybille , Harcourt Rebecca , Harry Jenny , Williams Keith L. , Gutstein Howard B. 
Published: May 2003

AbstractProteomic analysis of the brain is complicated by the need to obtain cells from specific anatomical regions, or nuclei. Laser capture microdissection (LCM) is a technique that is precise enough to dissect single cells within a tissue section, and thus could be useful for isolating specific brain nuclei for analysis. However, we and others have previously demonstrated that histological staining protocols used to guide LCM have detrimental effects on protein separation by two‐dimensional electrophoresis (2‐DE). Here we describe a new LCM method called navigated LCM. This microdissection method uses fixed but unstained tissue as starting material and thus enables us to avoid artifacts induced by tissue staining. By comparing 2‐DE results obtained from fixed, unstained LCM brain tissue samples to those obtained from manually dissected samples, we demonstrated that this microdissection process gave similar protein recovery rates and similar resolution of protein spots on 2‐DE gels. Moreover, matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry analysis of selected spots from gels derived from control and fixed, LCM samples revealed that the fixation‐LCM process had no effect on protein identification. Navigated LCM of tissue sections is therefore a practical and powerful method for performing proteomic studies in specifically defined brain regions.
A strategy for the comparative analysis of serum proteomes for the discovery of biomarkers for hepatocellular carcinoma
Authors: Steel Laura F. , Shumpert Donna , Trotter Michael , Seeholzer Steven H. , Evans Alison A. , London W. Thomas , Dwek Raymond , Block Timothy M. 
Published: May 2003

AbstractMany of the emerging technologies for the global evaluation of gene expression, at both the RNA and protein level, are being applied to the problem of finding biomarkers for human disease progression. These analyses can be made difficult, however, by variation between samples that arises from both technical and nondisease related physiological or genetic causes. In an effort to identify serum polypeptides whose presence or absence correlates with the clinical status of patients at high risk for hepatocellular carcinoma (HCC), we have developed a strategy that helps to focus the analysis on meaningful changes in protein levels above the background of variation. For the current study we divided the patient population into four clinically defined diagnostic groups that represent a generally increasing risk for HCC. Chronic infection with hepatitis B virus (HBV) is a major risk factor for HCC and our groups included patients with no indication of liver disease (healthy), those with inactive chronic HBV, those with active chronic HBV, and patients with a diagnosis of HCC and history of chronic HBV infection. Serum polypeptides from these patients were first analyzed in two‐dimensional gels by combining the serum from patients in each of the four groups to generate composite gel profiles. Analysis of these composite gels allowed us to identify two relatively abundant features that were reduced in the HCC group as compared to the healthy group. Tryptic fragment mass fingerprinting identified the features as a carboxy terminal fragment of complement C3 and an isoform of apolipoprotein A1. These two features were examined by two‐dimensional gel electrophoresis of serum from each individual in the four groups in order to verify that the inter‐group differences seen in composite gels reported changes in abundance for most members of the group, rather than extreme changes for a small fraction of the group. These preliminary studies suggest that a proteomic methodology can be used for the identification of serum biomarkers for HCC and other liver disease.
Analysis of the Shewanella oneidensis proteome by two‐dimensional gel electrophoresis under nondenaturing conditions
Authors: Giometti Carol S. , Khare Tripti , Tollaksen Sandra L. , Tsapin Alexandre , Zhu Wenhong , Yates John R. , Nealson Kenneth H. 
Published: May 2003

AbstractProteomes are dynamic, i.e., the protein components of living cells change in response to various stimuli. Protein changes can involve shifts in the abundance of protein components, in the interactions of protein components, and in the activity of protein components. Two‐dimensional gel electrophoresis (2‐DE) coupled with peptide mass spectrometry is useful for the analysis of relative protein abundance, but the denaturing conditions of classical 2‐DE do not allow analysis of protein interactions or protein function. We have developed a nondenaturing 2‐DE method that allows analysis of protein interactions and protein functions, as demonstrated in our analysis of the cytosol and crude membrane fractions of the facultative anaerobe Shewanella oneidensis MR‐1. Our experiments demonstrate that enzymatic activity is retained under the sample and protein separation methods described, as shown by positive malate dehydrogenase activity results. We have also found protein interactions within both the soluble and membrane fractions. The method described will be useful for the characterization of the functional proteomes of microbial systems.
A reference map of a human pituitary adenoma proteome
Authors: Zhan Xianquan , Desiderio Dominic M. 
Published: May 2003

AbstractIn order to compare the proteomes from different cell types of pituitary adenomas for our long‐term goal to clarify the molecular mechanisms that participate in the formation of pituitary adenoma, and to detect any tumor‐related marker for an “early‐stage” diagnosis, the two‐dimensional gel electrophoresis (2‐DE) reference map of a pituitary adenoma tissue proteome is described here. A vertical, two‐dimensional (2‐D) polyacrylamide gel electrophoresis system and PDQuest image analysis software have been used to provide a high level of between‐gel reproducibility and to accurately array each protein expressed in a pituitary adenoma tissue. Mass spectrometry (matrix‐assisted laser desorption/ionization‐time of flight MALDI‐TOF and liquid chromatography‐electrospray ionization‐quadrupole‐ion trap LC‐ESI‐Q‐IT) and protein databases were used to characterize each protein in the 2‐D gel. The results demonstrate that a good reproducibility of the 2‐D gel pattern was attained. The position deviation of matched spots among four 2‐D gels was 1.95 ± 0.45 mm in the isoelectric focusing direction, and 1.70 ± 0.53 mm in the sodium dodecyl sulfate‐polyacrylamide gel electrophoresis direction. A total of ca. 1000 protein spots were separated by 2‐DE, and 135 protein spots that represent 111 proteins were characterized with mass spectrometry (96 spots for MALDI‐TOF, 39 spots for LC‐ESI‐Q‐IT). The characterized proteins include pituitary hormones, cellular signals, enzymes, cellular‐defense proteins, cell‐structure proteins, transport proteins, etc. Those proteins were located in the cytoplasmic, cellular membrane, mitochondrial, endoplasmic reticulum, nuclear, ribonucleosome, extracellular fractions, or were secreted in plasma, etc. Those identified proteins contribute to a functional profile of the pituitary adenoma proteome. These data will be used to expand the proteome database of the human pituitary, which can be accessed in the website http://www.utmem.edu /proteomics.
Proteomic study of human umbilical vein endothelial cells in culture
Authors: Bruneel Arnaud , Labas Valérie , Mailloux Agnès , Sharma Sanjiv , Vinh Joelle , Vaubourdolle Michel , Baudin Bruno 
Published: May 2003

AbstractThe endothelium is a single layer of cells lining the inside face of all blood vessels. It constitutes a major metabolic organ which is critically involved in the generation and the regulation of multiple physiological and pathological processes such as coagulation, hemostasis, inflammation, atherosclerosis, angiogenesis and cancerous metastasis dissemination. In order to increase our knowledge about the protein content and the main biological pathways of human vascular endothelial cells, we have undertaken the proteomic analysis of the most explored present endothelial cell model, i.e. primocultures of human umbilical vein endothelial cells (HUVECs). Using low levels of protein loads (&#126; 30 νg), the association of two‐dimensional electrophoresis with matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry, liquid chromatography‐tandem mass spectrometry and database interrogations allowed us to identify 53 proteins of suspected endothelial origin in quiescent HUVECs. Beside cytoskeletal proteins such as actin, tubulin, tropomyosin and vimentin, we identified various proteins more especially implicated in cellular motility and plasticity (e.g. cofilin, F‐actin capping protein and prefoldin), in regulation of apoptosis and senescence (protease inhibitor 9, glucose related proteins, heat shock proteins, thioredoxin peroxidase, nucleophosmin) as well as other proteins implicated in coagulation (annexin V, high mobility group protein), antigen presentation (valosin containing protein and ubiquitin carboxyl terminal hydrolase isozyme L1) and enzymatic capabilities (glutathione‐S‐transferase, protein disulfide isomerases, lactate deshydrogenase). The presented annotated 2‐D maps of HUVECs will be soon available on the web at http://www. huvec.com.
Proteome analysis of secreted proteins during osteoclast differentiation using two different methods: Two‐dimensional electrophoresis and isotope‐coded affinity tags analysis with two‐dimensional chromatography
Authors: Kubota Kazuishi , Wakabayashi Kenji , Matsuoka Tatsuji 
Published: May 2003

AbstractBone is maintained by two cell types, bone‐forming osteoblasts and bone‐resorbing osteoclasts. Osteoblasts express two factors, osteoprotegerin and receptor activator of NF‐κB ligand (RANKL), inhibiting and promoting osteoclast differentiation, respectively. In contrast, modulators of bone resorption expressed by osteoclasts have not been so well studied enough. In the present study, we demonstrate proteome analysis of secreted proteins during osteoclast differentiation to elucidate the molecular mechanism of bone resorption and bone remodeling. To achieve this objective, we chose RAW264.7 cells with RANKL as a homogeneous osteoclast differentiation model and used two methods, two‐dimensional gel electrophoresis (2‐DE) and isotope‐coded affinity tags (ICAT) analysis with two‐dimensional liquid chromatography. We found 23 spots in 2‐DE and 19 proteins in ICAT analysis which were expressed differently during osteoclast differentiation. These two methods gave us closely related but different information about proteins, suggesting they are complementary or at least supplementary methods at present. Cathepsins, osteopontin, legumain, macrophage inflammatory protein‐1α, and other proteins were observed as up‐ or down‐regulated proteins and are discussed in the context of osteoclast differentiation and bone resorption. In addition to confirming previous observations, this study indicates novel proteins related to osteoclast differentiation which are potential therapeutic targets for the treatment of bone diseases, such as osteoporosis.
Identification of metastasis‐associated proteins by proteomic analysis and functional exploration of interleukin‐18 in metastasis
Authors: Jiang Daifeng , Ying Wantao , Lu Yinglin , Wan Jinghong , Zhai Yun , Liu Wanli , Zhu Yunping , Qiu Zongyin , Qian Xiaohong , He Fuchu 
Published: May 2003

AbstractVery little is currently known about mechanisms underlying cancer metastasis. In the present study, metastasis‐associated proteomes were separated and identified by comparative proteomic analysis, and the metastasis‐related function of candidate protein interleukin‐18 (IL‐18) was further elucidated. First, a pair of highly and poorly metastatic sublines (termed PLA801D and PLA801C, respectively), originating from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach was used to compare the protein expression profiles between PLA801C and PLA801D sublines. Eleven proteins were identified and further verified by one‐dimensional Western blotting, Northern blot and/or semiquantitative reverse transciptase polymerase chain reaction analysis. Compared with those in poorly metastatic PLA801C subline, cytokeratin 18, tissue transglutaminase, Rho GDP‐dissociation inhibitor 1, tropomyosin, fibroblast type, IL‐18 and annexin I were significantly up‐regulated, while protein disulfide isomerase, heat shock protein 60, peroxiredoxin 1, chlorine intracellular channel protein 1 (CLI1) and creatine kinase, B chain were significantly down‐regulated in the highly metastatic PLA801D subline. Intriguingly, all the identified candidate proteins except for CLI1 have been shown to be somehow associated with distinct aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. Considering that IL‐18 was present in highly metastatic PLA801D but absent in poorly metastatic PLA801C, the association of IL‐18 with metastasis was further elucidated by introducing IL‐18 sense/IL‐18 antisense into PLA801C/PLA801D sublines simultaneously. The results demonstrated that ectopically expressed IL‐18 promoted cell motility in vitro and down‐regulated E‐cadherin expression of PLA801C transfectants, while IL‐18 antisense remarkably decreased cell invasion potency in vitro and notably increased E‐cadherin expression of PLA801D transfectants, indicating that IL‐18 might play a role in metastasis by inhibiting E‐cadherin expression.
Susceptibility to oxidative stress: proteomic analysis of bronchoalveolar lavage from ozone‐sensitive and ozone‐resistant strains of mice
Authors: Wattiez Ruddy , Noël‐Georis Isabelle , Cruyt Christine , Broeckaert Fabrice , Bernard Alfred , Falmagne Paul 
Published: May 2003

AbstractPrevious studies have shown that the pulmonary response to ozone (O3) varies greatly among strains of mice, but the factor(s) and the mechanism(s) that are responsible for this differential susceptibility have not yet been clearly identified. The present study explores the molecular bases for this differential O3 susceptibility by studying the expression of proteins associated to the epithelial lining fluid (ELF) from two strains of mice, C57BL/6J and the C3H/HeJ, respectively described as O3‐sensitive and O3‐resistant. The ELF proteins of these two strains were displayed by two‐dimensional gel electrophoresis (2‐DE) of bronchoalveolar lavage fluids (BALFs) and the protein patterns obtained with BALF samples of both strains were compared. Two major differences were observed between the BALF 2‐DE protein maps obtained from C57BL/6J and C3H/HeJ strains. First, two isoforms of the antioxidant protein 2 (AOP2) were detected in a strain‐dependent manner: C3J/HeJ possesses only AOP2a (isoelectric point 5.7) and C57BL/6J exhibits only AOP2b (isoelectric point 6.0). Second, the levels of anti‐inflammatory and immunosuppressive Clara cell protein‐16 (CC16) were 1.3 times higher in the BALF from resistant C3H/HeJ than from sensitive C57BL/6 mice. Moreover, two 6 kDa isoforms of CC16 with isoelectric points of 4.9 (CC16a) and 5.2 (CC16b) are detected in both strains. Interestingly, the C57BL/6J strain had a twice decreased level of the acidic isoform of CC16 compared to C3H/HeJ. Our results suggest that AOP2 and CC16 might participate in the protection of the pulmonary tract to O3‐induced lung injury. The possible differential contribution of specific protein isoforms in the differential susceptibility to oxidative stress is discussed.