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K Ou

Publications and source records attributed to K Ou.

At least 19 recordsLinked to original sources
PubMed2001

An integrated approach in the discovery and characterization of a novel nuclear protein over-expressed in liver and pancreatic tumors.

An integrated approach in protein discovery through the use of multidisciplinary tools was reported. A novel protein, Hcc-1, was identified by analysis of the hepatocellular carcinoma (HCC)-M cell proteome. The assembled EST sequence of the 210 amino acid novel protein was subsequently confirmed by rapid amplification of cDNA ends (RACE). A total of 687 bp at the 5' untranslated region of Hcc-1 was identified. Promoter activity and several upstream open reading frames (uORFs) were demonstrated at this region. Bioinformatics prediction showed that the first 42 amino acids of the protein is a SAP domain with sequence matches to hnRNP from various vertebrate species. The Hcc-1 protein was localized to the cell nucleus while the gene was localized to chromosome 7q22.1. Hcc-1 cDNA level was increased in pancreatic adenocarcinoma. The level was also increased in well-differentiated hepatocellular carcinoma but decreases as the carcinoma progressed to a poorly differentiated stage.

5' Untranslated Regions↗
PubMed2001

Proteome analysis of a human heptocellular carcinoma cell line, HCC-M: an update.

Recently, we reported the proteome analysis of a human hepatocellular carcinoma cell line, HCC-M (Electrophoresis 2000, 21, 1787-1813), using two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). From a total of 408 unique spots excised from the 2-DE gel, 301 spots yielded good MALDI spectra. Out of these, 272 spots had matches returned from the database search leading to the identification of these proteins. Here, we report the results on the identification of the remaining 29 spots using nanoelectrospray ionization-tandem mass spectrometry (nESI-MS/MS). First, "peptide tag sequencing" was performed to obtain partial amino acid sequences of the peptides to search the SWISS-PROTand NCBI nonredundant protein databases. Spots that were still not able to find any matches from the databases were subjected to de novo peptide sequencing. The tryptic peptide sequences were used to search for homologues in the protein and nucleotide databases with the NCBI Basic Local Alignment Search Tool (BLAST), which was essential for the characterization of novel or post-translationally modified proteins. Using this approach, all the 29 spots were unambiguously identified. Among them, phosphotyrosyl phosphatase activator (PTPA), RNA-binding protein regulatory subunit, replication protein A 32 kDa subunit (RP-A) and N-acetylneuraminic acid phosphate synthase were reported to be cancer-related proteins.

Carcinoma, Hepatocellular↗
PubMed2001

Identification of a serine protease inhibitor homologue in Bird's Nest by an integrated proteomics approach.

For centuries, the edible nests of Collocalia spp. ("Bird's Nests") have been used as a Chinese delicacy that had been claimed to be an effective health-giving tonic. However, clinical studies indicated that in Singapore, Bird's Nest is the most common cause of food-induced anaphylaxis in children, which could lead to potentially life-threatening allergenic reactions. The purpose of this study was to characterize the major allergens in Bird's Nest by using the combined technologies of two-dimensional gel electrophoresis (2-DE), immunochemistry, N-terminal protein sequencing, and mass spectrometry. Results from the immunostaining of the Western blots of the Bird's Nest 2-DE separated proteins with the sera from allergic patients indicated the presence of a major allergen of 66 kDa. Initial searches of the matrix assisted laser desorption/ionization--time of flight--mass spectrometry (MALDI-TOF-MS) tryptic peptide masses of the allergen in the SWISS-PROT and NCBI nonredundant databases revealed that this protein was novel. Based on the partial protein sequence information obtained from N-terminal microsequencing and nanoelectrospray-tandem MS, the 66 kDa immunoreactive allergen was found to be homologous to ovoinhibitor, a Kazal-type serine protease inhibitor, which is one of the dominant allergens found in chicken egg white.

Allergens↗
PubMed2000

Two-dimensional electrophoresis map of the human hepatocellular carcinoma cell line, HCC-M, and identification of the separated proteins by mass spectrometry.

Currently, one of the most popular applications of proteomics is in the area of cancer research. In Africa, Southeast Asia, and China, hepatocellular carcinoma is one of the most common cancers, occurring as one of the top five cancers in frequency. This project was initiated with the purpose of separating and identifying the proteins of a human hepatocellular carcinoma cell line, HCC-M. After two-dimensional gel electrophoresis separation, silver staining, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses, tryptic peptide masses were searched for matches in the SWISS-PROT and NCBI nonredundant databases. Approximately 400 spots were analyzed using this approach. Among the proteins identified were housekeeping proteins such as alcohol dehydrogenase, alpha-enolase, asparagine synthetase, isocitrate dehydrogenase, and glucose-6-phosphate 1-dehydrogenase. In addition, we also identified proteins with expression patterns that have been postulated to be related to the process of carcinogenesis. These include 14-3-3 protein, annexin, prohibitin, and thioredoxin peroxidase. This study of the HCC-M proteome, coupled with similar proteome analyses of normal liver tissues, tumors, and other hepatocellular carcinoma cell lines, represents the first step towards the establishment of protein databases, which are valuable resources in studies on the differential protein expressions of human hepatocellular carcinoma.

Carcinoma, Hepatocellular↗
PubMed2000

[Distribution of peptidergic neurons in the rat suprachiasmatic nucleus].

The distribution of the peptidergic neurons in the suprachiasmatic nucleus(SCN) in five adult male SD rats was examined by multi-immunolabelling for vasoactive intestinal polypeptide(VIP), arginine vasopressin (AVP) and somatostatin(SOM). The immunoreactions were visualized with an Elite ABC Kit(Vector Lab). The substrates in the peroxidase reaction were diaminobenzidine(DAB), tetramethylbenzidine(TMB) and 4-chloro-1-naphtol. The results showed that the differences in distribution and population of VIP-, AVP- and SOM-like neurons existed in the rostral, intermediate and caudal regions of the SCN and these neurons were overlapped and SOM-like neurons were scattered among them in the area between VIP- and AVP-like neurons. These findings may provide new morphologic evidences for explaining the functional mechanism in the SCN.

Animals↗
PubMed1999

High-throughput mass spectrometric discovery of protein post-translational modifications.

The availability of genome sequences, affordable mass spectrometers and high-resolution two-dimensional gels has made possible the identification of hundreds of proteins from many organisms by peptide mass fingerprinting. However, little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we present an approach for the systematic characterisation of proteins using mass spectrometry and a software tool FindMod. This tool, available on the internet at http://www.expasy.ch/sprot/findmod.html , examines peptide mass fingerprinting data for mass differences between empirical and theoretical peptides. Where mass differences correspond to a post-translational modification, intelligent rules are applied to predict the amino acids in the peptide, if any, that might carry the modification. FindMod rules were constructed by examining 5153 incidences of post-translational modifications documented in the SWISS-PROT database, and for the 22 post-translational modifications currently considered (acetylation, amidation, biotinylation, C-mannosylation, deamidation, flavinylation, farnesylation, formylation, geranyl-geranylation, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N -acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone carboxylic acid, sulphation) a total of 29 different rules were made. These consider which amino acids can carry a modification, whether the modification occurs on N-terminal, C-terminal or internal amino acids, and the type of organisms on which the modification can be found. We illustrate the utility of the approach with proteins from 2-D gels of Escherichia coli and sheep wool, where post-translational modifications predicted by FindMod were confirmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a potentially large-scale means of protein characterisation in proteome projects.

Acetylation↗
PubMed1999

An improved synthesis of an umbelliferyl 5-thioxylopyranoside, precursor of the antithrombotic drug Iliparcil.

4-Ethyl-2-oxo-2H-1-benzopyran-7-yl 2,3,4-tri-O-acetyl-5-thio-beta-D-xylopyranoside, a synthetic intermediate of the orally active antithrombotic compound Iliparcil, has been prepared in 44-47% isolated yield. Different conditions were used for the glycosylation of 4-ethyl-2H-7-hydroxy-1-benzopyran-2-one 6 applying 2,3,4-tri-O-acetyl-5-thio-D-xylopyranosyl bromide (2), the analogous beta-chloride 3 or the alpha-trichloroacetimidate 5 as donors. With halides 2 and 3, the reaction was carried out in the presence of ZnO-ZnCl2 or ZnO alone. Both promoters are cheap, safe and therefore compatible with large-scale industrial processes.

Benzopyrans↗
PubMed1998

Protein identification with N and C-terminal sequence tags in proteome projects.

Genome sequences are available for increasing numbers of organisms. The proteomes (protein complement expressed by the genome) of many such organisms are being studied with two-dimensional (2D) gel electrophoresis. Here we have investigated the application of short N-terminal and C-terminal sequence tags to the identification of proteins separated on 2D gels. The theoretical N and C termini of 15, 519 proteins, representing all SWISS-PROT entries for the organisms Mycoplasma genitalium, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and human, were analysed. Sequence tags were found to be surprisingly specific, with N-terminal tags of four amino acid residues found to be unique for between 43% and 83% of proteins, and C-terminal tags of four amino acid residues unique for between 74% and 97% of proteins, depending on the species studied. Sequence tags of five amino acid residues were found to be even more specific. To utilise this specificity of sequence tags for protein identification, we created a world-wide web-accessible protein identification program, TagIdent (http://www.expasy.ch/www/tools.html), which matches sequence tags of up to six amino acid residues as well as estimated protein pI and mass against proteins in the SWISS-PROT database. We demonstrate the utility of this identification approach with sequence tags generated from 91 different E. coli proteins purified by 2D gel electrophoresis. Fifty-one proteins were unambiguously identified by virtue of their sequence tags and estimated pI and mass, and a further 11 proteins identified when sequence tags were combined with protein amino acid composition data. We conlcude that the TagIdent identification approach is best suited to the identification of proteins from prokaryotes whose complete genome sequences are available. The approach is less well suited to proteins from eukaryotes, as many eukaryotic proteins are not amenable to sequencing via Edman degradation, and tag protein identification cannot be unambiguous unless an organism's complete sequence is available.

Amino Acid Sequence↗
PubMed1998

Towards an automated approach for protein identification in proteome projects.

The development of automated, high throughput technologies for the rapid identification of proteins is essential for large-scale proteome projects. While a degree of automation already exists in some stages of the protein identification process, such as automated acquisition of matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectra, efficient interfaces between different stages are still lacking. We report the development of a highly automated, integrated system for large scale identification of proteins separated by two-dimensional gel electrophoresis (2-DE), based on peptide mass fingerprinting. A prototype robotic system was used to image and excise 288 protein spots from an amido black stained polyvinylidene difluoride (PVDF) blot. Protein samples were enzymatically digested with a commercial automated liquid handling system. MALDI-TOF mass spectrometry was used to acquire mass spectra automatically, and the data analysed with novel automated peptide mass fingerprinting database interrogation software. Using this highly automated system, we were able to identify 95 proteins on the basis of peptide mass fingerprinting, isoelectric point and molecular weight, in a period of less than ten working days. Advantages, problems, and future developments in robotic excision systems, liquid handling, and automated database interrogation software are discussed.

Automation↗
PubMed1998

'98 Escherichia coli SWISS-2DPAGE database update.

The combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), computer image analysis and several protein identification techniques allowed the Escherichia coli SWISS-2DPAGE database to be established. This is part of the ExPASy molecular biology server accessible through the WWW at the URL address http://www.expasy.ch/ch2d/ch2d-top.html . Here we report recent progress in the development of the E. coli SWISS-2DPAGE database. Proteins were separated with immobilized pH gradients in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. To increase the resolution of the separation and thus the number of identified proteins, a variety of wide and narrow range immobilized pH gradients were used in the first dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and spots were visualized by amido black staining. Protein identification techniques such as amino acid composition analysis, gel comparison and microsequencing were used, as well as a recently described Edman "sequence tag" approach. Some of the above identification techniques were coupled with database searching tools. Currently 231 polypeptides are identified on the E. coli SWISS-2DPAGE map: 64 have been identified by N-terminal microsequencing, 39 by amino acid composition, and 82 by sequence tag. Of 153 proteins putatively identified by gel comparison, 65 have been confirmed. Many proteins have been identified using more than one technique. Faster progress in the E. coli proteome project will now be possible with advances in biochemical methodology and with the completion of the entire E. coli genome.

Bacterial Proteins↗
PubMed1997

A role for Edman degradation in proteome studies.

Advances in protein database design and the software used to access the sequence data has led to progress in using protein attributes such as amino acid composition and peptide masses to identify proteins separated by two-dimensional electrophoresis. However, Edman degradation remains the principal technique for protein identification and it presents a significant bottleneck in the progress towards rapid protein identification. Simple modifications to the sequencing hardware, which automate the delivery of protein spots into the sequencer, and parallel sequencing of the protein spots represent a significant advance in the use of Edman degradation to rapidly generate the powerful protein attribute, an N-terminal sequence tag.

Amino Acid Sequence↗
PubMed1997

Establishment of the human reflex tear two-dimensional polyacrylamide gel electrophoresis reference map: new proteins of potential diagnostic value.

To understand the changes in protein expression associated with various physiological states as well as the development of pathological eye disease, we have begun to map the protein components of normal human reflex tears. An analytical reference map of normal human reflex tears was created using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) with pH 3.5-10 immobilized pH gradients (IPGs). Micropreparatively loaded gels were transferred to polyvinylidene difluoride (PVDF) and analysed by a combination of N-terminal sequence tagging and amino acid compositional analysis. Thirty spots were sequence tagged, resulting in identification of six different proteins (lipocalin, lysozyme, lactotransferrin, zinc-alpha-2 glycoprotein, cystatin S, cystatin SN) that matched to entries in the SWISS-PROT database. A group of N-terminally blocked proteins was clearly identified from SWISS-PROT by amino acid analysis, isoelectric point (pI) and molecular weight (Mr). A number of highly expressed protein components remain unidentified despite being subjected to amino acid analysis and Edman sequencing. A majority of the abundant proteins showed varying degrees of charge heterogeneity attributed to post-translational processing such as glycosylation and N-terminal truncation. We have identified a previously undescribed protein that we have named lacryglobin. This protein displays strong homology with mammaglobin, a protein overexpressed in breast cancer. The discovery of this homologue in tears offers the potential for disease diagnosis by screening tear fluid proteins.

Databases, Factual↗
PubMed1996

Two distinct forms of the peridinin-chlorophyll a-protein from Amphidinium carterae.

Peridinin-chlorophyll a-proteins (PCPs) have been purified by combination of ammonium sulphate precipitation and cation exchange chromatography. The amino acid sequences of several of the most abundant forms have been deduced by direct protein sequencing and from DNA and indicate a highly conserved multi-gene family. At least two of the PCP genes are tandemly arranged. A novel form of the protein was also obtained in low yield with fewer peridinins (six vs eight) per chlorophyll a and with a different molecular mass (34 kDa vs 32 kDa) of its apoprotein. It had only 31% sequence identity with any of the more abundant PCP forms but retained a two-domain structure.

Amino Acid Sequence↗
PubMed1996

Large-scale amino-acid analysis for proteome studies.

Amino-acid analysis is a relatively new method for identification of proteins separated by two-dimensional gel electrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes. This article describes modified amino-acid analysis methods for this purpose. Streamlined sample handling is a key feature of the process. To minimise sample manipulation, a single vial is used for hydrolysis and the protein hydrolysate on PVDF membrane is extracted by a one-step procedure. The hydrolysate should not be stored for long periods before analysis. Applications of the technique are presented to demonstrate the identification procedure. This approach is the most cost-effective and time-effective first step in mass protein screening for a large-scale proteome project.

Amino Acids↗
PubMed1996

Rapid protein identification using N-terminal "sequence tag" and amino acid analysis.

Proteins can be identified by amino acid analysis and database matching, but it is often desirable to increase the confidence in identity through the use of other techniques. Here we describe a rapid protein identification method that uses Edman degradation to create a 3 or 4 amino acid N-terminal "sequence tag," following which proteins are subjected to amino acid analysis protein identification procedures. Edman degradation methods have been modified to take only 23 min per cycle, and rapid amino acid analysis techniques are used. The Edman degradation and amino acid analysis is done on a single PVDF membrane-bound protein sample. A computer database matching program is also presented which uses both amino acid composition and "sequence tag" data for protein identification. This method represents the most inexpensive, accurate, and rapid means of protein identification, which is ideal for the screening of proteomes separated by 2-D gel electrophoresis. The creation of N-terminal Edman degradation "sequence-tags" prior to peptide mass fingerprinting of samples should also be useful.

Amino Acid Sequence↗
PubMed1996

Characterisation of proteins from two-dimensional electrophoresis gels by matrix-assisted laser desorption mass spectrometry and amino acid compositional analysis.

Amino acid compositional analysis and peptide mass fingerprinting by matrix assisted laser desorption mass spectrometry have been used to characterise proteins obtained from two-dimensional electrophoresis (2-DE) separations of human cardiac proteins. A group of twelve protein spots was selected for analysis. The identities of eight of the proteins had been determined by conventional protein characterisation methods, two were unknown proteins and two had putative identities from protein database spot comparison. Amino acid analysis and peptide mass fingerprinting gave corresponding identities for seven of the twelve proteins, which also agreed with our initial identifications. Three proteins which had been identified previously were not confirmed in this study and putative identities were obtained for the two unknown proteins. The advantages, problems and use of amino acid analysis and peptide mass fingerprinting for the analysis of proteins from 2-DE are discussed. The data highlight the need to use orthogonal techniques for the unequivocal identification of proteins from 2-DE gels.

Amino Acids↗
PubMed1996

From proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis.

Separation and identification of proteins by two-dimensional (2-D) electrophoresis can be used for protein-based gene expression analysis. In this report single protein spots, from polyvinylidene difluoride blots of micropreparative E. coli 2-D gels, were rapidly and economically identified by matching their amino acid composition, estimated pI and molecular weight against all E. coli entries in the SWISS-PROT database. Thirty proteins from an E. coli 2-D map were analyzed and identities assigned. Three of the proteins were unknown. By protein sequencing analysis, 20 of the 27 proteins were correctly identified. Importantly, correct identifications showed unambiguous "correct" score patterns. While incorrect protein identifications also showed distinctive score patterns, indicating that protein must be identified by other means. These techniques allow large-scale screening of the protein complement of simple organisms, or tissues in normal and disease states. The computer program described here is accessible via the World Wide Web at URL address (http:@expasy.hcuge.ch/).

Amino Acids↗