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PubMed2000

Identification of tumor-suppressor genes using human melanoma cell lines UACC903, UACC903(+6), and SRS3 by comparison of expression profiles.

The development and progression of cancer are believed to be due to multiple genetic alterations resulting in complex changes in expression of many genes. The parental malignant melanoma cell line UACC903 displays anchorage-independent growth, and the chromosome 6-suppressed subline UACC903(+6) displays anchorage-dependent growth. The anchorage-independent revertant cell line SRS3 derived from UACC903(+6) by retroviral transduction resembles the phenotype of UACC903. In this study, we first compared the expression profiles of 3317 genes between these three cell lines in pairs by cDNA microarrays, resulting in identification of genes with known suppressor activities. We then demonstrated connexin 43 (Cx43)-suppressing anchorage-independent growth of UACC903 on overexpression. Of 3317 genes with informative expression detected by cDNA microarray, 321 (9.68%) showed expression changes between at least one pair of the three cell lines. Notably, 12 genes displayed higher levels of expression in UACC903(+6) than in both UACC903 and SRS3, providing candidates for further identification of melanoma-suppressor genes. Genes encoding Cx43 (suppressor activity), monocyte chemotactic protein 1 (suppressor activity), and cysteine proteinase P32alpha (apoptotic activity) were all upregulated in UACC903(+6), in contrast to both UACC903 and SRS3. Transfection of Cx43, encoded on human chromosome 6q21-q23, a region frequently altered in malignant melanoma, resulted in its overexpression and the suppression of anchorage-independent growth of UACC903. Thus, our result proves the principle that the combination of the ability to alter cellular phenotype by successive genetic alterations and the ability to examine the global expression profiles facilitates the identification of tumor suppressor genes. Mol. Carcinog. 28:119-127, 2000.

Base Sequence↗

PubMed2002

Combinatorial peptide library-based identification of peptide ligands for tumor-reactive cytolytic T lymphocytes of unknown specificity.

A novel approach for the identification of tumor antigen-derived sequences recognized by CD8(+) cytolytic T lymphocytes (CTL) consists in using synthetic combinatorial peptide libraries. Here we have screened a library composed of 3.1 x 10(11) nonapeptides arranged in a positional scanning format, in a cytotoxicity assay, to search the antigen recognized by melanoma-reactive CTL of unknown specificity. The results of this analysis enabled the identification of several optimal peptide ligands, as most of the individual nonapeptides deduced from the primary screening were efficiently recognized by the CTL. The results of the library screening were also analyzed with a mathematical approach based on a model of independent and additive contribution of individual amino acids to antigen recognition. This biometrical data analysis enabled the retrieval, in public databases, of the native antigenic peptide SSX-2(41-49), whose sequence is highly homologous to the ones deduced from the library screening, among the ones with the highest stimulatory score. These results underline the high predictive value of positional scanning synthetic combinatorial peptide library analysis and encourage its use for the identification of CTL ligands.

Antigens, Neoplasm↗

PubMed2001

A combination of chemical derivatisation and improved bioinformatic tools optimises protein identification for proteomics.

The identification of individual protein species within an organism's proteome has been optimised by increasing the information produced from mass spectral analysis through the chemical derivatisation of tryptic peptides and the development of new software tools. Peptide fragments are subjected to two forms of derivatisation. First, lysine residues are converted to homoarginine moieties by guanidination. This procedure has two advantages, first, it usually identifies the C-terminal amino acid of the tryptic peptide and also greatly increases the total information content of the mass spectrum by improving the signal response of C-terminal lysine fragments. Second, an Edman-type phenylthiocarbamoyl (PTC) modification is carried out on the N-terminal amino acid. The renders the first peptide bond highly susceptible to cleavage during mass spectrometry (MS) analysis and consequently allows the ready identification of the N-terminal residue. The utility of the procedure has been demonstrated by developing novel bioinformatic tools to exploit the additional mass spectral data in the identification of proteome proteins from the yeast Saccharomyces cerevisiae. With this combination of novel chemistry and bioinformatics, it should be possible to identify unambiguously any yeast protein spot or band from either two-dimensional or one-dimensional electropheretograms.

Databases, Factual↗

PubMed2001

Identification of post-translationally modified proteins in proteome studies.

Proteome studies are powerful tools to solve many different problems in metabolism, signal transduction, drug discovery, and other areas of interest in life sciences. Up to now, high-sensitive methods for protein identification after two-dimensional gel electrophoresis using mass spectrometry are available. However, the identification of post-translational modifications after two-dimensional gel electrophoresis is still an unsolved problem. In this paper, we want to give several examples for the successful identification of post-translational modifications and point mutations.

Animals↗

PubMed2001

Strategy for protein isoform identification from expressed sequence tags and its application to peptide mass fingerprinting.

Expressed Sequence Tags (ESTs) are an invaluable resource for protein identification and characterisation in proteomics. They allow proteins to be identified in the absence of genome sequence data. When EST sequences are used for protein identification, they are usually first processed into contigs to reduce redundancy and generate longer sequences from the overlapping ESTs. However, the process of generating contigs may accidentally group biologically meaningful isoforms together. Here we report means of discovering isoforms in EST sequences and how to use this information in the framework of protein identification and characterisation with peptide mass fingerprinting. We illustrate our strategies with examples from the dbEST database as well as protein isoforms from two-dimensional polyacrylamide gels.

Amino Acid Sequence↗

PubMed2001

Multiple separations facilitate identification of protein variants by mass spectrometry.

Identification of variant proteins from complex biological samples promises to contribute much to our understanding the etiology of pathological states. Characterization of variants, either due to genetic mutations in protein sequences or to post-translational modifications, is considerably more difficult than the simple protein identifications typical of most current proteomic investigations. Identification of a few peptides by database retrieval is not adequate when the goal is to have a complete understanding of the modifications of the protein. Although one advantage of mass spectrometry is its ability to obtain specific responses to several components, the complexity of biological samples is often overwhelming, resulting in spectra lacking useful information. For complex mixtures, isolation procedures before mass spectrometric analysis may need to include a variety of chromatographic and electrophoretic separation techniques. In this report, we illustrate how several preparative steps were essential for obtaining information about modified human lens beta-crystallins. The preparative techniques prior to mass spectrometry included size exclusion chromatography, reversed phase chromatography, two-dimensional gel electrophoresis, in situ digestion of the proteins and peptide trapping and washing before a final reversed phase high performance liquid chromatographic separation on-line to the mass spectrometer. This approach for isolation and analysis, when customized for other proteins, should find application in many studies where protein variants of complex mixtures are to be identified.

Chromatography, Gel↗

PubMed2001

Simplified sample preparation method for protein identification by matrix-assisted laser desorption/ionization mass spectrometry: in-gel digestion on the probe surface.

Identification and detailed characterization of complex mixtures of proteins separated by polyacrylamide gel electrophoresis (PAGE) require optimized and robust methods for interfacing electrophoretic techniques to mass spectrometry. Peptide mapping by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) is used as the first protein screening method in many laboratories because of its inherent simplicity, mass accuracy, sensitivity and relatively high sample throughput. We present a simplified sample preparation method for MALDI-MS that enables in-gel digestion of protein samples directly on the MALDI-MS metal probe. Removal of detergent and reagents as well as protein reduction and S-alkylation were performed prior to cutting of protein samples from the polyacrylamide gel slab. The general utility of this approach was demonstrated by on-probe digestion and MALDI-MS peptide mapping of femtomole amounts of standard proteins isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A representative set of 47 human proteins obtained from a silver stained two-dimensional electrophoretic gel was analyzed by the new method and resulted in a success rate for protein identification similar to that obtained by the traditional protocols for in-gel digestion and MALDI peptide mass mapping of human proteins, i.e. approximately 60%. The overall performance of the novel on-probe digestion method is comparable with that of the standard in-gel sample preparation protocol while being less labour intensive and more cost-effective due to minimal consumption of reagents, enzymes and consumables. Preliminary data obtained on a MALDI quadrupole-TOF tandem mass spectrometer demonstrated the utility of the on-probe digestion protocol for peptide mass mapping and peptide sequencing on this instrument. Automation of the on-probe protein digestion procedure and its combination with automated MALDI tandem mass spectrometry should be advantageous in proteomics research aimed at the systematic identification and analysis of large sets of proteins from electrophoretic gels.

Amino Acid Sequence↗

PubMed2002

Stable isotope labelling in vivo as an aid to protein identification in peptide mass fingerprinting.

Peptide mass fingerprinting (PMF) is a powerful technique for identification of proteins derived from in-gel digests by virtue of their matrix-assisted laser desorption/ionization-time of flight mass spectra. However, there are circumstances where the under-representation of peptides in the mass spectrum and the complexity of the source proteome mean that PMF is inadequate as an identification tool. In this paper, we show that identification is substantially enhanced by inclusion of composition data for a single amino acid. Labelling in vivo with a stable isotope labelled amino acid (in this paper, decadeuterated leucine) identifies the number of such amino acids in each digest fragment, and show a considerable gain in the ability of PMF to identify the parent protein. The method is tolerant to the extent of labelling, and as such, may be applicable to a range of single cell systems.

Deuterium↗

PubMed2002

Identification of vaccine candidate antigens of Staphylococcus aureus by serological proteome analysis.

In this study we applied serological proteome analysis (Klade, C. S. et al. Proteomics 2001, 1, 890-898) for identification of bacterial vaccine candidate antigens. First, approximately one hundred sera from healthy individuals and patients suffering from Staphylococcus aureus infections were screened for antibodies against staphylococcal lysates and recombinant proteins representing surface antigens. Two pools (healthy donors, patients) each consisting of five sera with the highest antiproteinaceous IgG reactivity were selected. Second, S. aureus COL was grown under different conditions and the number of antigens expressed was monitored by Western blot analysis. Third, surface proteins were enriched by digesting the bacterial cell wall under isotonic conditions and subsequent removal of protoplasts. These protein preparations were resolved by two-dimensional electrophoresis (2-DE) (pI 4-7). 2-DE immunoblotting using the preselected serum pools at 1:10 000-1:100 000 dilutions revealed a number of highly immunogenic staphylococcal proteins. Twenty-one spots were isolated by preparative 2-DE, and analysed by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry sequencing of tryptic peptides. This led to the identification of 15 proteins including known and novel vaccine candidates. Seroreactivity of several antigens including serine-aspartate repeat containing protein D, immuno-dominant staphylococcal antigen and a novel 309 amino acid lipoprotein was independently confirmed by enzyme-linked immunosorbent assay and Western blot analysis of purified recombinant proteins. In conclusion, serological proteome analysis proved to be a powerful tool for the identification of novel staphylococcal antigens, which provide a basis for rational vaccine design.

Adult↗

PubMed2002

Identification of Staphylococcus aureus exotoxins by combined sodium dodecyl sulfate gel electrophoresis and matrix-assisted laser desorption/ ionization-time of flight mass spectrometry.

Staphylococcus aureus is an important human pathogen whose pathogenesis involves the synthesis of cell wall associated virulence factors and secreted toxins with damaging effects on the host cells. Most of these pathogenic factors are synthesized in a growth-phase dependent manner as a response to environmental stress like heat, lack of nutrients or other deleterious conditions. Conventional identification of these pathogenic factors is based on Western blot analysis or enzyme-linked immunosorbent assay (ELISA) and is limited by the commercial availability of antibodies against these toxins. We report here the use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry for monitoring the pathogenic factors of S. aureus. For the identification of pathogenic factors, a methicillin sensitive strain of S. aureus, ATCC-29213, was grown at 37 degrees C or 42 degrees C in brain-heart infusion broth and harvested during the early stationary phase of growth. Secreted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzymatically digested with trypsin and analyzed by MALDI-TOF mass spectrometry. When grown at 42 degrees C, alpha- and beta-hemolysins were found to accumulate in S. aureus supernatants while the concentration of protein A was slightly decreased. The identity of some of these toxins was confirmed by Western-blot analysis. MALDI-TOF mass spectrometry combined with sodium dodecyl sulfate gel electrophoresis represents a rapid and simple approach to characterize the virulence of S. aureus strains which seems to be particularly valuable for the identification of S. aureus exotoxins for which ELISA is not established.

Blotting, Western↗

PubMed2002

Identification of proteins from two-dimensional polyacrylamide gels using a novel acid-labile surfactant.

Protein identification by peptide mass mapping usually involves digestion of gel-separated proteins with trypsin, followed by mass measurement of the resulting peptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Positive identification requires measurement of enough peptide masses to obtain a definitive match with sequence information recorded in protein or DNA sequence databases. However, competitive binding and ionization of residual surfactant introduced during polyacrylamide gel electrophoresis (PAGE) can inhibit solid-phase extraction and MS analysis of tryptic peptides. We have evaluated a novel, acid-labile surfactant (ALS) as an alternative to sodium dodecylsulfate (SDS) for two-dimensional (2-D) PAGE separation and MALDI-MS mapping of proteins. ALS was substituted for SDS at the same concentration in buffers and gels used for 2-D PAGE. Manual and automated procedures for spot cutting and in-gel digestion were used to process Coomassie stained proteins for MS analysis. Results indicate that substituting ALS for SDS during PAGE can significantly increase the number of peptides detected by MALDI-MS, especially for proteins of relatively low abundance. This effect is attributed to decomposition of ALS under acidic conditions during gel staining, destaining, peptide extraction and MS sample preparation. Automated excision and digestion procedures reduce contamination by keratin and other impurities, further enhancing MS identification of gel separated proteins.

Alkanesulfonates↗

PubMed2002

Proteomic characterization of wheat amyloplasts using identification of proteins by tandem mass spectrometry.

We describe the initial characterization of the wheat amyloplast proteome, consisting of the identification and classification of 171 proteins. Whole amyloplasts and purified amyloplast membranes were prepared from wheat (Triticum aestivum). Protein extracts were examined by one-dimensional and two-dimensional electrophoresis, followed by high performance liquid chromatography-tandem mass spectrometry of separated proteins. Tandem mass spectrometry data of individual peptides was then searched by SEQUEST, using a database containing known protein sequences from both wheat and other homologous cereal crops. Using this approach we identified 108 proteins from whole amyloplasts and 63 proteins from purified amyloplast membranes. The majority of protein identifications were derived from protein sequences from cereal crops other than wheat, for which relatively little gene sequence data is available. The highest percentage of protein identifications obtained from any individual species was 46% of the total number of proteins identified, using sequence data found in our proprietary rice (Oryza sativa) genome database.

Chromatography, High Pressure Liquid↗

PubMed2002

Comparative bioinformatic analysis of complete proteomes and protein parameters for cross-species identification in proteomics.

Peptide mass fingerprinting (PMF) remains the most amenable technique for protein identification in proteomics, using mass spectrometry as the primary analytical technique coupled with bioinformatics. This relies on the presence of the amino acid sequence of the protein in the current databanks. Despite this, it is desirable to be able to use the technique for organisms whose genomes are not yet fully sequenced and apply cross-species protein identification. In this study, we have re-examined the feasibility of such approaches by considering the extent of protein similarity between genome sequences using a data set of 29 complete bacterial and two eukaryotic genomes. A range of protein and peptide features are considered, including protein isoelectric focussing point, protein mass, and amino acid conservation. The effectiveness of PMF approaches has then been tested with a series of computer simulations with varying peptide number and mass accuracy for several cross-species tests. The results show that PMF alone is unsuitable in general for divergent species jumps, or when protein similarity is less than 70% identity. Despite this, there exists a considerable enrichment above random of tryptic peptide conservation and PMF promises to remain useful when combined with other data than just peptide masses for cross-species protein identification.

Algorithms↗

PubMed2002

Molecular scanner experiment with human plasma: improving protein identification by using intensity distributions of matching peptide masses.

The development of high throughput utilities to identify proteins is a major challenge in present research in the field of proteomics. One such utility, the molecular scanner, uses proteins separated by two-dimensional polyacrylamide gel electrophoresis that are digested in the gel and during transfer onto a collecting membrane. After adding a matrix, the membrane is inserted into a matrix-assisted laser desorption/ionization-time of flight mass spectrometer and a peptide mass fingerprint (PMF) is measured for every scanned site. Since the spacing between scanned sites is much smaller than the size of the most abundant protein spots, there is a certain redundancy in the data that was used in an earlier experiment with Escherichia coli [1] to improve mass calibration and PMF identification results. It was observed that the signal intensity of a peptide mass as a function of the position on the membrane showed similar patterns if peptides stemmed from the same protein. Taking account of these similarities a clustering algorithm was used to find lists of experimental masses with similar intensity distributions, which provided clearer identification of the corresponding proteins. Here, these methods are applied to a human plasma scan, where proteins were highly modified and less separated. The presence of very abundant proteins like albumin and immunoglobulins added another difficulty. The calibration of the initial PMFs was not satisfactory and masses had to be recalibrated. After discarding chemical noise, the membrane was partitioned into regions and for each region protein identification was carried out separately. A new scoring method was used, where the PMF score was multiplied by a factor that measures the similarity of matching peptides. This method proved to be more robust than the method developed in [1] if the region where a protein was found had an extended, nonspherical shape and strong overlap with regions of other proteins. Many proteins annotated on the SWISS-2D PAGE human plasma master gel could be clearly identified and many interesting properties were observed.

Algorithms↗

PubMed2003

Brief communication: multiplex X/Y-PCR improves sex identification in aDNA analysis.

This study introduces a polymerase chain reaction (PCR)-based multiplex approach to improve the certainty of molecular sex identification on archaeological skeletal material. We coamplified amelogenin, two X-chromosomal short tandem repeats (STRs) (DXS6789 and DXS9898), and two Y-specific STRs (DYS391 and DYS392). The amplification results of this multiplex approach back each other up, and enable a reliable sex identification. This coamplification of X- and Y-specific markers in a multiplex assay combines the added advantage of positive identification of both female and male individuals with raising the validity of the diagnosis by obtaining multiple data simultaneously. This multiplex system was successfully applied to 3,000-year-old bone material.

Archaeology↗

PubMed1983

A numerical identification system for airways in the lung.

A numerical system for identifying airways at any level in the tracheobronchial tree has been developed which appears to be simple, logical, and accurate. The system has been successfully used in the authors' laboratory to expedite morphometric studies of the conducting airways in the tracheobronchial tree. Three simple and direct criteria or rules have been formulated for assignment of an airway identification number (AIN) to any specific pulmonary airway, according to this identification system. Some specific applications of this AIN system have been enumerated and several limitations that the authors have recognized have been described. Workers in the fields of bronchial morphometry and anatomy are invited to consider the possible theoretic and practical utility of this airway identification system.

Humans↗

PubMed2005

Dental morphology and variation in theropod dinosaurs: implications for the taxonomic identification of isolated teeth.

Isolated theropod teeth are common Mesozoic fossils and would be an important data source for paleoecology biogeography if they could be reliably identified as having come from particular taxa. However, obtaining identifications is confounded by a paucity of easily identifiable characters. Here we discuss a quantitative methodology designed to provide defensible identifications of isolated teeth using Tyrannosaurus as a comparison taxon. We created a standard data set based as much as possible on teeth of known taxonomic affinity against which to compare isolated crowns. Tooth morphology was described using measured variables describing crown length, base length and width, and derived variables related to basal shape, squatness, mesial curve shape, apex location with respect to base, and denticle size. Crown curves were described by fitting the power function Y = a + bX(0.5) to coordinate data collected from lateral-view images of mesial curve profiles. The b value from these analyses provides a measure of curvature. Discriminant analyses compared isolated teeth of various taxonomic affinities against the standard. The analyses classified known Tyrannosaurus teeth with Tyrannosaurus and separated most teeth known not to be Tyrannosaurus from Tyrannosaurus. They had trouble correctly classifying teeth that were very similar to Tyrannosaurus and for which there were few data in the standard. However, the results indicate that expanding the standard should facilitate the identification of numerous types of isolated theropod teeth.

Analysis of Variance↗

PubMed2001

Dynamical model development and parameter identification for an anaerobic wastewater treatment process.

This paper deals with the development and the parameter identification of an anaerobic digestion process model. A two-step (acidogenesis-methanization) mass-balance model has been considered. The model incorporates electrochemical equilibria in order to include the alkalinity, which has to play a central role in the related monitoring and control strategy of a treatment plant. The identification is based on a set of dynamical experiments designed to cover a wide spectrum of operating conditions that are likely to take place in the practical operation of the plant. A step by step identification procedure to estimate the model parameters is presented. The results of 70 days of experiments in a 1-m(3) fermenter are then used to validate the model.

Bacteria, Anaerobic↗