Search PubMed⌕ Search

Biomedical subjects

D F Hochstrasser

Publications and source records attributed to D F Hochstrasser.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1998

Multiple parameter cross-species protein identification using MultiIdent--a world-wide web accessible tool.

Recent increases in the number of genome sequencing projects means that the amount of protein sequence in databases is increasing at an astonishing pace. In proteome studies, this is facilitating the identification of proteins from molecularly well-defined organisms. However, in studies of proteins from the majority of organisms, proteins must be identified by comparing analytical data to sequences in databases from other species. This process is known as cross-species protein identification. Here we present a new program, MultiIdent, which uses multiple protein parameters such as amino acid composition, peptide masses, sequence tags, estimated protein pI and mass, to achieve cross-species protein identification. The program is structured so that protein amino acid composition, which is highly conserved across species boundaries, first generates a set of candidate proteins. These proteins are then queried with other protein parameters such as sequence tags and peptide masses. A final list of database entries which considers all analytical parameters is presented, ranked by an integrated score. We illustrate the power of the approach with the identification of a set of standard proteins, and the identification of proteins from dog heart separated by two-dimensional gel electrophoresis. The MultiIdent program is available on the world-wide web at: http://www.expasy.ch/sprot/multiident.h tml.

Amino Acid Sequence↗
PubMed1998

A gene encoding a novel RFX-associated transactivator is mutated in the majority of MHC class II deficiency patients.

Major histocompatibility class II (MHC-II) molecules are transmembrane proteins that have a central role in development and control of the immune system. They are encoded by a multigene family and their expression is tightly regulated. MHC-II deficiency (OMIM 209920) is an autosomal recessive immunodeficiency syndrome resulting from defects in trans-acting factors essential for transcription of MHC-II genes. There are four genetic complementation groups (A, B, C and D), reflecting the existence of four MHC-II regulators. The factors defective in groups A (CIITA), C (RFX5) and D (RFXAP) have been identified. CIITA is a non-DNA-binding co-activator that controls the cell-type specificity and inducibility of MHC-II expression. RFX5 and RFXAP are two subunits of RFX, a multi-protein complex that binds the X box motif of MHC-II promoters. Mutations in the genes encoding RFX5 (RFX5) or RFXAP (RFXAP) abolish binding of RFX (refs 7,8,12). Similar to groups C and D, group B is characterized by a defect in RFX binding, and although it accounts for the majority of patients, the factor defective in group B has remained unknown. We report here the isolation of RFX by a novel single-step DNA-affinity purification approach and the identification of RFXANK, the gene encoding a third subunit of RFX. RFXANK restores MHC-II expression in cell lines from patients in group B and is mutated in these patients. RFXANK contains a protein-protein interaction region consisting of three ankyrin repeats. Its interaction with RFX5 and RFXAP is essential for binding of the RFX complex to MHC-II promoters.

Amino Acid Sequence↗
PubMed1998

Identification of a basophil leukocyte interleukin-3-regulated protein that is identical to IgE-dependent histamine-releasing factor.

This study aimed to identify basophil leukocyte proteins associated with interleukin (IL)-3 and/or anti-IgE activation by two-dimensional (2-D) gel electrophoresis. We noticed one particular protein showing increased synthesis after recombinant human (rh)IL-3 and, to a lesser extent, anti-IgE stimulation. The protein was also present in the culture medium in increased amounts after rhIL-3 stimulation. On the basis of comigration with proteins in published 2-D gel electrophoresis databases and immunoblotting with a specific monoclonal antibody, we identified this protein as translationally controlled tumor protein (TCTP), also known as p23 or IgE-dependent histamine-releasing factor. The antibody was shown to be specific for TCTP/IgE-dependent histamine-releasing factor by blotting on 2-D gels of proteins from human lymphocytes and the human basophilic cell line KU812, followed by N-terminal amino-acid sequencing of the bound protein. Densitometric analysis of the gels showed that the synthesis of IgE-dependent histamine-releasing factor in human basophil leukocytes was dose dependent upon rhIL-3 stimulation with an optimum of 100 ng/ml. The level of the protein in the medium was also highest at an optimal rhIL-3 concentration of 100 ng/ml. Supernatants from cultured basophils were able to stimulate histamine release from other basophils. This histamine release was decreased by precipitation of TCTP/IgE-dependent histamine-releasing factor from these supernatants.

Amino Acid Sequence↗
PubMed1998

Proteome in perspective.

This review describes briefly proteome science. It explains why proteome science or proteomics emerged only recently and why a shift from genomics to proteomics is occurring. This review further illustrates that proteomics can unravel new domains in nature's complexity. Finally, it demonstrates that proteomics is offering new tools for the study of complex biological or medical problems.

Genome, Human↗
PubMed1997

Elevation of apolipoprotein E in the CSF of cattle affected by BSE.

The cerebrospinal fluid (CSF) of patients suffering from Creutzfeldt-Jakob disease (CJD) display two unique polypeptide chains by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). In the absence of a well-defined ante-mortem diagnostic test for bovine spongiform encephalopathy (BSE), spinal fluid samples of eight normal cows and eight cows known to carry BSE by post-mortem histological analysis were investigated to verify if equivalent polypeptides were present. Proteins with similar migration to human CJD polypeptides were not detected. But surprisingly, a cluster of polypeptide spots that was faint or not detected in normal bovine CSF samples was found to be elevated or massively increased in BSE CSF samples (more than 10-fold increase). These elevated polypeptide chains were identified as apolipoprotein E.

Amino Acid Sequence↗
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

Melanie II--a third-generation software package for analysis of two-dimensional electrophoresis images: I. Features and user interface.

Although two-dimensional electrophoresis (2-DE) computer analysis software packages have existed ever since 2-DE technology was developed, it is only now that the hardware and software technology allows large-scale studies to be performed on low-cost personal computers or workstations, and that setting up a 2-DE computer analysis system in a small laboratory is no longer considered a luxury. After a first attempt in the seventies and early eighties to develop 2-DE analysis software systems on hardware that had poor or even no graphical capabilities, followed in the late eighties by a wave of innovative software developments that were possible thanks to new graphical interface standards such as XWindows, a third generation of 2-DE analysis software packages has now come to maturity. It can be run on a variety of low-cost, general-purpose personal computers, thus making the purchase of a 2-DE analysis system easily attainable for even the smallest laboratory that is involved in proteome research. Melanie II 2-D PAGE, developed at the University Hospital of Geneva, is such a third-generation software system for 2-DE analysis. Based on unique image processing algorithms, this user-friendly object-oriented software package runs on multiple platforms, including Unix, MS-Windows 95 and NT, and Power Macintosh. It provides efficient spot detection and quantitation, state-of-the-art image comparison, statistical data analysis facilities, and is Internet-ready. Linked to proteome databases such as those available on the World Wide Web, it represents a valuable tool for the "Virtual Lab" of the post-genome area.

Algorithms↗
PubMed1997

Melanie II--a third-generation software package for analysis of two-dimensional electrophoresis images: II. Algorithms.

After two generations of software systems for the analysis of two-dimensional electrophoresis (2-DE) images, a third generation of such software packages has recently emerged that combines state-of-the-art graphical user interfaces with comprehensive spot data analysis capabilities. A key characteristic common to most of these software packages is that many of their tools are implementations of algorithms that resulted from research areas such as image processing, vision, artificial intelligence or machine learning. This article presents the main algorithms implemented in the Melanie II 2-D PAGE software package. The applications of these algorithms, embodied as the feature of the program, are explained in an accompanying article (R. D. Appel et al.; Electrophoresis 1997, 18, 2724-2734).

Algorithms↗
PubMed1997

Make2ddb: a simple package to set up a two-dimensional electrophoresis database for the World Wide Web.

Two-dimensional electrophoresis (2-DE) has become a highly reproducible protein separation technique that currently serves as the main basis for proteome research and in particular for protein identification. Also, the Internet provides large utilities for exchanging data, and we can observe increased interest among scientists to build remote 2-DE databases, since many members of the concerned community are now able to access the data. By preparing the data and programs that are required to create a federated 2-DE database, the Make2ddb package, described here, helps to build such a database on the user own World Wide Web site.

Computer Communication Networks↗
PubMed1997

A two-dimensional protein map of human amniotic fluid at 17 weeks' gestation.

Using updated technical procedures (immobilized pH gradients for isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: IPG/SDS-PAGE) we provide a two-dimensional (2-D) map of amniotic fluid (AF) proteins. This map comprises over 800 silver-stained spots. Over 150 spots have been identified by matching on the net with human plasma and cerebrospinal fluid maps available from SWISS 2DPAGE database; several additional spots were assigned by immunoblotting and/or microanalytical techniques. This report details our investigation on AF proteins focusing on the 17th week of gestation, when AF is most commonly used for clinical evaluation of fetal disorders. As a whole, the map displays a number of potential markers for fetal development and for gestation abnormalities. The 2-D electrophoretic technique allows the monitoring of all these proteins at the same time along with additional spots that may prove of diagnostic significance.

Adult↗
PubMed1997

Protein expression profiles in human breast ductal carcinoma and histologically normal tissue.

Reference two-dimensional (2-D) gels are presented for human breast ductal carcinoma and histologically normal tissue. Whole biopsy fragments were analyzed, including epithelial and nonepithelial components. Thirty-five spots have been assigned by gel matching to the human liver SWISS-2DPAGE reference map and/or to the human primary keratinocyte IPG map from the Danish Center for Human Genome. N-terminal microsequencing was applied to confirm randomly chosen matching assignments and to identify six new spots. Protein expression profiles in ductal carcinoma and in normal breast tissue appeared to be similar, except for a pattern consisting of 32 spots, which were highly expressed in all carcinoma specimens, and less intense and occasionally undetectable in normal tissue. This difference was statistically significant. Assignment has been obtained for several spots, namely GRP94, GRP78, GRP75, mitochondrial HSP60, calreticulin, protein disulfide isomerase, peptidyl-prolyl cis-trans isomerase, collagen-binding protein 2, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, thioredoxin, cytochrome c oxidase VA subunit, tubulin beta isoform and macrophage migration inhibitory factor (MIF). The cancer- and tissue-specificity of the described pattern was assessed by matching to the Swiss-2DPAGE human liver, hepatoma, lymphoma, erythroleukemia reference maps. The pattern of 32 spots was found to be indicative of epithelial neoplasia.

Aged↗
PubMed1997

Standardized characterization of gene expression in human colorectal epithelium by two-dimensional electrophoresis.

New diagnostic and prognostic markers are needed in colorectal cancer. They can be found by differential analysis at DNA, RNA or protein level. The accuracy of phenotypic comparisons of tumor and normal tissues depends on the purity of the samples. We present an effective method to identify and isolate proteins that are differentially expressed under altered conditions, and a two-dimensional reference protein map of the normal human colonic epithelium. Normal colonic mucosa, primary tumors and liver metastases were prepared in the operating room. After washing in an ice-cold medium containing protease inhibitors, crypts were isolated by mechanical preparation without using metalloproteinases. Epithelial cells were then selected using Ber-EP4 Dynabeads. The samples were denaturated before processing for immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis according to SWISS-2DPAGE standards. The samples contained more than 95% epithelial cells as confirmed by fluorescence-activated cell sorting using pan-anticytokeratin antibodies. Cell surfaces were not damaged, as assessed by scanning electronic microscope. A protein reference map of the normal colonic epithelium was defined. Using gel matching, N-terminal sequencing and/or immunoblotting techniques, 60 polypeptides - including proteins specifically expressed in colorectal epithelium - have now been identified. This reproducible method of sample preparation permits the comparison of protein patterns found in various pathological states with the present reference map (http://www.expasy.ch). Some of these patterns might provide diagnostic or prognostic markers, or even molecular targets for therapy in the future.

Colorectal Neoplasms↗
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

New alterations of serum glycoproteins in alcoholic and cirrhotic patients revealed by high resolution two-dimensional gel electrophoresis.

Plasma protein are synthesized and secreted by the liver. Several reports have shown that excessive consumption of ethanol interferes with the hepatic protein synthesis and/or secretion. This study was undertaken to identify the plasma/serum proteins altered in two groups of patients with different alcohol-related diseases: actively drinking alcoholic patients group without liver disease and alcohol cirrhotic patients group. Two-dimensional gel electrophoresis was used to separate proteins with high resolution. Proteins were detected by silver staining and glycoproteins were specifically visualized and analyzed after lectin blotting followed by chemiluminescence detection. Different protein alterations were identified in each group of patients. In the alcoholic group, two new glycosylation modifications of serum proteins were identified. An abnormal microheterogeneity of haptoglobin and alpha1-antitrypsin was detected in the serum of all alcoholic patients. We also characterized by two-dimensional gel electrophoresis the carbohydrate deficient transferrin. The modifications of haptoglobin, alpha1-antitrypsin and transferrin present a similar change of charge and molecular weight in the two-dimensional gel electrophoresis pattern. These qualitative estimations support the hypothesis of a general mechanism of liver glycosylation alteration of serum proteins induced by excessive alcohol consumption. The immunoglobulin alterations were easily visualized and identified for the cirrhotic and the alcoholic patients. And finally, the decrease of haptoglobin and albumin spots for cirrhotic patients was confirmed.

Alcoholism↗