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Sample records for rapid protein identification

  1. Method for Rapid Protein Identification in a Large Database

    PubMed Central

    Zhang, Wenli; Zhao, Xiaofang

    2013-01-01

    Protein identification is an integral part of proteomics research. The available tools to identify proteins in tandem mass spectrometry experiments are not optimized to face current challenges in terms of identification scale and speed owing to the exponential growth of the protein database and the accelerated generation of mass spectrometry data, as well as the demand for nonspecific digestion and post-modifications in complex-sample identification. As a result, a rapid method is required to mitigate such complexity and computation challenges. This paper thus aims to present an open method to prevent enzyme and modification specificity on a large database. This paper designed and developed a distributed program to facilitate application to computer resources. With this optimization, nearly linear speedup and real-time support are achieved on a large database with nonspecific digestion, thus enabling testing with two classical large protein databases in a 20-blade cluster. This work aids in the discovery of more significant biological results, such as modification sites, and enables the identification of more complex samples, such as metaproteomics samples. PMID:24000323

  2. Small acid soluble proteins for rapid spore identification.

    SciTech Connect

    Branda, Steven S.; Lane, Todd W.; VanderNoot, Victoria A.; Jokerst, Amanda S.

    2006-12-01

    This one year LDRD addressed the problem of rapid characterization of bacterial spores such as those from the genus Bacillus, the group that contains pathogenic spores such as B. anthracis. In this effort we addressed the feasibility of using a proteomics based approach to spore characterization using a subset of conserved spore proteins known as the small acid soluble proteins or SASPs. We proposed developing techniques that built on our previous expertise in microseparations to rapidly characterize or identify spores. An alternative SASP extraction method was developed that was amenable to both the subsequent fluorescent labeling required for laser-induced fluorescence detection and the low ionic strength requirements for isoelectric focusing. For the microseparations, both capillary isoelectric focusing and chip gel electrophoresis were employed. A variety of methods were evaluated to improve the molecular weight resolution for the SASPs, which are in a molecular weight range that is not well resolved by the current methods. Isoelectric focusing was optimized and employed to resolve the SASPs using UV absorbance detection. Proteomic signatures of native wild type Bacillus spores and clones genetically engineered to produce altered SASP patterns were assessed by slab gel electrophoresis, capillary isoelectric focusing with absorbance detection as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection.

  3. Web-based software for rapid "top-down" proteomic identification of protein biomarkers with implications for bacterial identification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have developed web-based software for the rapid identification of protein biomarkers of bacterial microorganisms. Proteins from bacterial cell lysates were ionized by matrix-assisted laser desorption/ionization (MALDI), mass-isolated and fragmented using a time-of-flight/time-of-flight (TOF-TOF)...

  4. Rapid identification of fluorochrome modification sites in proteins by LC ESI-Q-TOF mass spectrometry.

    PubMed

    Manikwar, Prakash; Zimmerman, Tahl; Blanco, Francisco J; Williams, Todd D; Siahaan, Teruna J

    2011-07-20

    Conjugation of either a fluorescent dye or a drug molecule to the ε-amino groups of lysine residues of proteins has many applications in biology and medicine. However, this type of conjugation produces a heterogeneous population of protein conjugates. Because conjugation of fluorochrome or drug molecule to a protein may have deleterious effects on protein function, the identification of conjugation sites is necessary. Unfortunately, the identification process can be time-consuming and laborious; therefore, there is a need to develop a rapid and reliable way to determine the conjugation sites of the fluorescent label or drug molecule. In this study, the sites of conjugation of fluorescein-5'-isothiocyanate and rhodamine-B-isothiocyanate to free amino groups on the insert-domain (I-domain) protein derived from the α-subunit of lymphocyte function-associated antigen-1 (LFA-1) were determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) along with peptide mapping using trypsin digestion. A reporter fragment of the fluorochrome moiety that is generated in the collision cell of the Q-TOF without explicit MS/MS precursor selection was used to identify the conjugation site. Selected ion plots of the reporter ion readily mark modified peptides in chromatograms of the complex digest. Interrogation of theses spectra reveals a neutral loss/precursor pair that identifies the modified peptide. The results show that one to seven fluorescein molecules or one to four rhodamine molecules were attached to the lysine residue(s) of the I-domain protein. No modifications were found in the metal ion-dependent adhesion site (MIDAS), which is an important binding region of the I-domain. PMID:21612301

  5. Rapid Identification of Novel Immunodominant Proteins and Characterization of a Specific Linear Epitope of Campylobacter jejuni

    PubMed Central

    Hoppe, Sebastian; Bier, Frank F.; Nickisch-Rosenegk, Markus v.

    2013-01-01

    Campylobacter jejuni remains one of the major gut pathogens of our time. Its zoonotic nature and wide-spread distribution in industrialized countries calls for a quick and reliable diagnostic tool. Antibody-based detection presents a suitable means to identify pathogenic bacteria. However, the knowledge about immunodominant targets is limited. Thus, an approach is presented, which allows for the rapid screening of numerous cDNA derived expression clones to identify novel antigens. The deeper understanding of immunodominant proteins assists in the design of diagnostic tools and furthers the insight into the bacterium’s pathogenicity as well as revealing potential candidates for vaccination. We have successfully screened 1536 clones of an expression library to identify 22 proteins that have not been described as immunodominant before. After subcloning the corresponding 22 genes and expression of full-length proteins, we investigated the immunodominant character by microarrays and ELISA. Subsequently, seven proteins were selected for epitope mapping. For cj0669 and cj0920c linear epitopes were identified. For cj0669, specificity assays revealed a specific linear epitope site. Consequently, an eleven amino acid residue sequence TLIKELKRLGI was analyzed via alanine scan, which revealed the glycine residue to be significant for binding of the antibody. The innovative approach presented herein of generating cDNAs of prokaryotes in combination with a microarray platform rendering time-consuming purification steps obsolete has helped to illuminate novel immunodominant proteins of C.jejuni. The findings of a specific linear epitope pave the way for a plethora of future research and the potential use in diagnostic applications such as serological screenings. Moreover, the current approach is easily adaptable to other highly relevant bacteria making it a formidable tool for the future discovery of antigens and potential biomarkers. Consequently, it is desirable to simplify

  6. Microbial Protein-Antigenome Determination (MAD) Technology: A Proteomics-Based Strategy for Rapid Identification of Microbial Targets of Host Humoral Immune Responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunogenic, pathogen-specific proteins have excellent potential for development of novel management modalities. Here, we describe an innovative application of proteomics called Microbial protein-Antigenome Determination (MAD) Technology for rapid identification of native microbial proteins that eli...

  7. Microbial Protein-Antigenome Determination (MAD) Technology: A Proteomics-Based Strategy for Rapid Identification of Microbial Targets of Host Humoral Immune Responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunogenic, pathogen-specific proteins have excellent potential for development of novel management modalities. Here, we describe an innovative application of proteomics called Microbial protein-Antigenome Determination (MAD) Technology for rapid identification of native microbial proteins that el...

  8. Rapid Identification of Malaria Vaccine Candidates Based on α-Helical Coiled Coil Protein Motif

    PubMed Central

    Villard, Viviane; Agak, George W.; Frank, Géraldine; Jafarshad, Ali; Servis, Catherine; Nébié, Issa; Sirima, Sodiomon B.; Felger, Ingrid; Arevalo-Herrera, Myriam; Herrera, Socrates; Heitz, Frederic; Bäcker, Volker; Druilhe, Pierre; Kajava, Andrey V.; Corradin, Giampietro

    2007-01-01

    To identify malaria antigens for vaccine development, we selected α-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally “native” epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high α-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens. PMID:17653272

  9. A rapid extraction procedure of human hair proteins and identification of phosphorylated species.

    PubMed

    Nakamura, Akira; Arimoto, Makoto; Takeuchi, Keiji; Fujii, Toshihiro

    2002-05-01

    We developed a rapid and convenient extraction procedure of human hair proteins to examine their biochemical properties in detail. This procedure is based upon the fact that the combination of thiourea and urea in the presence of a reductant can effectively remove proteins from the cortex part of human hair. The extracted fraction mainly consisted of hard alpha-keratins with molecular masses of 40-60 kDa, matrix proteins with 12-18kDa, and minor components with 110-115kDa and 125-135kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein phosphorylation in human hair was investigated by immunoblotting with antibodies against phosphoserine, phosphothreonine and phosphotyrosine. We found serine phosphorylation in alpha-keratins and matrix proteins and threonine phosphorylation in alpha-keratins. The extraction was also found to be effective when wool, chicken feathers, rat hair and human nails were used as starting materials. PMID:12033494

  10. Identification of Thermostabilizing Mutations for Membrane Proteins: Rapid Method Based on Statistical Thermodynamics.

    PubMed

    Yasuda, Satoshi; Kajiwara, Yuta; Takamuku, Yuuki; Suzuki, Nanao; Murata, Takeshi; Kinoshita, Masahiro

    2016-04-28

    Membrane proteins are responsible for the communication between cells and their environments. They are indispensable to the expression of life phenomena and also implicated in a number of diseases. Nevertheless, the studies on membrane proteins are far behind those on water-soluble proteins, primarily due to their low structural stability. Introduction of mutations can enhance their thermostability and stability in detergents, but the stabilizing mutations are currently identified by experiments. The recently reported computational methods suffer such drawbacks as the exploration of only limited mutational space and the empiricism whose results are difficult to physically interpret. Here we develop a rapid method that allows us to treat all of the possible mutations. It employs a free-energy function (FEF) that takes into account the translational entropy of hydrocarbon groups within the lipid bilayer as well as the protein intramolecular hydrogen bonding. The method is illustrated for the adenosine A2a receptor whose wild-type structure is known and utilized. We propose a reliable strategy of finding key residues to be mutated and selecting their mutations, which will lead to considerably higher stability. Representative single mutants predicted to be stabilizing or destabilizing were experimentally examined and the success rate was found to be remarkably high. The melting temperature Tm for two of them was substantially higher than that of the wild type. A double mutant with even higher Tm was also obtained. Our FEF captures the essential physics of the stability changes upon mutations. PMID:27056055

  11. Identification and characterization of a novel GGA/C-binding protein, GBP-i, that is rapidly inducible by cytokines.

    PubMed Central

    Raj, G V; Khalili, K

    1994-01-01

    Immunosuppressive states with accompanying alterations in cytokine profiles have been postulated to play a vital role in the reactivation of viruses from latency. Cytokines regulate gene expression by activating transcription factors via well-characterized signal transduction pathways. In this study, we report the identification of a novel inducible protein, GBP-i, that binds to a double-stranded GGA/C-rich region of the transcriptional control region of the human papovavirus JC virus (JCV), specifically within the origin of viral DNA replication. GBP-i is distinct from previously characterized GC-box-binding proteins with respect to both its sequence specificity and its electrophoretic mobility on native and denaturing gels. GBP-i responds within 90 min to phorbol myristate acetate stimulation; however, unlike typical phorbol myristate acetate-inducible factors, this rapid induction is regulated primarily at the transcriptional level. Further, the induction of GBP-i appears to be widespread and mediated by many inflammatory cytokines, including interleukin-1 beta, tumor necrosis factor alpha, gamma interferon, and transforming growth factor beta. Interestingly, the induced protein acts as a transcriptional repressor in its native context in the JCVL promoter. However, when its binding sequence is transposed to a heterologous promoter, GBP-i appears to function as a transcriptional activator. The data presented here suggest a role for GBP-i in cytokine-mediated induction of viral and cellular genes. Images PMID:7969118

  12. A rapid identification of hit molecules for target proteins via physico-chemical descriptors.

    PubMed

    Mukherjee, Goutam; Jayaram, B

    2013-06-21

    We report here a novel computationally fast protocol (RASPD) for identifying good candidates for any target protein from any molecule/million molecule database. A QSAR-type equation sets up the extent of complementarity of the physico-chemical properties of the target protein and the candidate molecule and an estimate of the binding energy is generated. A correlation coefficient of 0.84 and an average error ±1.45 kcal mol(-1) are obtained for the calculated protein-ligand binding energies against experiment for more than 380 protein-ligand complexes. RASPD is seen to perform better than other popular scoring functions in predicting binding energies. The most interesting feature of this methodology is that it takes only a fraction of a second for calculating the binding energy of any ligand without docking in the active site of the target protein as opposed to several minutes for regular docking and scoring methods, while the accuracy in sorting good candidates remains comparable to that of conventional techniques. An entire million compound library, a (~10(5) compound) natural product library and a (~10(5) compound) NCI database can be scanned against a specified target protein within a few minutes for identifying hit molecules. The RASPD methodology is freely accessible at . PMID:23646352

  13. Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates

    PubMed Central

    Ju, Jung Won; Kim, Ho-Cheol; Shin, Hyun-Il; Kim, Yu Jung; Kim, Dong-Myung

    2015-01-01

    Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA. PMID:26599101

  14. Rapid identification of amino acid types in proteins using phase modulated 2D HN(CACB) and 2D HN(COCACB)

    NASA Astrophysics Data System (ADS)

    Dubey, Abhinav; Mondal, Somnath; Chandra, Kousik; Atreya, Hanudatta S.

    2016-06-01

    We present a simple approach to rapidly identify amino acid types in proteins from a 2D spectrum. The method is based on the fact that 13Cβ chemical shifts of different amino acid types fall in distinct spectral regions. By evolving the 13C chemical shifts in the conventional HNCACB or HN(CO)CACB type experiment for a single specified delay period, the phase of the cross peaks of different amino acid residues are modulated depending on their 13Cβ shift values. Following this specified evolution period, the 2D HN projections of these experiments are acquired. The 13C evolution period can be chosen such that all residues belonging to a given set of amino acid types have the same phase pattern (positive or negative) facilitating their identification. This approach does not require the preparation of any additional samples, involves the analysis of 2D [15N-1H] HSQC-type spectra obtained from the routinely used triple resonance experiments with minor modifications, and is applicable to deuterated proteins. The method will be useful for quick assignment of signals that shift during ligand binding or in combination with selective labeling/unlabeling approaches for identification of amino acid types to aid the sequential assignment process.

  15. Rapid identification of amino acid types in proteins using phase modulated 2D HN(CACB) and 2D HN(COCACB).

    PubMed

    Dubey, Abhinav; Mondal, Somnath; Chandra, Kousik; Atreya, Hanudatta S

    2016-06-01

    We present a simple approach to rapidly identify amino acid types in proteins from a 2D spectrum. The method is based on the fact that (13)C(β) chemical shifts of different amino acid types fall in distinct spectral regions. By evolving the (13)C chemical shifts in the conventional HNCACB or HN(CO)CACB type experiment for a single specified delay period, the phase of the cross peaks of different amino acid residues are modulated depending on their (13)C(β) shift values. Following this specified evolution period, the 2D HN projections of these experiments are acquired. The (13)C evolution period can be chosen such that all residues belonging to a given set of amino acid types have the same phase pattern (positive or negative) facilitating their identification. This approach does not require the preparation of any additional samples, involves the analysis of 2D [(15)N-(1)H] HSQC-type spectra obtained from the routinely used triple resonance experiments with minor modifications, and is applicable to deuterated proteins. The method will be useful for quick assignment of signals that shift during ligand binding or in combination with selective labeling/unlabeling approaches for identification of amino acid types to aid the sequential assignment process. PMID:27078090

  16. A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus.

    PubMed

    Lund, Christian H; Bromley, Jennifer R; Stenbæk, Anne; Rasmussen, Randi E; Scheller, Henrik V; Sakuragi, Yumiko

    2015-01-01

    A growing body of evidence suggests that protein-protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. We tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. Our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta. PMID:25326916

  17. A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus

    SciTech Connect

    Lund, C. H.; Bromley, J. R.; Stenbaek, A.; Rasmussen, R. E.; Scheller, H. V.; Sakuragi, Y.

    2014-10-18

    A growing body of evidence suggests that protein–protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. We tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. In conclusion, our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta.

  18. A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus

    DOE PAGESBeta

    Lund, C. H.; Bromley, J. R.; Stenbaek, A.; Rasmussen, R. E.; Scheller, H. V.; Sakuragi, Y.

    2014-10-18

    A growing body of evidence suggests that protein–protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. Wemore » tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. In conclusion, our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta.« less

  19. Rapid methods for identification of yeasts.

    PubMed Central

    Huppert, M; Harper, G; Sun, S H; Delanerolle, V

    1975-01-01

    Opportunistic infections by yeasts have been implicated as one of the major causes of complications in the compromised patient. Rapid recognition and identification of these yeasts is essential for patient management, but conventional liquid medium methods for completing identification tests are cumbersome and time consuming. Rapid tests have been devised based on modifications of methods commonly used in bacteriology. These rapid methods included tests for carbohydrate and nitrate assimilation, fermentation, and urease production. These were compared with several current methods for accuracy of results, for time to final identification, and for economy of time and reagents. In addition, the usual tests for pseudogerm tube formation, for production of hyphae or pseudohyphae, and for growth temperatures were included. The rapid tests achieved 96% or better accuracy compared with expected results, and 46 species of yeasts were identified in 1 to 2 days compared with the 10 to 14 days required by conventional liquid culture methods. Images PMID:1241586

  20. Rapid identification of microorganisms by intrinsic fluorescence

    NASA Astrophysics Data System (ADS)

    Bhatta, Hemant; Goldys, Ewa M.; Learmonth, Robert

    2005-03-01

    Microbial contamination has serious consequences for the industries that use fermentation processes. Common contaminants such as faster growing lactic acid bacteria or wild yeast can rapidly outnumber inoculated culture yeast and produce undesirable end products. Our study focuses on a rapid method of identification of such contaminants based on autofluorescence spectroscopy of bacterial and yeast species. Lactic acid bacteria (Lac-tobacillus casei), and yeast (Saccharomyces cerevisiae) were cultured under controlled conditions and studied for variations in their autofluorescence. We observed spectral differences in the spectral range representative of tryptophan residues of proteins, with excitation at 290 nm and emission scanned in the 300 nm - 440 nm range. Excitation scans between 240 nm and 310 nm were also performed for the emission at 340 nm. Moreover, we observed clearly pronounced differences in the excitation and emission in the visible range, with 410 nm excitation. These results demonstrate that bacterial and yeast species can be differentiated using their intrinsic fluorescence both in UV and in the visible region. The comparative spectroscopic study of selected strains of Saccharomyces yeast showed clear differences between strains. Spectrally-resolved laser scanning microscopy was carried out to link the results obtained using ensembles of cells with spectral properties of individual cells. Strongly fluorescent subpopulation were observed for all yeast strains with excitation at 405 nm. The fluorescence spectra showed variations correlated with cell brightness. The presented results demonstrate that using autofluorescence, it is possible to differentiate between yeast and lactic acid bacteria and between different yeast species.

  1. Rapid identification of Streptomyces isolates by MALDI-TOF MS.

    PubMed

    Loucif, Lotfi; Bendjama, Esma; Gacemi-Kirane, Djamila; Rolain, Jean-Marc

    2014-12-01

    The recent emergence of multidrug-resistant bacteria over the last decade has led to a renewal in the discovery of new antimicrobial drugs. Streptomyces members are practically unlimited sources of new antibiotics. However, the identification of Streptomyces species is difficult and time-consuming. Therefore, there is a need for alternative methods for their rapid identification. In this study, an efficient protocol of identification using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) was developed and applied for the rapid identification of Streptomyces isolates from the El Kala lakes in northeastern Algeria. A collection of 48 Streptomyces isolates were used for this study. The optimized procedure allowed us to obtain specific and reproducible protein spectra for each Streptomyces isolate tested. The spectra generated were used to build a preliminary local database based on their initial 16S rRNA identification. The blind test used for the identification of 20 Streptomyces strains already available in our created database and 20 unknown Streptomyces isolates showed that all (100%) of the Streptomyces strains listed in the database were rapidly (<30min) identified with high scores of up to 2.8. Here, for the first time we showed that MALDI-TOF MS could be used as a cost-effective tool for the rapid identification of Streptomyces isolates. PMID:24862894

  2. Transcriptomics-assisted quantitative trait locus fine mapping for the rapid identification of a nodulin 26-like intrinsic protein gene regulating boron efficiency in allotetraploid rapeseed.

    PubMed

    Hua, Yingpeng; Zhang, Didi; Zhou, Ting; He, Mingliang; Ding, Guangda; Shi, Lei; Xu, Fangsen

    2016-07-01

    Allotetraploid rapeseed (Brassica napus L., An An Cn Cn , 2n = 4x = 38) is extraordinarily susceptible to boron (B) deficiency, a ubiquitous problem causing severe losses in seed yield. The breeding of B-efficient rapeseed germ plasm is a cost-effective and environmentally friendly strategy for the agricultural industry; however, genes regulating B efficiency in allotetraploid rapeseed have not yet been isolated. In this research, quantitative trait locus (QTL) fine mapping and digital gene expression (DGE) profiling were combined to identify the candidate genes underlying the major-effect QTL qBEC-A3a, which regulates B efficiency. Comparative phenotype analyses of the near-isogenic lines (NILs) indicated that qBEC-A3a plays a significant role in improving B efficiency under B deficiency. Exploiting QTL fine mapping and DGE analyses revealed a nodulin 26-like intrinsic protein (NIP) gene, which encodes a likely boric acid channel. The gene co-expression network for putative B transporters also highlighted its central role in the efficiency of B uptake. An integration of whole-genome re-sequencing (WGS) with bulked segregant analysis (BSA) authenticated the emerging availability of QTL-seq for the QTL analyses in allotetraploid rapeseed. Transcriptomics-assisted QTL mapping and comparative genomics provided novel insights into the rapid identification of quantitative trait genes (QTGs) in plant species with complex genomes. PMID:26934080

  3. Rapid Cellular Identification by Dynamic Electromechanical Response

    SciTech Connect

    Nikiforov, Maxim; Jesse, Stephen; Kalinin, Sergei V; Reukov, Vladimir V; Vertegel, Alexey; Thompson, Gary L

    2009-01-01

    Coupling between electrical and mechanical phenomena is ubiquitous in living systems. Here, we demonstrate rapid identification of cellular organisms using difference in electromechanical activity in a broad frequency range. Principal component analysis of the dynamic electromechanical response spectra bundled with neural network based recognition provides a robust identification algorithm based on their electromechanical signature, and allows unambiguous differentiation of model Micrococcus Lysodeikticus and Pseudomonas Fluorescens system. This methodology provides a universal pathway for biological identification obviating the need for well-defined analytical models of Scanning Probe Microscopy response.

  4. Rapid detection and identification of infectious agents

    SciTech Connect

    Kingsbury, D.T.; Falkow, S.

    1985-01-01

    This book contains papers divided among five sections. Some of the paper titles are: Aspects of Using Nucleic Acid Filter Hybridization to Characterize and Detect Enteroviral RNAs; Rapid Identification of Lesihmania Species using Specific Hybridization of Kinetoplast DNA Sequences; Selection of DNA Probes for use in the Diagnosis of Infectious Disease; and Summary of DNA Probes.

  5. Protein identification and Peptide expression resolver: harmonizing protein identification with protein expression data.

    PubMed

    Kearney, Paul; Butler, Heather; Eng, Kevin; Hugo, Patrice

    2008-01-01

    Proteomic discovery platforms generate both peptide expression information and protein identification information. Peptide expression data are used to determine which peptides are differentially expressed between study cohorts, and then these peptides are targeted for protein identification. In this paper, we demonstrate that peptide expression information is also a powerful tool for enhancing confidence in protein identification results. Specifically, we evaluate the following hypothesis: tryptic peptides originating from the same protein have similar expression profiles across samples in the discovery study. Evidence supporting this hypothesis is provided. This hypothesis is integrated into a protein identification tool, PIPER (Protein Identification and Peptide Expression Resolver), that reduces erroneous protein identifications below 5%. PIPER's utility is illustrated by application to a 72-sample biomarker discovery study where it is demonstrated that false positive protein identifications can be reduced below 5%. Consequently, it is recommended that PIPER methodology be incorporated into proteomic studies where both protein expression and identification data are collected. PMID:18062667

  6. Identification of a receptor-like protein kinase gene rapidly induced by abscisic acid, dehydration, high salt, and cold treatments in Arabidopsis thaliana.

    PubMed Central

    Hong, S W; Jon, J H; Kwak, J M; Nam, H G

    1997-01-01

    A cDNA clone for a receptor-like protein kinase gene (RPK1) was isolated from Arabidopsis thaliana. The clone is 1952 bp long with 1623 bp of an open reading frame encoding a peptide of 540 amino acids. The deduced peptide (RPK1) contains four distinctive domains characteristic of receptor kinases: (a) a putative amino-terminal signal sequence domain; (b) a domain with five extracellular leucine-rich repeat sequences; (c) a membrane-spanning domain; and (d) a cytoplasmic protein kinase domain that contains all of the 11 subdomains conserved among protein kinases. The RPK1 gene is expressed in flowers, stems, leaves, and roots. Expression of the RPK1 gene is induced within 1 h after treatment with abscisic acid (ABA). The gene is also rapidly induced by several environmental stresses such as dehydration, high salt, and low temperature, suggesting that the gene is involved in a general stress response. The dehydration-induced expression is not impaired in aba-1, abi1-1, abi2-1, and abi3-1 mutants, suggesting that the dehydration-induced expression of the RPK1 gene is ABA-independent. A possible role of this gene in the signal transduction pathway of ABA and the environmental stresses is discussed. PMID:9112773

  7. Rapid Identification of Emerging Pathogens: Coronavirus

    PubMed Central

    Hofstadler, Steven A.; Blyn, Lawrence B.; Eshoo, Mark W.; Hall, Thomas A.; Massire, Christian; Levene, Harold M.; Hannis, James C.; Harrell, Patina M.; Neuman, Benjamin; Buchmeier, Michael J.; Jiang, Yun; Ranken, Raymond; Drader, Jared J.; Samant, Vivek; Griffey, Richard H.; McNeil, John A.; Crooke, Stanley T.; Ecker, David J.

    2005-01-01

    We describe a new approach for infectious disease surveillance that facilitates rapid identification of known and emerging pathogens. The process uses broad-range polymerase chain reaction (PCR) to amplify nucleic acid targets from large groupings of organisms, electrospray ionization mass spectrometry for accurate mass measurements of PCR products, and base composition signature analysis to identify organisms in a sample. We demonstrate this principle by using 14 isolates of 9 diverse Coronavirus spp., including the severe acute respiratory syndrome–associated coronavirus (SARS-CoV). We show that this method could identify and distinguish between SARS and other known CoV, including the human CoV 229E and OC43, individually and in a mixture of all 3 human viruses. The sensitivity of detection, measured by using titered SARS-CoV spiked into human serum, was ≈1 PFU/mL. This approach, applicable to the surveillance of bacterial, viral, fungal, or protozoal pathogens, is capable of automated analysis of >900 PCR reactions per day. PMID:15757550

  8. Rapid identification of Listeria spp.: an AOAC performance test of the MIT 1000 rapid microbial identification system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methods that rapidly confirm the identification of foodborne pathogens are highly desired. The Micro Imaging Technology (MIT) 1000 Rapid Microbial Identification (RMID) System is a benchtop instrument that detects laser light scattered from individual bacterial cells in solution with an array of 35 ...

  9. Rapid visco analysis of food protein pastes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Whey protein isolate (WPI) powders are used in many formulations to boost nutrients. To predict the pasting behavior of proteins, WPI was tested under varying temperatures, using the Rapid-Visco-Analyzer (RVA), under pasting temperatures from 65 to 75 degrees'C, RVA speeds from 100 to 500 rpm, and ...

  10. Rapid Identification of Protein Biomarkers of E. coli O157:H7 by MALDI-TOF-TOF Mass Spectrometry and Top-Down Proteomics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have identified six protein biomarkers from two strains of E. coli O157:H7 and one non-pathogenic E. coli strain by matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight tandem mass spectrometry (TOF/TOF-MS/MS) and top-down proteomics. Mature, intact proteins were ext...

  11. [Rapid identification of Enterobacteriaceae: evaluation of 3 commercial systems].

    PubMed

    Sala, A; Dore, R; Raffaele, L; Cappello, P

    1985-06-01

    We have compared three rapid systems for the identification of Enterobacteriaceae: MS-2, Rapid 20E, Micro-ID. These methods allows to identifications of bacteria within 4-5 hours. We have chosen API 20E as reference system; because it is normally used in the clinical microbiology laboratories. We have noted good agreement of concordance for MS-2, Micro-ID and Rapid 20E towards API 20E, respectively 95, 90, 84%. We have, moreover, analysed significative difference about three systems biochemical tests in comparison with the same of API 20E. PMID:4080964

  12. [Study of Rapid Species Identification of Bacteria in Water].

    PubMed

    Wang, Jiu-yue; Zhao, Nan-jing; Duan, Jing-bo; Fang, Li; Meng, De-shuo; Yang, Rui-fang; Xiao, Xue; Liu, Jian-guo; Liu, Wen-qing

    2015-09-01

    Multi-wavelength ultraviolet visible (UV-Vis) transmission spectra of bacteria combined the forward scattering and absorption properties of microbes, contains substantial information on size, shape, and the other chemical, physiological character of bacterial cells, has the bacterial species specificity, which can be applied to rapid species identification of bacterial microbes. Four different kinds of bacteria including Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Klebsiella pneumonia which were commonly existed in water were researched in this paper. Their multi-wavelength UV-Vis transmission spectra were measured and analyzed. The rapid identification method and model of bacteria were built which were based on support vector machine (SVM) and multi-wavelength UV-Vis transmission spectra of the bacteria. Using the internal cross validation based on grid search method of the training set for obtaining the best penalty factor C and the kernel parameter g, which the model needed. Established the bacteria fast identification model according to the optimal parameters and one-against-one classification method included in LibSVM. Using different experimental bacteria strains of transmission spectra as a test set of classification accuracy verification of the model, the analysis results showed that the bacterial rapid identification model built in this paper can identification the four kinds bacterial which chosen in this paper as the accuracy was 100%, and the model also can identified different subspecies of E. coli test set as the accuracy was 100%, proved the model had a good stability in identification bacterial species. In this paper, the research results of this study not only can provide a method for rapid identification and early warning of bacterial microbial in drinking water sources, but also can be used as the microbes identified in biomedical a simple, rapid and accurate means. PMID:26669181

  13. catRAPID signature: identification of ribonucleoproteins and RNA-binding regions

    PubMed Central

    Livi, Carmen Maria; Klus, Petr; Delli Ponti, Riccardo; Tartaglia, Gian Gaetano

    2016-01-01

    Motivation: Recent technological advances revealed that an unexpected large number of proteins interact with transcripts even if the RNA-binding domains are not annotated. We introduce catRAPID signature to identify ribonucleoproteins based on physico-chemical features instead of sequence similarity searches. The algorithm, trained on human proteins and tested on model organisms, calculates the overall RNA-binding propensity followed by the prediction of RNA-binding regions. catRAPID signature outperforms other algorithms in the identification of RNA-binding proteins and detection of non-classical RNA-binding regions. Results are visualized on a webpage and can be downloaded or forwarded to catRAPID omics for predictions of RNA targets. Availability and implementation: catRAPID signature can be accessed at http://s.tartaglialab.com/new_submission/signature. Contact: gian.tartaglia@crg.es or gian@tartaglialab.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26520853

  14. Rapid identification of viridans streptococci by mass spectrometric discrimination.

    PubMed

    Friedrichs, C; Rodloff, A C; Chhatwal, G S; Schellenberger, W; Eschrich, K

    2007-08-01

    Viridans streptococci (VS) are responsible for several systemic diseases, such as endocarditis, abscesses, and septicemia. Unfortunately, species identification by conventional methods seems to be more difficult than species identification of other groups of bacteria. The aim of the present study was to evaluate the use of cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) for the rapid identification of 10 different species of VS. A total of 99 VS clinical isolates, 10 reference strains, and 20 strains from our in-house culture collection were analyzed by MALDI-TOF-MS. To evaluate the mass-spectrometric discrimination results, all strains were identified in parallel by phenotypic and genotypic methods. MALDI-TOF-MS identified 71 isolates as the mitis group, 23 as the anginosus group, and 5 as Streptococcus salivarius. Comparison of the species identification results obtained by the MALDI-TOF-MS analyses and with the phenotypic/genotypic identification systems showed 100% consistency at the species level. Thus, MALDI-TOF-MS seems to be a rapid and reliable method for the identification of species of VS from clinical samples. PMID:17553974

  15. Protein Identification Using Top-Down

    SciTech Connect

    Liu, Xiaowen; Sirotkin, Yakov; Shen, Yufeng; Anderson, Gordon A.; Tsai, Yi-Hsuan S.; Ting, Ying S.; Goodlett, David R.; Smith, Richard D.; Bafna, Vineet; Pevzner, Pavel A.

    2012-06-01

    In the last two years, due to advances in protein separation and mass spectrometry, top-down mass spectrometry moved from analyzing single proteins to analyzing complex samples and identifying hundreds and even thousands of proteins. However, computational tools for database search of top-down spectra against protein databases are still in infancy. We describe MS-Align+, a fast algorithm for top-down protein identification based on spectral alignment that enables searches for unexpected post-translational modifications (PTMs). We also propose a method for evaluating statistical significance of top-down protein identifications and further benchmark MS-Align+ along with PIITA, ProSightPTM and SEQUEST, which were previously used for top-down MS/MS database searches. We demonstrate that MS-Align+ and PIITA significantly increase the number of identified proteins as compared to ProSightPTM and SEQUEST.

  16. HRR length and velocity decision regions for rapid target identification

    NASA Astrophysics Data System (ADS)

    Hussain, Moayyed A.

    1999-09-01

    Effective theater defense requires rapid target identification with ground sensors. Modern radar performs target recognition and target imaging tasks, in addition to conventional tasks of detection and tracking. New processing techniques, like stepped frequency waveforms and RF hardware are now becoming available and will soon result in lower- cost high resolution rate. Additional feature extraction, namely length and velocity obtained from tracker can be used to design an efficient and a rapid ID after a preliminary recognition is performed. Prior information of these features for critical set of targets can be used to design decision regions for a given SNR value.

  17. Identification of Post-translational Modifications of Plant Protein Complexes

    PubMed Central

    Piquerez, Sophie J. M.; Balmuth, Alexi L.; Sklenář, Jan; Jones, Alexandra M.E.; Rathjen, John P.; Ntoukakis, Vardis

    2014-01-01

    Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved. Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs. This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein. PMID:24637539

  18. Rapid identification of single microbes by various Raman spectroscopic techniques

    NASA Astrophysics Data System (ADS)

    Rösch, Petra; Harz, Michaela; Schmitt, Michael; Peschke, Klaus-Dieter; Ronneberger, Olaf; Burkhardt, Hans; Motzkus, Hans-Walter; Lankers, Markus; Hofer, Stefan; Thiele, Hans; Popp, Jürgen

    2006-02-01

    A fast and unambiguous identification of microorganisms is necessary not only for medical purposes but also in technical processes such as the production of pharmaceuticals. Conventional microbiological identification methods are based on the morphology and the ability of microbes to grow under different conditions on various cultivation media depending on their biochemical properties. These methods require pure cultures which need cultivation of at least 6 h but normally much longer. Recently also additional methods to identify bacteria are established e.g. mass spectroscopy, polymerase chain reaction (PCR), flow cytometry or fluorescence spectroscopy. Alternative approaches for the identification of microorganisms are vibrational spectroscopic techniques. With Raman spectroscopy a spectroscopic fingerprint of the microorganisms can be achieved. Using UV-resonance Raman spectroscopy (UVRR) macromolecules like DNA/RNA and proteins are resonantly enhanced. With an excitation wavelength of e.g. 244 nm it is possible to determine the ratio of guanine/cytosine to all DNA bases which allows a genotypic identification of microorganisms. The application of UVRR requires a large amount of microorganisms (> 10 6 cells) e.g. at least a micro colony. For the analysis of single cells micro-Raman spectroscopy with an excitation wavelength of 532 nm can be used. Here, the obtained information is from all type of molecules inside the cells which lead to a chemotaxonomic identification. In this contribution we show how wavelength dependent Raman spectroscopy yields significant molecular information applicable for the identification of microorganisms on a single cell level.

  19. Identification of essential proteins based on edge clustering coefficient.

    PubMed

    Wang, Jianxin; Li, Min; Wang, Huan; Pan, Yi

    2012-01-01

    Identification of essential proteins is key to understanding the minimal requirements for cellular life and important for drug design. The rapid increase of available protein-protein interaction (PPI) data has made it possible to detect protein essentiality on network level. A series of centrality measures have been proposed to discover essential proteins based on network topology. However, most of them tended to focus only on the location of single protein, but ignored the relevance between interactions and protein essentiality. In this paper, a new centrality measure for identifying essential proteins based on edge clustering coefficient, named as NC, is proposed. Different from previous centrality measures, NC considers both the centrality of a node and the relationship between it and its neighbors. For each interaction in the network, we calculate its edge clustering coefficient. A node’s essentiality is determined by the sum of the edge clustering coefficients of interactions connecting it and its neighbors. The new centrality measure NC takes into account the modular nature of protein essentiality. NC is applied to three different types of yeast protein-protein interaction networks, which are obtained from the DIP database, the MIPS database and the BioGRID database, respectively. The experimental results on the three different networks show that the number of essential proteins discovered by NC universally exceeds that discovered by the six other centrality measures: DC, BC, CC, SC, EC, and IC. Moreover, the essential proteins discovered by NC show significant cluster effect. PMID:22084147

  20. Rapid bacterial identification using evanescent-waveguide oligonucleotide microarray classification.

    PubMed

    Francois, Patrice; Charbonnier, Yvan; Jacquet, Jean; Utinger, Dominic; Bento, Manuela; Lew, Daniel; Kresbach, Gerhard M; Ehrat, Markus; Schlegel, Werner; Schrenzel, Jacques

    2006-06-01

    Bacterial identification relies primarily on culture-based methodologies and requires 48-72 h to deliver results. We developed and used i) a bioinformatics strategy to select oligonucleotide signature probes, ii) a rapid procedure for RNA labelling and hybridization, iii) an evanescent-waveguide oligoarray with exquisite signal/noise performance, and iv) informatics methods for microarray data analysis. Unique 19-mer signature oligonucleotides were selected in the 5'-end of 16s rDNA genes of human pathogenic bacteria. Oligonucleotides spotted onto a Ta(2)O(5)-coated microarray surface were incubated with chemically labelled total bacterial RNA. Rapid hybridization and stringent washings were performed before scanning and analyzing the slide. In the present paper, the eight most abundant bacterial pathogens representing >54% of positive blood cultures were selected. Hierarchical clustering analysis of hybridization data revealed characteristic patterns, even for closely related species. We then evaluated artificial intelligence-based approaches that outperformed conventional threshold-based identification schemes on cognate probes. At this stage, the complete procedure applied to spiked blood cultures was completed in less than 6 h. In conclusion, when coupled to optimal signal detection strategy, microarrays provide bacterial identification within a few hours post-sampling, allowing targeted antimicrobial prescription. PMID:16216356

  1. Identification of extracellularly phosphorylated membrane proteins.

    PubMed

    Burghoff, Sandra; Willberg, Wibke; Schrader, Jürgen

    2015-10-01

    Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling. PMID:26152529

  2. Rapid Molecular Identification of Human Taeniid Cestodes by Pyrosequencing Approach

    PubMed Central

    Thanchomnang, Tongjit; Tantrawatpan, Chairat; Intapan, Pewpan M.; Sanpool, Oranuch; Janwan, Penchom; Lulitanond, Viraphong; Tourtip, Somjintana; Yamasaki, Hiroshi; Maleewong, Wanchai

    2014-01-01

    Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse. PMID:24945530

  3. Rapid molecular identification of human taeniid cestodes by pyrosequencing approach.

    PubMed

    Thanchomnang, Tongjit; Tantrawatpan, Chairat; Intapan, Pewpan M; Sanpool, Oranuch; Janwan, Penchom; Lulitanond, Viraphong; Tourtip, Somjintana; Yamasaki, Hiroshi; Maleewong, Wanchai

    2014-01-01

    Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse. PMID:24945530

  4. Emerging technologies for rapid identification of bloodstream pathogens.

    PubMed

    Kothari, Atul; Morgan, Margie; Haake, David A

    2014-07-15

    Technologies for rapid microbial identification are poised to revolutionize clinical microbiology and enable informed decision making for patients with life-threatening bloodstream infections. Species identification of microorganisms in positive blood cultures can be performed in minutes using commercial fluorescence in situ hybridization tests or mass spectroscopy. Microorganisms in positive blood cultures can also be identified within 1-2.5 hours using automated polymerase chain reaction-based systems that can also detect selected antibiotic resistance markers, such as methicillin resistance. When combined with antibiotic stewardship programs, these approaches improve clinical outcomes and reduce healthcare expenditures. Tests for direct detection in whole blood samples are highly desirable because of their potential to identify bloodstream pathogens without waiting 1-2 days for blood cultures to become positive. However, results for pathogen detection in whole blood do not overlap with those of conventional blood culture techniques and we are still learning how best to use these approaches. PMID:24771332

  5. Rapid identification of cytokinins by an immunological method

    SciTech Connect

    Morris, R.O.; Jameson, P.E.; Morris, J.W. ); Laloue, M. )

    1991-04-01

    A method for rapid identification of bacterial cytokinins has been developed in which cultures are fed ({sup 3}H)adenine, the cytokinins (including, {sup 3}H-labeled cytokinins) are isolated by immunoaffinity chromatography, and analyzed by HPLC with on-line scintillation counting. Analysis of Agrobacterium tumefaciens strains showed that some produced primarily trans-zeatin, whereas others produced primarily trans-zeatin riboside. Pseudomonas syringae pv savastanoi produced mixtures of transzeatin, dihydrozeatin, 1{double prime}-methyl-trans-zeatin riboside, and other unknown cytokinin-like substances. Corynebacterium fascians, produced cis-zeatin, isopentenyladenine and isopentenyladenosine. The technique is designed for qualitative rather than quantitative studies and allows ready identification of bacterial cytokinins. It may also have utility in the study of plant cytokinins if adequate incorporation of label into cytokinin precursor pools can be achieved.

  6. Autonomous Metabolomics for Rapid Metabolite Identification in Global Profiling

    PubMed Central

    2015-01-01

    An autonomous metabolomic workflow combining mass spectrometry analysis with tandem mass spectrometry data acquisition was designed to allow for simultaneous data processing and metabolite characterization. Although previously tandem mass spectrometry data have been generated on the fly, the experiments described herein combine this technology with the bioinformatic resources of XCMS and METLIN. As a result of this unique integration, we can analyze large profiling datasets and simultaneously obtain structural identifications. Validation of the workflow on bacterial samples allowed the profiling on the order of a thousand metabolite features with simultaneous tandem mass spectra data acquisition. The tandem mass spectrometry data acquisition enabled automatic search and matching against the METLIN tandem mass spectrometry database, shortening the current workflow from days to hours. Overall, the autonomous approach to untargeted metabolomics provides an efficient means of metabolomic profiling, and will ultimately allow the more rapid integration of comparative analyses, metabolite identification, and data analysis at a systems biology level. PMID:25496351

  7. Autonomous metabolomics for rapid metabolite identification in global profiling.

    PubMed

    Benton, H Paul; Ivanisevic, Julijana; Mahieu, Nathaniel G; Kurczy, Michael E; Johnson, Caroline H; Franco, Lauren; Rinehart, Duane; Valentine, Elizabeth; Gowda, Harsha; Ubhi, Baljit K; Tautenhahn, Ralf; Gieschen, Andrew; Fields, Matthew W; Patti, Gary J; Siuzdak, Gary

    2015-01-20

    An autonomous metabolomic workflow combining mass spectrometry analysis with tandem mass spectrometry data acquisition was designed to allow for simultaneous data processing and metabolite characterization. Although previously tandem mass spectrometry data have been generated on the fly, the experiments described herein combine this technology with the bioinformatic resources of XCMS and METLIN. As a result of this unique integration, we can analyze large profiling datasets and simultaneously obtain structural identifications. Validation of the workflow on bacterial samples allowed the profiling on the order of a thousand metabolite features with simultaneous tandem mass spectra data acquisition. The tandem mass spectrometry data acquisition enabled automatic search and matching against the METLIN tandem mass spectrometry database, shortening the current workflow from days to hours. Overall, the autonomous approach to untargeted metabolomics provides an efficient means of metabolomic profiling, and will ultimately allow the more rapid integration of comparative analyses, metabolite identification, and data analysis at a systems biology level. PMID:25496351

  8. Rapid Detection and Identification of Biogenic Aerosol Releases and Sources

    NASA Astrophysics Data System (ADS)

    Wagner, J.; Macher, J.; Ghosal, S.; Ahmed, K.; Hemati, K.; Wall, S.; Kumagai, K.

    2011-12-01

    Biogenic aerosols can be important contributors to aerosol chemistry, cloud droplet and ice nucleation, absorption and scattering of radiation, human health and comfort, and plant, animal, and microbial ecology. Many types of bioaerosols, e.g., fungal spores, are released into the atmosphere in response to specific climatological and meteorological conditions. The rapid identification of bioaerosol releases is thus important for better characterization of the above phenomena, as well as enabling public officials to respond quickly and appropriately to releases of infectious agents or biological toxins. One approach to rapid and accurate bioaerosol detection is to employ sequential, automated samples that can be fed directly into an image acquisition and data analysis device. Raman spectroscopy-based identification of bioaerosols, automated analysis of microscopy images, and automated detection of near-monodisperse peaks in aerosol size-distribution data were investigated as complementary approaches to traditional, manual methods for the identification and counting of fungal and actinomycete spores. Manual light microscopy is a widely used analytical technique that is compatible with a number of air sample formats and requires minimal sample preparation. However, a major drawback is its dependence on a human analyst's ability to distinguish particles and accurately count, size, and identify them. Therefore, automated methods, such as those evaluated in this study, have the potential to provide cost-effective and rapid alternatives if demonstrated to be accurate and reliable. An exploratory examination of individual spores for several macro- and microfungi (those with and without large fruiting bodies) by Raman microspectroscopy found unique spectral features that were used to identify fungi to the genus level. Automated analyses of digital spore images accurately recognized and counted single fungal spores and clusters. An automated procedure to discriminate near

  9. Rapid identification of bacteria with a disposable colorimetric sensing array.

    PubMed

    Carey, James R; Suslick, Kenneth S; Hulkower, Keren I; Imlay, James A; Imlay, Karin R C; Ingison, Crystal K; Ponder, Jennifer B; Sen, Avijit; Wittrig, Aaron E

    2011-05-18

    Rapid identification of both species and even specific strains of human pathogenic bacteria grown on standard agar has been achieved from the volatiles they produce using a disposable colorimetric sensor array in a Petri dish imaged with an inexpensive scanner. All 10 strains of bacteria tested, including Enterococcus faecalis and Staphylococcus aureus and their antibiotic-resistant forms, were identified with 98.8% accuracy within 10 h, a clinically important time frame. Furthermore, the colorimetric sensor arrays also proved useful as a simple research tool for the study of bacterial metabolism and as an easy method for the optimization of bacterial production of fine chemicals or other fermentation processes. PMID:21524080

  10. Evaluation of a rapid polymerase chain reaction based identification technique for Vibrio cholerae isolates.

    PubMed

    le Roux, W J; Masoabi, D; de Wet, C M E; Venter, S N

    2004-01-01

    Rapid and accurate identification of waterborne pathogens, such as Vibrio cholerae, in drinking-water sources is important to enable effective resource management and public health protection. Phenotypic systems currently being used for the identification of Vibrio cholerae isolates are time-consuming and the need exists for the development of suitable molecular techniques that can offer both fast and reliable identification. During this study, isolates identified as Vibrio cholerae by means of two different biochemical test systems (API 20E and VITEK 32) were analysed with the polymerase chain reaction (PCR) to compare the reliability of the various identification systems. The selected PCR technique amplified a sequence within the outer membrane protein of Vibrio cholerae, a gene specific for V. cholerae. It was found that out of 243 isolates biochemically identified as V. cholerae with either the API or VITEK system, 21 isolates did not give a positive result with the PCR detection method. Sequencing the 16S rDNA of more than half of these isolates and comparison of the sequences with Internet databases indicated that most of the isolates belonged to the genus Aeromonas. The results indicated that the rapid PCR procedure was more accurate than the API or VITEK systems currently being used for the phenotypic identification of Vibrio cholerae isolates. PMID:15318514

  11. Rapid Accurate Identification of Bacterial and Viral Pathogens

    SciTech Connect

    Dunn, John

    2007-03-09

    The goals of this program were to develop two assays for rapid, accurate identification of pathogenic organisms at the strain level. The first assay "Quantitative Genome Profiling or QGP" is a real time PCR assay with a restriction enzyme-based component. Its underlying concept is that certain enzymes should cleave genomic DNA at many sites and that in some cases these cuts will interrupt the connection on the genomic DNA between flanking PCR primer pairs thereby eliminating selected PCR amplifications. When this occurs the appearance of the real-time PCR threshold (Ct) signal during DNA amplification is totally eliminated or, if cutting is incomplete, greatly delayed compared to an uncut control. This temporal difference in appearance of the Ct signal relative to undigested control DNA provides a rapid, high-throughput approach for DNA-based identification of different but closely related pathogens depending upon the nucleotide sequence of the target region. The second assay we developed uses the nucleotide sequence of pairs of shmi identifier tags (-21 bp) to identify DNA molecules. Subtle differences in linked tag pair combinations can also be used to distinguish between closely related isolates..

  12. Rapid experimental SAD phasing and hot-spot identification with halogenated fragments

    PubMed Central

    Bauman, Joseph D.; Harrison, Jerry Joe E. K.; Arnold, Eddy

    2016-01-01

    Through X-ray crystallographic fragment screening, 4-bromopyrazole was discovered to be a ‘magic bullet’ that is capable of binding at many of the ligand ‘hot spots’ found in HIV-1 reverse transcriptase (RT). The binding locations can be in pockets that are ‘hidden’ in the unliganded crystal form, allowing rapid identification of these sites for in silico screening. In addition to hot-spot identification, this ubiquitous yet specific binding provides an avenue for X-ray crystallographic phase determination, which can be a significant bottleneck in the determination of the structures of novel proteins. The anomalous signal from 4-bromopyrazole or 4-iodopyrazole was sufficient to determine the structures of three proteins (HIV-1 RT, influenza A endonuclease and proteinase K) by single-wavelength anomalous dispersion (SAD) from single crystals. Both compounds are inexpensive, readily available, safe and very soluble in DMSO or water, allowing efficient soaking into crystals. PMID:26870381

  13. Comprehensive Identification of Proteins from MALDI Imaging*

    PubMed Central

    Maier, Stefan K.; Hahne, Hannes; Gholami, Amin Moghaddas; Balluff, Benjamin; Meding, Stephan; Schoene, Cédrik; Walch, Axel K.; Kuster, Bernhard

    2013-01-01

    Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful tool for the visualization of proteins in tissues and has demonstrated considerable diagnostic and prognostic value. One main challenge is that the molecular identity of such potential biomarkers mostly remains unknown. We introduce a generic method that removes this issue by systematically identifying the proteins embedded in the MALDI matrix using a combination of bottom-up and top-down proteomics. The analyses of ten human tissues lead to the identification of 1400 abundant and soluble proteins constituting the set of proteins detectable by MALDI IMS including >90% of all IMS biomarkers reported in the literature. Top-down analysis of the matrix proteome identified 124 mostly N- and C-terminally fragmented proteins indicating considerable protein processing activity in tissues. All protein identification data from this study as well as the IMS literature has been deposited into MaTisse, a new publically available database, which we anticipate will become a valuable resource for the IMS community. PMID:23782541

  14. Heat shock triggers rapid protein phosphorylation in soybean seedings

    SciTech Connect

    Krishnan, H.B.; Pueppke, S.G.

    1987-10-29

    Heat shock arrests the synthesis of many cellular proteins and simultaneously initiates expression of a unique set of proteins, termed heat shock proteins. We have found that heat shock rapidly triggers phosphorylation of a set of proteins in soybean seedlings. Although the kinetics of phosphorylation and the heat shock response are similar, the major identified phosphorylation products do not comigrate with heat shock proteins on polyacrylamide gels. Cadmium, which is known to induce the heat shock response, stimulates phosphorylation of the same set of proteins. The rapidity of phosphorylation suggests that it may play a pivotal role in sensing and transducing elevated temperature stress in plants.

  15. Passive IFF: Autonomous Nonintrusive Rapid Identification of Friendly Assets

    NASA Technical Reports Server (NTRS)

    Moynihan, Philip; Steenburg, Robert Van; Chao, Tien-Hsin

    2004-01-01

    A proposed optoelectronic instrument would identify targets rapidly, without need to radiate an interrogating signal, apply identifying marks to the targets, or equip the targets with transponders. The instrument was conceived as an identification, friend or foe (IFF) system in a battlefield setting, where it would be part of a targeting system for weapons, by providing rapid identification for aimed weapons to help in deciding whether and when to trigger them. The instrument could also be adapted to law-enforcement and industrial applications in which it is necessary to rapidly identify objects in view. The instrument would comprise mainly an optical correlator and a neural processor (see figure). The inherent parallel-processing speed and capability of the optical correlator would be exploited to obtain rapid identification of a set of probable targets within a scene of interest and to define regions within the scene for the neural processor to analyze. The neural processor would then concentrate on each region selected by the optical correlator in an effort to identify the target. Depending on whether or not a target was recognized by comparison of its image data with data in an internal database on which the neural processor was trained, the processor would generate an identifying signal (typically, friend or foe ). The time taken for this identification process would be less than the time needed by a human or robotic gunner to acquire a view of, and aim at, a target. An optical correlator that has been under development for several years and that has been demonstrated to be capable of tracking a cruise missile might be considered a prototype of the optical correlator in the proposed IFF instrument. This optical correlator features a 512-by-512-pixel input image frame and operates at an input frame rate of 60 Hz. It includes a spatial light modulator (SLM) for video-to-optical image conversion, a pair of precise lenses to effect Fourier transforms, a filter SLM

  16. Rapid Bacterial Identification Using Fourier Transform Infrared Spectroscopy

    SciTech Connect

    Valentine, Nancy B.; Johnson, Timothy J.; Su, Yin-Fong; Forrester, Joel B.

    2007-02-01

    Recent studies at Pacific Northwest National Laboratory (PNNL) using infrared spectroscopy combined with statistical analysis have shown the ability to identify and discriminate vegetative bacteria, bacterial spores and background interferents from one another. Since the anthrax releases in 2001, rapid identification of unknown powders has become a necessity. Bacterial endospores are formed by some Bacillus species as a result of the vegetative bacteria undergoing environmental stress, e.g. a lack of nutrients. Endospores are formed as a survival mechanism and are extremely resistant to heat, cold, sunlight and some chemicals. They become airborne easily and are thus readily dispersed which was demonstrated in the Hart building. Fourier Transform Infrared (FTIR) spectroscopy is one of several rapid analytical methods used for bacterial endospore identification. The most common means of bacterial identification is culturing, but this is a time-consuming process, taking hours to days. It is difficult to rapidly identify potentially harmful bacterial agents in a highly reproducible way. Various analytical methods, including FTIR, Raman, photoacoustic FTIR and Matrix Assisted Laser Desorption/Ionization (MALDI) have been used to identify vegetative bacteria and bacterial endospores. Each has shown certain areas of promise, but each has shortcomings in terms of sensitivity, measurement time or portability. IR spectroscopy has been successfully used to distinguish between the sporulated and vegetative state. [1,2] It has also shown its utility at distinguishing between the spores of different species. [2-4] There are several Bacillus species that occur commonly in nature, so it is important to be able to distinguish between the many different species versus those that present an imminent health threat. The spectra of the different sporulated species are all quite similar, though there are some subtle yet reproducible spectroscopic differences. Thus, a more robust and

  17. Rapid Protein Separations in Microfluidic Devices

    NASA Technical Reports Server (NTRS)

    Fan, Z. H.; Das, Champak; Xia, Zheng; Stoyanov, Alexander V.; Fredrickson, Carl K.

    2004-01-01

    This paper describes fabrication of glass and plastic microfluidic devices for protein separations. Although the long-term goal is to develop a microfluidic device for two-dimensional gel electrophoresis, this paper focuses on the first dimension-isoelectric focusing (IEF). A laser-induced fluorescence (LIF) imaging system has been built for imaging an entire channel in an IEF device. The whole-channel imaging eliminates the need to migrate focused protein bands, which is required if a single-point detector is used. Using the devices and the imaging system, we are able to perform IEF separations of proteins within minutes rather than hours in traditional bench-top instruments.

  18. Insect Seminal Fluid Proteins: Identification and Function

    PubMed Central

    Avila, Frank W.; Sirot, Laura K.; LaFlamme, Brooke A.; Rubinstein, C. Dustin; Wolfner, Mariana F.

    2014-01-01

    Seminal fluid proteins (SFPs) produced in reproductive tract tissues of male insects and transferred to females during mating induce numerous physiological and behavioral post-mating changes in females. These changes include decreasing receptivity to re-mating, affecting sperm storage parameters, increasing egg production, modulating sperm competition, feeding behaviors, and mating plug formation. In addition, SFPs also have anti-microbial functions and induce expression of anti-microbial peptides in at least some insects. Here, we review recent identification of insect SFPs and discuss the multiple roles these proteins play in the post-mating processes of female insects. PMID:20868282

  19. Rapid identification of Zygosaccharomyces with genus-specific primers.

    PubMed

    Hulin, Michelle; Wheals, Alan

    2014-03-01

    There has been a recent and rapid increase in the number of species of the genus Zygosaccharomyces which now comprises Z. bailii, Z. bisporus, Z. gambellarensis, Z. kombuchaensis, Z. lentus, Z. machadoi, Z. mellis, Z. parabaillii, Z. pseudobailii, Z. pseudorouxii, Z. rouxii, Z. sapae, and Z. siamensis. Z. pseudorouxii is an unofficial name given to isolates closely related to the newly-described species Z. sapae. The Zygosaccharomyces genus contains species that are important as food and beverage spoilage organisms and others are associated with fermentations and sweet foodstuffs, such as honey. Their economic significance means that the ability to identify them rapidly is of significant importance. Although Z. rouxii and Z. bailii have been genome-sequenced the extent of sequence data for the others, especially the newly-discovered species, is sometimes extremely limited which makes identification slow. However, parts of the ITS1/5.8S/ITS2 rDNA region contain sequences of sufficient similarity within the genus and of sufficient difference with outgroups, to be potential regions for the design of genus-wide specific primers. We report here the development of genus-specific primers that can detect all the major Zygosaccharomyces species including all those associated with foods; the rare and localised species Z. machadoi and Z. gambellarensis are not detected. The size of the single amplicon produced varies between species and in some cases is sufficiently different to assign provisional species identification. Sequence data from rDNA regions are available for virtually all described yeast species in all genera, thus, prior to having sufficient sequence data from structural genes, rDNA regions may provide more generally suitable candidates for both genus-specific and species-specific primer design. PMID:24382328

  20. Rapid identification of bacteria with miniaturized pyrolysis/GC analysis

    NASA Astrophysics Data System (ADS)

    Morgan, Catherine H.; Mowry, Curtis; Manginell, Ronald P.; Frye-Mason, Gregory C.; Kottenstette, Richard J.; Lewis, Patrick

    2001-02-01

    Identification of bacteria and other biological moieties finds a broad range of applications in the environmental, biomedical, agricultural, industrial, and military arenas. Linking these applications are biological markers such as fatty acids, whose mass spectral profiles can be used to characterize biological samples and to distinguish bacteria at the gram-type, genera, and even species level. Common methods of sample analysis require sample preparation that is both lengthy and labor intensive, especially for whole cell bacteria. The background technique relied on here utilizes chemical derivatization of fatty acids to the more volatile fatty acid methyl esters (FAMEs), which can be separated on a gas chromatograph column or input directly into a mass spectrometer. More recent publications demonstrate improved sample preparation time with in situ derivatization of whole bacterial samples using pyrolysis at the inlet; although much faster than traditional techniques, these systems still rely on bench-top analytical equipment and individual sample preparation. Development of a miniaturized pyrolysis/GC instrument by this group is intended to realize the benefits of FAME identification of bacteria and other biological samples while further facilitating sample handling and instrument portability. The technologies being fabricated and tested have the potential of achieving pyrolysis and FAME separation on a very small scale, with rapid detection time (1-10 min from introduction to result), and with a modular sample inlet. Performance results and sensor characterization will be presented for the first phase of instrument development, encompassing the microfabricated pyrolysis and gas chromatograph elements.

  1. A Rapid Screening Assay to Search for Phosphorylated Proteins in Tissue Extracts

    PubMed Central

    Garaguso, Ignazio; Borlak, Juergen

    2012-01-01

    Reversible protein phosphorylation is an essential mechanism in the regulation of diverse biological processes, nonetheless is frequently altered in disease. As most phosphoproteome studies are based on optimized in-vitro cell culture studies new methods are in need to improve de novo identification and characterization of phosphoproteins in extracts from tissues. Here, we describe a rapid and reliable method for the detection of phosphoproteins in tissue extract based on an experimental strategy that employs 1D and 2D SDS PAGE, Western immunoblotting of phosphoproteins, in-gel protease digestion and enrichment of phosphorpeptides using metal oxide affinity chromatography (MOAC). Subsequently, phosphoproteins are identified by MALDI-TOF-MS/MS with the CHCA-TL or DHB ML sample matrix preparation method and further characterized by various bioinformatic software tools to search for candidate kinases and phosphorylation-dependent binding motifs. The method was applied to mouse lung tissue extracts and resulted in an identification of 160 unique phosphoproteins. Notably, TiO2 enrichment of pulmonary protein extracts resulted in an identification of additional 17 phosphoproteins and 20 phosphorylation sites. By use of MOAC, new phosphorylation sites were identified as evidenced for the advanced glycosylation end product-specific receptor. So far this protein was unknown to be phosphorylated in lung tissue of mice. Overall the developed methodology allowed efficient and rapid screening of phosphorylated proteins and can be employed as a general experimental strategy for an identification of phosphoproteins in tissue extracts. PMID:23166814

  2. Influenza viruses in birds: rapid identification by counterimmunoelectrophoresis.

    PubMed Central

    Lecomte, J; Berthiaume, L; Boudreault, A

    1979-01-01

    Counterimmunoelectrophoresis with an antiserum raised in rabbits against the M protein of the avian N virus proved to be particularly useful for large-scale identification of influenza A virus isolates. Of a total of 231 hemagglutinating agents isolated from 1,656 rectal swabs collected from shore and open-country birds, 158 could be identified as influenza A viruses by counterimmunoelectrophoresis, and 75 were serologically related to Newcastle disease virus by hemagglutination inhibition with an antiserum to Newcastle disease virus. Two isolates contained a mixture of influenza A virus and Newcastle disease virus; although the Newcastle disease virus virus particles outnumbered the influenza A virus particles in a ratio of 1,000:1, as seen by electron microscopy, the latter could be readily detected by counterimmunoelectrophoresis. This type of assay appears to be of potential use for epidemiological surveillance of influenza virus isolated from humans and animals. It combines specificity, sensitivity, and simplicity. Images PMID:85632

  3. Development of an antigen-based rapid diagnostic test for the identification of blowfly (Calliphoridae) species of forensic significance.

    PubMed

    McDonagh, Laura; Thornton, Chris; Wallman, James F; Stevens, Jamie R

    2009-06-01

    In this study we examine the limitations of currently used sequence-based approaches to blowfly (Calliphoridae) identification and evaluate the utility of an immunological approach to discriminate between blowfly species of forensic importance. By investigating antigenic similarity and dissimilarity between the first instar larval stages of four forensically important blowfly species, we have been able to identify immunoreactive proteins of potential use in the development of species-specific immuno-diagnostic tests. Here we outline our protein-based approach to species determination, and describe how it may be adapted to develop rapid diagnostic assays for the 'on-site' identification of blowfly species. PMID:19414163

  4. YahO protein as a calibrant for top-down proteomic identification of Shiga toxin using MALDI-TOF-TOF-MS/MS and post-source decay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF-TOF) mass spectrometry is increasingly utilized for rapid top-down proteomic identification of proteins. This identification may involve analysis of either a pure protein or a protein mixture. For analysis of a pure protein...

  5. Rapid presumptive identification of Cryptococcus neoformans by staphylococcal coagglutination.

    PubMed Central

    Maccani, J E

    1981-01-01

    A coagglutination reagent was prepared by sensitizing the Cowan I strain of Staphylococcus aureus with rabbit immune globulin directed against Cryptococcus neofromans A15 and absorbed with C. laurentii. This reagent was evaluated for its usefulness in differentiating C. neoformans from other yeast colonies rapidly. Antigen-containing extracts were prepared form Sabouraud dextrose agar cultures of 48 C. neoformans, 33 other Cryptococcus species, 21 Candida, 4 Torulopsis, 3 Saccharomyces, and 2 Rhodotorula strains. This was done by suspending a 0.001-ml loopful of colony growth in 0.5 ml of phenolized saline, mixing for 30 s, and then centrifuging. Equal volumes (50 microliters) of coagglutination reagent and yeast extract were mixed within marked circles on a glass slide and then mechanically rotated at 180 rpm for 8 min. Forty-five of the 48 strains of C. neoformans produced strong (3+ to 4+) agglutination, and 3 strains of serotype C produced weak (1+ to 2+) agglutination with the reagent. Other Cryptococcus species which reacted positively were 4 C. albidus subsp. diffluens, 7 C. albidus subsp. albidus, and 2 C. terreus strains; however, false-positive errors in identification were circumvented by performing a supplemental rapid test for nitrate utilization which differentiated these yeasts from C. neoformans. None of the other yeasts tested (including 14 C. laurentii, 2 C. luteolus, and 2 C. uniguttulatus strains) produced any degree of agglutination with the reagent. A commercial cryptococcal latex agglutination reagent (Crypto-Test, Microbiological Associates, Walkersville, Md.) proved less reliable for identifying C. neoformans yeast colonies because of cross-reactions which occurred with all other species of Cryptococcus tested. PMID:7016909

  6. A genetically encoded aldehyde for rapid protein labelling.

    PubMed

    Tuley, Alfred; Lee, Yan-Jiun; Wu, Bo; Wang, Zhiyong U; Liu, Wenshe R

    2014-07-18

    Using a mutant pyrrolysyl-tRNA synthetase-tRNA(Pyl)(CUA) pair, 3-formyl-phenylalanine is genetically incorporated into proteins at amber mutation sites in Escherichia coli. This non-canonical amino acid readily reacts with hydroxylamine dyes, leading to rapid and site-selective protein labelling. PMID:24756176

  7. Epock: rapid analysis of protein pocket dynamics

    PubMed Central

    Laurent, Benoist; Chavent, Matthieu; Cragnolini, Tristan; Dahl, Anna Caroline E.; Pasquali, Samuela; Derreumaux, Philippe; Sansom, Mark S.P.; Baaden, Marc

    2015-01-01

    Summary: The volume of an internal protein pocket is fundamental to ligand accessibility. Few programs that compute such volumes manage dynamic data from molecular dynamics (MD) simulations. Limited performance often prohibits analysis of large datasets. We present Epock, an efficient command-line tool that calculates pocket volumes from MD trajectories. A plugin for the VMD program provides a graphical user interface to facilitate input creation, run Epock and analyse the results. Availability and implementation: Epock C++ source code, Python analysis scripts, VMD Tcl plugin, documentation and installation instructions are freely available at http://epock.bitbucket.org. Contact: benoist.laurent@gmail.com or baaden@smplinux.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25505095

  8. Rapid flow cytometric measurement of protein inclusions and nuclear trafficking

    PubMed Central

    Whiten, D. R.; San Gil, R.; McAlary, L.; Yerbury, J. J.; Ecroyd, H.; Wilson, M. R.

    2016-01-01

    Proteinaceous cytoplasmic inclusions are an indicator of dysfunction in normal cellular proteostasis and a hallmark of many neurodegenerative diseases. We describe a simple and rapid new flow cytometry-based method to enumerate, characterise and, if desired, physically recover protein inclusions from cells. This technique can analyse and resolve a broad variety of inclusions differing in both size and protein composition, making it applicable to essentially any model of intracellular protein aggregation. The method also allows rapid quantification of the nuclear trafficking of fluorescently labelled molecules. PMID:27516358

  9. Rapid flow cytometric measurement of protein inclusions and nuclear trafficking.

    PubMed

    Whiten, D R; San Gil, R; McAlary, L; Yerbury, J J; Ecroyd, H; Wilson, M R

    2016-01-01

    Proteinaceous cytoplasmic inclusions are an indicator of dysfunction in normal cellular proteostasis and a hallmark of many neurodegenerative diseases. We describe a simple and rapid new flow cytometry-based method to enumerate, characterise and, if desired, physically recover protein inclusions from cells. This technique can analyse and resolve a broad variety of inclusions differing in both size and protein composition, making it applicable to essentially any model of intracellular protein aggregation. The method also allows rapid quantification of the nuclear trafficking of fluorescently labelled molecules. PMID:27516358

  10. Rapid identification of chemical genetic interactions in Saccharomyces cerevisiae.

    PubMed

    Dilworth, David; Nelson, Christopher J

    2015-01-01

    Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described. PMID:25867090

  11. Identification, Analysis and Prediction of Protein Ubiquitination Sites

    PubMed Central

    Radivojac, Predrag; Vacic, Vladimir; Haynes, Chad; Cocklin, Ross R.; Mohan, Amrita; Heyen, Joshua W.; Goebl, Mark G.; Iakoucheva, Lilia M.

    2009-01-01

    Summary Ubiquitination plays an important role in many cellular processes and is implicated in many diseases. Experimental identification of ubiquitination sites is challenging due to rapid turnover of ubiquitinated proteins and the large size of the ubiquitin modifier. We identified 141 new ubiquitination sites using a combination of liquid chromatography, mass spectrometry and mutant yeast strains. Investigation of the sequence biases and structural preferences around known ubiquitination sites indicated that their properties were similar to those of intrinsically disordered protein regions. Using a combined set of new and previously known ubiquitination sites, we developed a random forest predictor of ubiquitination sites, UbPred. The class-balanced accuracy of UbPred reached 72%, with the area under the ROC curve at 80%. The application of UbPred showed that high confidence Rsp5 ubiquitin ligase substrates and proteins with very short half-lives were significantly enriched in the number of predicted ubiquitination sites. Proteome-wide prediction of ubiquitination sites in Saccharomyces cerevisiae indicated that highly ubiquitinated substrates were prevalent among transcription/enzyme regulators and proteins involved in cell cycle control. In the human proteome, cytoskeletal, cell cycle, regulatory and cancer-associated proteins display higher extent of ubiquitination than proteins from other functional categories. We show that gain and loss of predicted ubiquitination sites may likely represent a molecular mechanism behind a number of disease-associated mutations. UbPred is available at http://www.ubpred.org PMID:19722269

  12. Rapid identification of female Culexmosquito species using Expert System in the South East Asian region

    PubMed Central

    Murty, Upadhyayula Suryanarayana; Kumar, Duvvuri Venkata Rama Satya; Rao, Mutheneni Srinivasa; Reuben, Rachel; Tewari, Satish Chandra; Hiriyan, J; Akiyama, J; Akavaram, Deepa

    2005-01-01

    Rapid identification of mosquito (vector) species is critical for vector control and disease management. Pictorial keys of mosquito species are currently used for the identification of new mosquito species. However, this approach is not very effective. Here, we describe the use of an ID3 algorithm (part of artificial intelligence) for the rapid identification of the South East Asian female Culex mosquito species. Availability http://www.envisiict.org/ PMID:17597850

  13. Identification of ligands for bacterial sensor proteins.

    PubMed

    Fernández, Matilde; Morel, Bertrand; Corral-Lugo, Andrés; Rico-Jiménez, Miriam; Martín-Mora, David; López-Farfán, Diana; Reyes-Darias, José Antonio; Matilla, Miguel A; Ortega, Álvaro; Krell, Tino

    2016-02-01

    Bacteria have evolved a variety of different signal transduction mechanisms. However, the cognate signal molecule for the very large amount of corresponding sensor proteins is unknown and their functional annotation represents a major bottleneck in the field of signal transduction. The knowledge of the signal molecule is an essential prerequisite to understand the signalling mechanisms. Recently, the identification of signal molecules by the high-throughput protein screening of commercially available ligand collections using differential scanning fluorimetry has shown promise to resolve this bottleneck. Based on the analysis of a significant number of different ligand binding domains (LBDs) in our laboratory, we identified two issues that need to be taken into account in the experimental design. Since a number of LBDs require the dimeric state for ligand recognition, it has to be assured that the protein analysed is indeed in the dimeric form. A number of other examples demonstrate that purified LBDs can contain bound ligand which prevents further binding. In such cases, the apo-form can be generated by denaturation and subsequent refolding. We are convinced that this approach will accelerate the functional annotation of sensor proteins which will help to understand regulatory circuits in bacteria. PMID:26511375

  14. Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology

    NASA Astrophysics Data System (ADS)

    Tenzer, Stefan; Docter, Dominic; Kuharev, Jörg; Musyanovych, Anna; Fetz, Verena; Hecht, Rouven; Schlenk, Florian; Fischer, Dagmar; Kiouptsi, Klytaimnistra; Reinhardt, Christoph; Landfester, Katharina; Schild, Hansjörg; Maskos, Michael; Knauer, Shirley K.; Stauber, Roland H.

    2013-10-01

    In biological fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living systems. As physiological systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biological relevancy. Here we show that label-free snapshot proteomics can be used to obtain quantitative time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to form rapidly (<0.5 minutes) and, over time, to change significantly in terms of the amount of bound protein, but not in composition. Rapid corona formation is found to affect haemolysis, thrombocyte activation, nanoparticle uptake and endothelial cell death at an early exposure time.

  15. Identification of protein interacting partners using tandem affinity purification.

    PubMed

    Bailey, Dalan; Urena, Luis; Thorne, Lucy; Goodfellow, Ian

    2012-01-01

    A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification(1). Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast(2,3) but more recently has been adapted to use in mammalian cells(4-8). As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E(9,10).The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation(10). The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence(8). To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter. Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous

  16. Applications of graph theory in protein structure identification.

    PubMed

    Yan, Yan; Zhang, Shenggui; Wu, Fang-Xiang

    2011-01-01

    There is a growing interest in the identification of proteins on the proteome wide scale. Among different kinds of protein structure identification methods, graph-theoretic methods are very sharp ones. Due to their lower costs, higher effectiveness and many other advantages, they have drawn more and more researchers' attention nowadays. Specifically, graph-theoretic methods have been widely used in homology identification, side-chain cluster identification, peptide sequencing and so on. This paper reviews several methods in solving protein structure identification problems using graph theory. We mainly introduce classical methods and mathematical models including homology modeling based on clique finding, identification of side-chain clusters in protein structures upon graph spectrum, and de novo peptide sequencing via tandem mass spectrometry using the spectrum graph model. In addition, concluding remarks and future priorities of each method are given. PMID:22165974

  17. Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing: identification of novel zinc finger proteins

    PubMed Central

    Hughes, Marcus D.; Zhang, Zhan-Ren; Sutherland, Andrew J.; Santos, Albert F.; Hine, Anna V.

    2005-01-01

    We have successfully linked protein library screening directly with the identification of active proteins, without the need for individual purification, display technologies or physical linkage between the protein and its encoding sequence. By using ‘MAX’ randomization we have rapidly constructed 60 overlapping gene libraries that encode zinc finger proteins, randomized variously at the three principal DNA-contacting residues. Expression and screening of the libraries against five possible target DNA sequences generated data points covering a potential 40 000 individual interactions. Comparative analysis of the resulting data enabled direct identification of active proteins. Accuracy of this library analysis methodology was confirmed by both in vitro and in vivo analyses of identified proteins to yield novel zinc finger proteins that bind to their target sequences with high affinity, as indicated by low nanomolar apparent dissociation constants. PMID:15722478

  18. A method to rapidly create protein aggregates in living cells

    PubMed Central

    Miyazaki, Yusuke; Mizumoto, Kota; Dey, Gautam; Kudo, Takamasa; Perrino, John; Chen, Ling-chun; Meyer, Tobias; Wandless, Thomas J.

    2016-01-01

    The accumulation of protein aggregates is a common pathological hallmark of many neurodegenerative diseases. However, we do not fully understand how aggregates are formed or the complex network of chaperones, proteasomes and other regulatory factors involved in their clearance. Here, we report a chemically controllable fluorescent protein that enables us to rapidly produce small aggregates inside living cells on the order of seconds, as well as monitor the movement and coalescence of individual aggregates into larger structures. This method can be applied to diverse experimental systems, including live animals, and may prove valuable for understanding cellular responses and diseases associated with protein aggregates. PMID:27229621

  19. Lattice model for rapidly folding protein-like heteropolymers.

    PubMed Central

    Shrivastava, I; Vishveshwara, S; Cieplak, M; Maritan, A; Banavar, J R

    1995-01-01

    Protein folding is a relatively fast process considering the astronomical number of conformations in which a protein could find itself. Within the framework of a lattice model, we show that one can design rapidly folding sequences by assigning the strongest attractive couplings to the contacts present in a target native state. Our protein design can be extended to situations with both attractive and repulsive contacts. Frustration is minimized by ensuring that all the native contacts are again strongly attractive. Strikingly, this ensures the inevitability of folding and accelerates the folding process by an order of magnitude. The evolutionary implications of our findings are discussed. PMID:7568102

  20. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis.

    PubMed Central

    Telenti, A; Marchesi, F; Balz, M; Bally, F; Böttger, E C; Bodmer, T

    1993-01-01

    A method for the rapid identification of mycobacteria to the species level was developed on the basis of evaluation by the polymerase chain reaction (PCR) of the gene encoding for the 65-kDa protein. The method involves restriction enzyme analysis of PCR products obtained with primers common to all mycobacteria. Using two restriction enzymes, BstEII and HaeIII, medically relevant and other frequent laboratory isolates were differentiated to the species or subspecies level by PCR-restriction enzyme pattern analysis. PCR-restriction enzyme pattern analysis was performed on isolates (n = 330) from solid and fluid culture media, including BACTEC, or from frozen and lyophilized stocks. The procedure does not involve hybridization steps or the use of radioactivity and can be completed within 1 working day. Images PMID:8381805

  1. Identification and Molecular Mechanisms of the Rapid Tonicity-induced Relocalization of the Aquaporin 4 Channel.

    PubMed

    Kitchen, Philip; Day, Rebecca E; Taylor, Luke H J; Salman, Mootaz M; Bill, Roslyn M; Conner, Matthew T; Conner, Alex C

    2015-07-01

    The aquaporin family of integral membrane proteins is composed of channels that mediate cellular water flow. Aquaporin 4 (AQP4) is highly expressed in the glial cells of the central nervous system and facilitates the osmotically driven pathological brain swelling associated with stroke and traumatic brain injury. Here we show that AQP4 cell surface expression can be rapidly and reversibly regulated in response to changes of tonicity in primary cortical rat astrocytes and in transfected HEK293 cells. The translocation mechanism involves PKA activation, influx of extracellular calcium, and activation of calmodulin. We identify five putative PKA phosphorylation sites and use site-directed mutagenesis to show that only phosphorylation at one of these sites, serine 276, is necessary for the translocation response. We discuss our findings in the context of the identification of new therapeutic approaches to treating brain edema. PMID:26013827

  2. Identification and Molecular Mechanisms of the Rapid Tonicity-induced Relocalization of the Aquaporin 4 Channel*

    PubMed Central

    Kitchen, Philip; Day, Rebecca E.; Taylor, Luke H. J.; Salman, Mootaz M.; Bill, Roslyn M.; Conner, Matthew T.; Conner, Alex C.

    2015-01-01

    The aquaporin family of integral membrane proteins is composed of channels that mediate cellular water flow. Aquaporin 4 (AQP4) is highly expressed in the glial cells of the central nervous system and facilitates the osmotically driven pathological brain swelling associated with stroke and traumatic brain injury. Here we show that AQP4 cell surface expression can be rapidly and reversibly regulated in response to changes of tonicity in primary cortical rat astrocytes and in transfected HEK293 cells. The translocation mechanism involves PKA activation, influx of extracellular calcium, and activation of calmodulin. We identify five putative PKA phosphorylation sites and use site-directed mutagenesis to show that only phosphorylation at one of these sites, serine 276, is necessary for the translocation response. We discuss our findings in the context of the identification of new therapeutic approaches to treating brain edema. PMID:26013827

  3. Identification of essential proteins based on ranking edge-weights in protein-protein interaction networks.

    PubMed

    Wang, Yan; Sun, Huiyan; Du, Wei; Blanzieri, Enrico; Viero, Gabriella; Xu, Ying; Liang, Yanchun

    2014-01-01

    Essential proteins are those that are indispensable to cellular survival and development. Existing methods for essential protein identification generally rely on knock-out experiments and/or the relative density of their interactions (edges) with other proteins in a Protein-Protein Interaction (PPI) network. Here, we present a computational method, called EW, to first rank protein-protein interactions in terms of their Edge Weights, and then identify sub-PPI-networks consisting of only the highly-ranked edges and predict their proteins as essential proteins. We have applied this method to publicly-available PPI data on Saccharomyces cerevisiae (Yeast) and Escherichia coli (E. coli) for essential protein identification, and demonstrated that EW achieves better performance than the state-of-the-art methods in terms of the precision-recall and Jackknife measures. The highly-ranked protein-protein interactions by our prediction tend to be biologically significant in both the Yeast and E. coli PPI networks. Further analyses on systematically perturbed Yeast and E. coli PPI networks through randomly deleting edges demonstrate that the proposed method is robust and the top-ranked edges tend to be more associated with known essential proteins than the lowly-ranked edges. PMID:25268881

  4. Identification of Posttranslational Modification-Dependent Protein Interactions Using Yeast Surface Displayed Human Proteome Libraries

    PubMed Central

    Bidlingmaier, Scott; Liu, Bin

    2016-01-01

    The identification of proteins that interact specifically with posttranslational modifications such as phosphorylation is often necessary to understand cellular signaling pathways. Numerous methods for identifying proteins that interact with posttranslational modifications have been utilized, including affinity-based purification and analysis, protein microarrays, phage display, and tethered catalysis. Although these techniques have been used successfully, each has limitations. Recently, yeast surface-displayed human proteome libraries have been utilized to identify protein fragments with affinity for various target molecules, including phosphorylated peptides. When coupled with fluorescently activated cell sorting and high throughput methods for the analysis of selection outputs, yeast surface-displayed human proteome libraries can rapidly and efficiently identify protein fragments with affinity for any soluble ligand that can be fluorescently detected, including posttranslational modifications. In this review we compare the use of yeast surface display libraries to other methods for the identification of interactions between proteins and posttranslational modifications and discuss future applications of the technology. PMID:26060076

  5. Identification of Posttranslational Modification-Dependent Protein Interactions Using Yeast Surface Displayed Human Proteome Libraries.

    PubMed

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    The identification of proteins that interact specifically with posttranslational modifications such as phosphorylation is often necessary to understand cellular signaling pathways. Numerous methods for identifying proteins that interact with posttranslational modifications have been utilized, including affinity-based purification and analysis, protein microarrays, phage display, and tethered catalysis. Although these techniques have been used successfully, each has limitations. Recently, yeast surface-displayed human proteome libraries have been utilized to identify protein fragments with affinity for various target molecules, including phosphorylated peptides. When coupled with fluorescently activated cell sorting and high throughput methods for the analysis of selection outputs, yeast surface-displayed human proteome libraries can rapidly and efficiently identify protein fragments with affinity for any soluble ligand that can be fluorescently detected, including posttranslational modifications. In this review we compare the use of yeast surface display libraries to other methods for the identification of interactions between proteins and posttranslational modifications and discuss future applications of the technology. PMID:26060076

  6. Rapid purification of protein complexes from mammalian cells

    PubMed Central

    Medina, Dan; Moskowitz, Neal; Khan, Subarna; Christopher, Scott; Germino, Joseph

    2000-01-01

    The evaluation of the protein binding partner(s) of biologically important proteins is currently an area of intense research, especially since the development of the yeast two-hybrid assay. However, not all protein–protein interactions uncovered by this assay are biologically relevant and another confirmatory assay must be performed. Ideally, this assay should be rapid, versatile and performed under conditions which mimic the ‘normal’ physiological state as closely as possible. Towards this goal, we have constructed two eukaryotic expression vectors that facilitate the purification of a protein of interest, along with any associated proteins, from mammalian cells. These vectors incorporate the following features: (i) a tetracycline-responsive promoter so that the level of protein production can be regulated; (ii) an N-terminal glutathione S-transferase tag or a triple repeat of the HA1 epitope, to facilitate purification of the protein either by glutathione affinity chromatography or immunoprecipitation, respectively, followed by a multiple cloning site; (iii) the gene for the enhanced green fluorescent protein (for detection of the presence of the fusion protein and subcellular localization); (iv) a puromycin marker for the selection of stable transformants; (v) a truncated EBNA protein and oriP sequence for episomal replication of the vector. These latter two features permit expansion of small cultures of transfected cells under puromycin selection, thereby increasing the amount of tagged protein that can be purified. We show that these vectors can be used to direct the doxycycline-inducible expresssion of tagged proteins and to recover tagged CIP1–p21 protein complexes from HeLa cells. Furthermore, we show that these tagged p21-purified complexes contain both cyclin A and Cdk2, which are known to interact with p21, but not β-actin. PMID:10871384

  7. Rapid and Accurate Identification of Candida albicans Isolates by Use of PNA FISHFlow▿

    PubMed Central

    Trnovsky, Jan; Merz, William; Della-Latta, Phyllis; Wu, Fann; Arendrup, Maiken Cavling; Stender, Henrik

    2008-01-01

    We developed the simple, rapid (1 h), and accurate PNA FISHFlow method for the identification of Candida albicans. The method exploits unique in solution in situ hybridization conditions under which the cells are simultaneously fixed and hybridized. This method facilitates the accurate identification of clinical yeast isolates using two scoring techniques: flow cytometry and fluorescence microscopy. PMID:18287325

  8. Rapid Confirmation of Listeria spp. with the MIT 1000 Microbial Identification System

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methods that can rapidly confirm the identification of foodborne pathogens are highly desired. The USDA has recently entered into a collaborative research agreement with Micro Imaging Technology to evaluate their MIT 1000 microbial identification system for its ability to identify Listeria species ...

  9. A Collective Variable for the Rapid Exploration of Protein Druggability.

    PubMed

    Cuchillo, Rémi; Pinto-Gil, Kevin; Michel, Julien

    2015-03-10

    An efficient molecular simulation methodology has been developed for the evaluation of the druggability (ligandability) of a protein. Previously proposed techniques were designed to assess the druggability of crystallographic structures and cannot be tightly coupled to molecular dynamics (MD) simulations. By contrast, the present approach, JEDI (Just Exploring Druggability at protein Interfaces), features a druggability potential made of a combination of empirical descriptors that can be collected "on-the-fly" during MD simulations. Extensive validation studies indicate that JEDI analyses discriminate druggable and nondruggable protein binding site conformations with accuracy similar to alternative methodologies, and at a fraction of the computational cost. Since the JEDI function is continuous and differentiable, the druggability potential can be used as collective variable to rapidly detect cryptic druggable binding sites in proteins with a variety of MD free energy methods. Protocols for applications to flexible docking problems are outlined. PMID:26579775

  10. Byonic: Advanced Peptide and Protein Identification Software

    PubMed Central

    Bern, Marshall; Kil, Yong J.; Becker, Christopher

    2013-01-01

    Byonic™ is the name of a software package for peptide and protein identification by tandem mass spectrometry. This software, which has only recently become commercially available, facilitates a much wider range of search possibilities than previous search software such as SEQUEST and Mascot. Byonic allows the user to define an essentially unlimited number of variable modification types. Byonic also allows the user to set a separate limit on the number of occurrences of each modification type, so that a search may consider only one or two chance modifications such as oxidations and deamidations per peptide, yet allow three or four biological modifications such as phosphorylations, which tend to cluster together. Hence Byonic can search for 10s or even 100s of modification types simultaneously without a prohibitively large combinatorial explosion. Byonic’s Wildcard Search™ allows the user to search for unanticipated or even unknown modifications alongside known modifications. Finally, Byonic’s Glycopeptide Search allows the user to identify glycopeptides without prior knowledge of glycan masses or glycosylation sites. PMID:23255153

  11. Rapid turbidimetric detection of milk powder adulteration with plant proteins.

    PubMed

    Scholl, Peter F; Farris, Samantha M; Mossoba, Magdi M

    2014-02-19

    Development of assays to screen milk for economically motivated adulteration with foreign proteins has been stalled since 2008 due to strong international reactions to the melamine poisoning incident in China and the surveillance emphasis placed on low molecular weight nitrogen-rich adulterants. New screening assays are still needed to detect high molecular weight foreign protein adulterants and characterize this understudied potential risk. A rapid turbidimetric method was developed to screen milk powder for adulteration with insoluble plant proteins. Milk powder samples spiked with 0.03-3% by weight of soy, pea, rice, and wheat protein isolates were extracted in 96-well plates, and resuspended pellet solution absorbance was measured. Limits of detection ranged from 100 to 200 μg, or 0.1-0.2% of the sample weight, and adulterant pellets were visually apparent even at ∼0.1%. Extraction recoveries ranged from 25 to 100%. Assay sensitivity and simplicity indicate that it would be ideally suitable to rapidly screen milk samples in resource poor environments where adulteration with plant protein is suspected. PMID:24484379

  12. Rapid thermal tuning of chromophore structure in membrane protein.

    PubMed

    Wang, Jianping; El-Sayed, Mostafa A

    2009-04-01

    We show that the configuration and the optical property of the retinal chromophore in bacteriorhodopsin (bR) can be tuned dynamically from the all-trans configuration to the 13-cis by using a nanosecond laser-induced temperature-jump. The rapid bleach in the visible absorption optical density of retinal has an apparent formation time of ca. 170 ns, whereas the relaxation process finishes within tens of ms. The dynamical transition of retinal from the all-trans to 13-cis species is believed to occur as a result of rapid protein conformational change especially in the vicinity of retinal binding site. Our study reveals the intrinsic dynamical aspect of the retinal chromophore with respect to the protein structure. PMID:19275202

  13. Rapid Identification of Genes Contributing to FH Resistance in Wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Development of wheat and barley with improved Fusarium head blight resistance will be greatly aided by knowledge of the plant genes that make essential contributions to the FHB resistance mechanism. This knowledge will permit identification of the best naturally occurring variants for use in breedin...

  14. Protein identification problem from a Bayesian point of view

    PubMed Central

    Li, Yong Fuga; Arnold, Randy J.; Radivojac, Predrag

    2012-01-01

    We present a generic Bayesian framework for the peptide and protein identification in proteomics, and provide a unified interpretation for the database searching and the de novo peptide sequencing approaches that are used in peptide identification. We describe several probabilistic graphical models and a variety of prior distributions that can be incorporated into the Bayesian framework to model different types of prior information, such as the known protein sequences, the known protein abundances, the peptide precursor masses, the estimated peptide retention time and the peptide detectabilities. Various applications of the Bayesian framework are discussed theoretically, including its application to the identification of peptides containing mutations and post-translational modifications. PMID:24761189

  15. Identification of Foodborne Bacteria by High Energy Collision-Induced Dissociation of Their Protein Biomarkers by MALDI Tandem-Time-of-Flight Mass Spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Development of methods for rapid identification of foodborne bacteria is an important area of analytical science and food safety. MALDI-TOF-MS has been utilized to rapidly identify pathogens including foodborne bacteria. Identification typically involves detection of high copy cytosolic proteins i...

  16. Mass Spectrometry and Next-Generation Sequencing Reveal an Abundant and Rapidly Evolving Abalone Sperm Protein

    PubMed Central

    Palmer, Melody R.; McDowall, Margo H.; Stewart, Lia; Ouaddi, Aleena; MacCoss, Michael J.; Swanson, Willie J.

    2014-01-01

    SUMMARY Abalone, a broadcast spawning marine mollusk, is an important model for molecular interactions and positive selection in fertilization, but the focus has previously been on only two sperm proteins, lysin and sp18.We used genomic and proteomic techniques to bring new insights to this model by characterizing the testis transcriptome and sperm proteome of the Red abalone Haliotis rufescens. One pair of homologous, testis-specific proteins contains a secretion signal and is small, abundant, and associated with the acrosome. Comparative analysis revealed that homologs are extremely divergent between species, and show strong evidence for positive selection. The acrosomal localization and rapid evolution of these proteins indicates that they play an important role in fertilization, and could be involved in the species-specificity of sperm-egg interactions in abalone. Our genomic and proteomic characterization of abalone fertilization resulted in the identification of interesting, novel peptides that have eluded detection in this important model system for 20 years. PMID:23585193

  17. Comparison of Rapid NFT system and conventional methods for identification of nonsaccharolytic gram-negative bacteria.

    PubMed

    Martin, R; Siavoshi, F; McDougal, D L

    1986-12-01

    This study examined the Rapid NFT system (Analytab Products, Plainview, N.Y.) to determine its ability to accurately identify 229 clinical isolates of mostly nonsaccharolytic gram-negative rods. Identifications were classified by the following scheme: correct (corresponding to excellent, very good, good, or acceptable identification as listed in the code book); low discrimination (correct identification among a range of listed possibilities, with additional tests necessary for accurate identification); incorrect. Correct identification was considered correct to species and subspecies for all organisms except Alcaligenes faecalis and "Alcaligenes odorans"; "A. faecalis/odorans" was considered a correct response. By using these criteria, 71.6% of the strains were correctly identified, 17.9% were identified with low discrimination, and 10.5% were incorrectly identified. When consideration was made for incorrect identification resulting from taxonomic problems (e.g., Alcaligenes and Moraxella spp.), incorrect identifications fell to 5.2%. The Rapid NFT system was truly rapid and was easy to use and interpret. Its use of carbon substrate assimilation enables it to provide more accurate identification of medically important nonsaccharolytic bacteria than do other commercially available systems. PMID:3536999

  18. Seed Storage Proteins as a System for Teaching Protein Identification by Mass Spectrometry in Biochemistry Laboratory

    ERIC Educational Resources Information Center

    Wilson, Karl A.; Tan-Wilson, Anna

    2013-01-01

    Mass spectrometry (MS) has become an important tool in studying biological systems. One application is the identification of proteins and peptides by the matching of peptide and peptide fragment masses to the sequences of proteins in protein sequence databases. Often prior protein separation of complex protein mixtures by 2D-PAGE is needed,…

  19. Rapid microbiochemical method for presumptive identification of gastroenteritis-associated members of the family Enterobacteriaceae.

    PubMed Central

    Yong, D C; Thompson, J S; Prytula, A

    1985-01-01

    A method for rapid screening of isolates of pathogenic members of the family Enterobacteriaceae is described. Flow charts are used in conjunction with triple sugar iron agar, o-nitrophenyl-beta-D-galactopyranoside-phenylalanine-motility sulfate screening media, oxidase test, and six rapid biochemical tests, namely, lysine decarboxylase, urease, indole, esculin hydrolysis, malonate, and xylose. This scheme is used to provide an inexpensive but rapid presumptive identification of Salmonella, Shigella, Edwardsiella, Aeromonas, Plesiomonas, Vibrio, and Yersinia isolates from stool cultures. PMID:4008622

  20. Essential protein identification based on essential protein-protein interaction prediction by Integrated Edge Weights.

    PubMed

    Jiang, Yuexu; Wang, Yan; Pang, Wei; Chen, Liang; Sun, Huiyan; Liang, Yanchun; Blanzieri, Enrico

    2015-07-15

    Essential proteins play a crucial role in cellular survival and development process. Experimentally, essential proteins are identified by gene knockouts or RNA interference, which are expensive and often fatal to the target organisms. Regarding this, an alternative yet important approach to essential protein identification is through computational prediction. Existing computational methods predict essential proteins based on their relative densities in a protein-protein interaction (PPI) network. Degree, betweenness, and other appropriate criteria are often used to measure the relative density. However, no matter what criterion is used, a protein is actually ordered by the attributes of this protein per se. In this research, we presented a novel computational method, Integrated Edge Weights (IEW), to first rank protein-protein interactions by integrating their edge weights, and then identified sub PPI networks consisting of those highly-ranked edges, and finally regarded the nodes in these sub networks as essential proteins. We evaluated IEW on three model organisms: Saccharomyces cerevisiae (S. cerevisiae), Escherichia coli (E. coli), and Caenorhabditis elegans (C. elegans). The experimental results showed that IEW achieved better performance than the state-of-the-art methods in terms of precision-recall and Jackknife measures. We had also demonstrated that IEW is a robust and effective method, which can retrieve biologically significant modules by its highly-ranked protein-protein interactions for S. cerevisiae, E. coli, and C. elegans. We believe that, with sufficient data provided, IEW can be used to any other organisms' essential protein identification. A website about IEW can be accessed from http://digbio.missouri.edu/IEW/index.html. PMID:25892709

  1. Improved Protocol for Rapid Identification of Certain Spa Types Using High Resolution Melting Curve Analysis

    PubMed Central

    Mayerhofer, Benjamin; Stöger, Anna; Pietzka, Ariane T.; Fernandez, Haizpea Lasa; Prewein, Bernhard; Sorschag, Sieglinde; Kunert, Renate; Allerberger, Franz; Ruppitsch, Werner

    2015-01-01

    Methicillin-resistant Staphylococcus aureus is one of the most significant pathogens associated with health care. For efficient surveillance, control and outbreak investigation, S. aureus typing is essential. A high resolution melting curve analysis was developed and evaluated for rapid identification of the most frequent spa types found in an Austrian hospital consortium covering 2,435 beds. Among 557 methicillin-resistant Staphylococcus aureus isolates 38 different spa types were identified by sequence analysis of the hypervariable region X of the protein A gene (spa). Identification of spa types through their characteristic high resolution melting curve profiles was considerably improved by double spiking with genomic DNA from spa type t030 and spa type t003 and allowed unambiguous and fast identification of the ten most frequent spa types t001 (58%), t003 (12%), t190 (9%), t041 (5%), t022 (2%), t032 (2%), t008 (2%), t002 (1%), t5712 (1%) and t2203 (1%), representing 93% of all isolates within this hospital consortium. The performance of the assay was evaluated by testing samples with unknown spa types from the daily routine and by testing three different high resolution melting curve analysis real-time PCR instruments. The ten most frequent spa types were identified from all samples and on all instruments with 100% specificity and 100% sensitivity. Compared to classical spa typing by sequence analysis, this gene scanning assay is faster, cheaper and can be performed in a single closed tube assay format. Therefore it is an optimal screening tool to detect the most frequent endemic spa types and to exclude non-endemic spa types within a hospital. PMID:25768007

  2. Improved protocol for rapid identification of certain spa types using high resolution melting curve analysis.

    PubMed

    Mayerhofer, Benjamin; Stöger, Anna; Pietzka, Ariane T; Fernandez, Haizpea Lasa; Prewein, Bernhard; Sorschag, Sieglinde; Kunert, Renate; Allerberger, Franz; Ruppitsch, Werner

    2015-01-01

    Methicillin-resistant Staphylococcus aureus is one of the most significant pathogens associated with health care. For efficient surveillance, control and outbreak investigation, S. aureus typing is essential. A high resolution melting curve analysis was developed and evaluated for rapid identification of the most frequent spa types found in an Austrian hospital consortium covering 2,435 beds. Among 557 methicillin-resistant Staphylococcus aureus isolates 38 different spa types were identified by sequence analysis of the hypervariable region X of the protein A gene (spa). Identification of spa types through their characteristic high resolution melting curve profiles was considerably improved by double spiking with genomic DNA from spa type t030 and spa type t003 and allowed unambiguous and fast identification of the ten most frequent spa types t001 (58%), t003 (12%), t190 (9%), t041 (5%), t022 (2%), t032 (2%), t008 (2%), t002 (1%), t5712 (1%) and t2203 (1%), representing 93% of all isolates within this hospital consortium. The performance of the assay was evaluated by testing samples with unknown spa types from the daily routine and by testing three different high resolution melting curve analysis real-time PCR instruments. The ten most frequent spa types were identified from all samples and on all instruments with 100% specificity and 100% sensitivity. Compared to classical spa typing by sequence analysis, this gene scanning assay is faster, cheaper and can be performed in a single closed tube assay format. Therefore it is an optimal screening tool to detect the most frequent endemic spa types and to exclude non-endemic spa types within a hospital. PMID:25768007

  3. Progress towards rapid identification of phytochemicals in plant extracts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    New mass spectrometry equipment is bringing closer to reality the rapid accurate assessment of chemical composition of extracts from a variety of plant materials. Using a variety of plant sources, we are using HPLC separation, UV-VIS spectrometry, ion trap mass fragmentation and accurate mass deter...

  4. Rapid Temporal Dynamics of Transcription, Protein Synthesis, and Secretion during Macrophage Activation*

    PubMed Central

    Eichelbaum, Katrin; Krijgsveld, Jeroen

    2014-01-01

    Macrophages provide the first line of host defense with their capacity to react to an array of cytokines and bacterial components requiring tight regulation of protein expression and secretion to invoke a properly tuned innate immune response. To capture the dynamics of this system, we introduce a novel method combining pulsed stable isotope labeling with amino acids in cell culture (SILAC) with pulse labeling using the methionine analog azidohomoalanine that allows the enrichment of newly synthesized proteins via click-chemistry followed by their identification and quantification by mass spectrometry. We show that this permits the analysis of proteome changes on a rapid time scale, as evidenced by the detection of 4852 newly synthesized proteins after only a 20-min SILAC pulse. We have applied this methodology to study proteome response during macrophage activation in a time-course manner. We have combined this with full proteome, transcriptome, and secretome analyses, producing an integrative analysis of the first 3 h of lipopolysaccharide-induced macrophage activation. We observed the rapid induction of multiple processes well known to TLR4 signaling, as well as anti-inflammatory proteins and proteins not previously associated with immune response. By correlating transcriptional, translational, and secretory events, we derived novel mechanistic principles of processes specifically induced by lipopolysaccharides, including ectodomain shedding and proteolytic processing of transmembrane and extracellular proteins and protein secretion independent of transcription. In conclusion, we demonstrate that the combination of pulsed azidohomoalanine and pulsed SILAC permits the detailed characterization of proteomic events on a rapid time scale. We anticipate that this approach will be very useful in probing the immediate effects of cellular stimuli and will provide mechanistic insight into cellular perturbation in multiple biological systems. The data have been deposited

  5. Detection and identification of protein interactions of S100 proteins by ProteinChip technology.

    PubMed

    Lehmann, Roland; Melle, Christian; Escher, Niko; von Eggeling, Ferdinand

    2005-01-01

    The aim of this work was to establish an approach for identification of protein interactions. This assay used an anti-S100A8 antibody coupled on beads and incubated with cell extract. The bead eluates were analyzed using ProteinChip technology and subsequently subjected to an appropriate digestion. Molecular masses of digestion fragments were determined by SELDI-MS, and database analysis revealed S100A10 as interacting protein. This result was confirmed by co-immunoprecipitation and immunocapturing. Using S100A10 as new bait, a specific interaction with S100A7 was detectable. PMID:16212425

  6. Rapid Cell Population Identification in Flow Cytometry Data*

    PubMed Central

    Aghaeepour, Nima; Nikolic, Radina; Hoos, Holger H.; Brinkman, Ryan R.

    2011-01-01

    We have developed flowMeans, a time-efficient and accurate method for automated identification of cell populations in flow cytometry (FCM) data based on K-means clustering. Unlike traditional K-means, flowMeans can identify concave cell populations by modelling a single population with multiple clusters. flowMeans uses a change point detection algorithm to determine the number of sub-populations, enabling the method to be used in high throughput FCM data analysis pipelines. Our approach compares favourably to manual analysis by human experts and current state-of-the-art automated gating algorithms. flowMeans is freely available as an open source R package through Bioconductor. PMID:21182178

  7. Ultrasensitive isolation, identification and quantification of DNA-protein adducts by ELISA-based RADAR assay.

    PubMed

    Kiianitsa, Kostantin; Maizels, Nancy

    2014-07-01

    Enzymes that form transient DNA-protein covalent complexes are targets for several potent classes of drugs used to treat infectious disease and cancer, making it important to establish robust and rapid procedures for analysis of these complexes. We report a method for isolation of DNA-protein adducts and their identification and quantification, using techniques compatible with high-throughput screening. This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA-protein covalent complexes in living cells. Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA-protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA-protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA-protein adducts by direct ELISA assay. The ELISA-based RADAR assay can detect Top1-DNA and Top2a-DNA adducts in human cells, and gyrase-DNA adducts in Escherichia coli. This approach will be useful for discovery and characterization of new drugs to treat infectious disease and cancer, and for development of companion diagnostics assays for individualized medicine. PMID:24914050

  8. Micro-apparatus for rapid determinations of protein solubilities

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Munson, Sibyl

    1991-01-01

    We have developed a column-based micro-technique for rapid determinations of protein solubilities. While retaining a large crystal surface area, the column dead volume has been reduced to equal to or less than 5 micro liters. The technique was tested with tetragonal lysozyme (pH 4.5, 0.1 M acetate, 3.0 percent NaCl, 5-25 C) and column volumes of about 60, 300, and 900 micro liters. Identical solubility data were obtained, indicating that equilibration was obtained even in the smallest columns. In addition, solubility data for Br- and I- salts of lysozyme (pH 4.5, 0.1 M acetate buffer, 0.5 M salt concentrations) were obtained. It appears that the technique can be further miniaturized. The limit in further reducing the crystalline column volume is determined by the minimum solution sample size needed to determine the protein concentration.

  9. A rapid and sensitive assay for DNA-protein covalent complexes in living cells.

    PubMed

    Kiianitsa, Kostantin; Maizels, Nancy

    2013-05-01

    A number of proteins form covalent bonds with DNA as obligatory transient intermediates in normal nuclear transactions. Drugs that trap these complexes have proven to be potent therapeutics in both cancer and infectious disease. Nonetheless, current assays for DNA-protein adducts are cumbersome, limiting both mechanistic studies and translational applications. We have developed a rapid and sensitive assay that enables quantitative immunodetection of protein-DNA adducts. This new 'RADAR' (rapid approach to DNA adduct recovery) assay accelerates processing time 4-fold, increases sample throughput 20-fold and requires 50-fold less starting material than the current standard. It can be used to detect topoisomerase 1-DNA adducts in as little as 60 ng of DNA, corresponding to 10 000 human cells. We apply the RADAR assay to demonstrate that expression of SLFN11 does not increase camptothecin sensitivity by promoting accumulation of topoisomerase 1-DNA adducts. The RADAR assay will be useful for analysis of the mechanisms of formation and resolution of DNA-protein adducts in living cells, and identification and characterization of reactions in which covalent DNA adducts are transient intermediates. The assay also has potential application to drug discovery and individualized medicine. PMID:23519618

  10. Computational Methods for Protein Identification from Mass Spectrometry Data

    PubMed Central

    McHugh, Leo; Arthur, Jonathan W

    2008-01-01

    Protein identification using mass spectrometry is an indispensable computational tool in the life sciences. A dramatic increase in the use of proteomic strategies to understand the biology of living systems generates an ongoing need for more effective, efficient, and accurate computational methods for protein identification. A wide range of computational methods, each with various implementations, are available to complement different proteomic approaches. A solid knowledge of the range of algorithms available and, more critically, the accuracy and effectiveness of these techniques is essential to ensure as many of the proteins as possible, within any particular experiment, are correctly identified. Here, we undertake a systematic review of the currently available methods and algorithms for interpreting, managing, and analyzing biological data associated with protein identification. We summarize the advances in computational solutions as they have responded to corresponding advances in mass spectrometry hardware. The evolution of scoring algorithms and metrics for automated protein identification are also discussed with a focus on the relative performance of different techniques. We also consider the relative advantages and limitations of different techniques in particular biological contexts. Finally, we present our perspective on future developments in the area of computational protein identification by considering the most recent literature on new and promising approaches to the problem as well as identifying areas yet to be explored and the potential application of methods from other areas of computational biology. PMID:18463710

  11. Rapid Evolution of Virus Sequences in Intrinsically Disordered Protein Regions

    PubMed Central

    Gitlin, Leonid; Hagai, Tzachi; LaBarbera, Anthony; Solovey, Mark; Andino, Raul

    2014-01-01

    Nodamura Virus (NoV) is a nodavirus originally isolated from insects that can replicate in a wide variety of hosts, including mammals. Because of their simplicity and ability to replicate in many diverse hosts, NoV, and the Nodaviridae in general, provide a unique window into the evolution of viruses and host-virus interactions. Here we show that the C-terminus of the viral polymerase exhibits extreme structural and evolutionary flexibility. Indeed, fewer than 10 positively charged residues from the 110 amino acid-long C-terminal region of protein A are required to support RNA1 replication. Strikingly, this region can be replaced by completely unrelated protein sequences, yet still produce a functional replicase. Structure predictions, as well as evolutionary and mutational analyses, indicate that the C-terminal region is structurally disordered and evolves faster than the rest of the viral proteome. Thus, the function of an intrinsically unstructured protein region can be independent of most of its primary sequence, conferring both functional robustness and sequence plasticity on the protein. Our results provide an experimental explanation for rapid evolution of unstructured regions, which enables an effective exploration of the sequence space, and likely function space, available to the virus. PMID:25502394

  12. Rapidly learned identification of epileptic seizures from sonified EEG.

    PubMed

    Loui, Psyche; Koplin-Green, Matan; Frick, Mark; Massone, Michael

    2014-01-01

    Sonification refers to a process by which data are converted into sound, providing an auditory alternative to visual display. Currently, the prevalent method for diagnosing seizures in epilepsy is by visually reading a patient's electroencephalogram (EEG). However, sonification of the EEG data provides certain advantages due to the nature of human auditory perception. We hypothesized that human listeners will be able to identify seizures from EEGs using the auditory modality alone, and that accuracy of seizure identification will increase after a short training session. Here, we describe an algorithm that we have used to sonify EEGs of both seizure and non-seizure activity, followed by a training study in which subjects listened to short clips of sonified EEGs and determined whether each clip was of seizure or normal activity, both before and after a short training session. Results show that before training subjects performed at chance level in differentiating seizures from non-seizures, but there was a significant improvement of accuracy after the training session. After training, subjects successfully distinguished seizures from non-seizures using the auditory modality alone. Further analyses using signal detection theory demonstrated improvement in sensitivity and reduction in response bias as a result of training. This study demonstrates the potential of sonified EEGs to be used for the detection of seizures. Future studies will attempt to increase accuracy using novel training and sonification modifications, with the goals of managing, predicting, and ultimately controlling seizures using sonification as a possible biofeedback-based intervention for epilepsy. PMID:25352802

  13. Rapid identification of chromosomal rearrangements by PRINS technique

    SciTech Connect

    Pellestor, F.; Giradet, A.; Andreo, B.

    1994-09-01

    Chromosomal rearrangements contribute significantly to human reproductive failure, malformation/mental retardation syndromes and carcinogenesis. The variety of structural rearrangements is almost infinite and an identification by conventional cytogenetics is often labor intensive and may remain doubtful. Recent advances in molecular cytogenetics have provided new tools for detecting chromosomal abnormalities. The fluorescence in situ hybridization (FISH) procedure is actually the most employed technique and has led to numerous clinical applications. However, techniques required to produce suitable probes are time consuming and not accessible to all cytogenetics laboratories. The PRimed In Situ labeling (PRINS) method provides an alternate way for in situ chromosome screening. In this procedure, the chromosomal detection is performed by in situ annealing of a specific primer and subsequent primer extension by a Taq DNA polymerase in the presence of labeled nucleotides. Application of PRINS in clinical diagnosis is still limited. We have developed a semi-automatic PRINS protocol and used it to identify the origin of several chromosomal abnormalities. We report here the results of studies of three structural rearrangements: a translocation t(21;21), a supernumerary ring marker chromosome 18 and a complex chromosome 13 mosaicism involving a 13;13 Robertsonian translocation and a ring chromosome 13.

  14. Rapidly Learned Identification of Epileptic Seizures from Sonified EEG

    PubMed Central

    Loui, Psyche; Koplin-Green, Matan; Frick, Mark; Massone, Michael

    2014-01-01

    Sonification refers to a process by which data are converted into sound, providing an auditory alternative to visual display. Currently, the prevalent method for diagnosing seizures in epilepsy is by visually reading a patient’s electroencephalogram (EEG). However, sonification of the EEG data provides certain advantages due to the nature of human auditory perception. We hypothesized that human listeners will be able to identify seizures from EEGs using the auditory modality alone, and that accuracy of seizure identification will increase after a short training session. Here, we describe an algorithm that we have used to sonify EEGs of both seizure and non-seizure activity, followed by a training study in which subjects listened to short clips of sonified EEGs and determined whether each clip was of seizure or normal activity, both before and after a short training session. Results show that before training subjects performed at chance level in differentiating seizures from non-seizures, but there was a significant improvement of accuracy after the training session. After training, subjects successfully distinguished seizures from non-seizures using the auditory modality alone. Further analyses using signal detection theory demonstrated improvement in sensitivity and reduction in response bias as a result of training. This study demonstrates the potential of sonified EEGs to be used for the detection of seizures. Future studies will attempt to increase accuracy using novel training and sonification modifications, with the goals of managing, predicting, and ultimately controlling seizures using sonification as a possible biofeedback-based intervention for epilepsy. PMID:25352802

  15. Functional recognition imaging using artificial neural networks: applications to rapid cellular identification via broadband electromechanical response

    NASA Astrophysics Data System (ADS)

    Nikiforov, M. P.; Reukov, V. V.; Thompson, G. L.; Vertegel, A. A.; Guo, S.; Kalinin, S. V.; Jesse, S.

    2009-10-01

    Functional recognition imaging in scanning probe microscopy (SPM) using artificial neural network identification is demonstrated. This approach utilizes statistical analysis of complex SPM responses at a single spatial location to identify the target behavior, which is reminiscent of associative thinking in the human brain, obviating the need for analytical models. We demonstrate, as an example of recognition imaging, rapid identification of cellular organisms using the difference in electromechanical activity over a broad frequency range. Single-pixel identification of model Micrococcus lysodeikticus and Pseudomonas fluorescens bacteria is achieved, demonstrating the viability of the method.

  16. Dealing with the identification of protein species in ancient amphorae.

    PubMed

    Dallongeville, Sophie; Garnier, Nicolas; Casasola, Dario Bernal; Bonifay, Michel; Rolando, Christian; Tokarski, Caroline

    2011-03-01

    This manuscript deals with the identification of protein residues in amphorae, including particularly identification of protein species. The work described was performed on fishes, the anchovy (Engraulis encrasicolus) and bonito (Sarda sarda) species frequently found in the Mediterranean area. Based on proteomic techniques, the analytical strategy was adapted to analysis of protein residues from tiny ceramic fragments. The major difficulty was to extract proteins and limit their hydrolysis during the sample preparation; consequently, multiple soft extraction techniques were evaluated. The most valuable results were obtained using a solution containing high amounts of denaturing agents, urea and thiourea, reducing agent, dithiothreitol, and detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The analysis using nano liquid chromatography-nano electrospray ionization double quadrupole time-of-flight mass spectrometry resulted in the identification of up to 200 proteins for the anchovy and bonito species, among which 73 peptides were found to be fish-specific. Because bonito and anchovy species are not documented and fully sequenced in genomic databases, the preliminary protein identification was realized via sequence homology to other fish sequenced species. Amino acid substitutions of peptides were assigned on the basis of the interpretation of tandem mass spectrometry spectra using de novo sequencing; these peptides, not reported up to now in databases, constitute species-specific markers. The method developed was finally applied to an archaeological sample replica impregnated with a mixture of fish tissue from both species; this experiment successfully led to the identification of 17 fish proteins, including 33 fish-specific peptides. This work shows that the analytical method developed has great potential for the identification of protein species in complex archaeological samples. PMID:20890751

  17. Novel method for rapid identification of Nocardia species by detection of preformed enzymes.

    PubMed Central

    Biehle, J R; Cavalieri, S J; Felland, T; Zimmer, B L

    1996-01-01

    The purpose of the present study was to devise a method for the identification of Nocardia species that is more technically simple, accurate, and rapid than current standard methods of identification. We focused on a commercial bacteria identification system that contained chromogenic test substrates. Two MicroScan products were selected for use in the study on the basis of their content of chromogenic and conventional substrates. They were the Rapid Anaerobe Identification and the HNID panels. A total of 85 strains of Nocardia representing five species were used in the study. All isolates were identified as Nocardia species by the use of standard methods. The beta-naphthylamide-labeled substrate L-pyrrolidonyl-beta-naphthylamide (PYR), the nitrophenyl-labeled substrate p-nitrophenyl-alpha-D-mannopyranoside (MNP), and indoxyl phosphate were found to be useful for identification purposes. N. farcinica and N. nova were the only species positive for PYR, whereas N. brasiliensis was the only species that hydrolyzed MNP. All strains of N. brasiliensis, N. otitidiscavarium, and N. farcinica were positive for indoxyl phosphate, whereas strains of N. nova and N. asteroides sensu stricto were always negative. Agreement between the standard and enzymatic identification methods was 100%. In summary, detection of preformed enzymes appears to be a simple and reproducible method for the identification of Nocardia spp. PMID:8748283

  18. Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

    PubMed Central

    Wildner, Letícia Muraro; Bazzo, Maria Luiza; Liedke, Susie Coutinho; Nogueira, Christiane Lourenço; Segat, Gabriela; Senna, Simone Gonçalves; Schlindwein, Aline Daiane; de Oliveira, Jaquelline Germano; Rovaris, Darcita B; Bonjardim, Claudio A; Kroon, Erna G; Ferreira, Paulo CP

    2014-01-01

    The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria. PMID:24821059

  19. Fluorescent, bioactive protein nanoparticles (prodots) for rapid, improved cellular uptake.

    PubMed

    Deshapriya, Inoka K; Stromer, Bobbi S; Pattammattel, Ajith; Kim, Christina S; Iglesias-Bartolome, Ramiro; Gonzalez-Fajardo, Laura; Patel, Vyomesh; Gutkind, J Silvio; Lu, Xiuling; Kumar, Challa V

    2015-03-18

    A simple and effective method for synthesizing highly fluorescent, protein-based nanoparticles (Prodots) and their facile uptake into the cytoplasm of cells is described here. Prodots made from bovine serum albumin (nBSA), glucose oxidase (nGO), horseradish peroxidase (nHRP), catalase (nCatalase), and lipase (nLipase) were found to be 15-50 nm wide and have been characterized by gel electrophoresis, transmission electron microscopy (TEM), circular dichroism (CD), fluorescence spectroscopy, dynamic light scattering (DLS), and optical microscopic methods. Data showed that the secondary structure of the protein in Prodots is retained to a significant extent and specific activities of nGO, nHRP, nCatalase, and nLipase were 80%, 70%, 65%, and 50% of their respective unmodified enzyme activities. Calorimetric studies indicated that the denaturation temperatures of nGO and nBSA increased while those of other Prodots remained nearly unchanged, and accelerated storage half-lives of Prodots at 60 °C increased by 4- to 8-fold. Exposure of nGO and nBSA+ nGO to cells indicated rapid uptake within 1-3 h, accompanied by significant blebbing of the plasma membrane, but no uptake has been noted in the absence of nGO. The presence of nGO/glucose in the media facilitated the uptake, and hydrogen peroxide induced membrane permeability could be responsible for this rapid uptake of Prodots. In control studies, FITC alone did not enter the cell, BSA-FITC was not internalized even in the presence of nGO, and there has been no uptake of nBSA-FITC in the absence of nGO. These are the very first examples of very rapid cellular uptake of fluorescent nanoparticles into cells, particularly nanoparticles made from pure proteins. The current approach is a simple and efficient method for the preparation of bioactive, fluorescent protein nanoparticles of controllable size for cellular imaging, and cell uptake is under the control of two separate chemical triggers. PMID:25642999

  20. Plant protein kinase substrates identification using protein microarrays.

    PubMed

    Ma, Shisong; Dinesh-Kumar, Savithramma P

    2015-01-01

    Protein kinases regulate signaling pathways by phosphorylating their targets. They play critical roles in plant signaling networks. Although many important protein kinases have been identified in plants, their substrates are largely unknown. We have developed and produced plant protein microarrays with more than 15,000 purified plant proteins. Here, we describe a detailed protocol to use these microarrays to identify plant protein kinase substrates via in vitro phosphorylation assays on these arrays. PMID:25930701

  1. Stable isotope, site-specific mass tagging for protein identification

    DOEpatents

    Chen, Xian

    2006-10-24

    Proteolytic peptide mass mapping as measured by mass spectrometry provides an important method for the identification of proteins, which are usually identified by matching the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. The present invention describes a method for increasing the specificity, accuracy and efficiency of the assignments of particular proteolytic peptides and consequent protein identification, by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence without the need for ultrahigh instrumental accuracy. Selected amino acid(s) are labeled with .sup.13C/.sup.15N/.sup.2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tag(s) can then be readily distinguished from other peptides in mass spectra. The present method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency and accuracy for protein identifications.

  2. Clinical impact of rapid in vitro susceptibility testing and bacterial identification.

    PubMed Central

    Doern, G V; Vautour, R; Gaudet, M; Levy, B

    1994-01-01

    During the past decade, a variety of instrument-assisted bacterial identification and antimicrobial susceptibility test systems have been developed which permit provision of test results in a matter of hours rather than days, as has been the case with traditional overnight procedures. These newer rapid techniques are much more expensive than older methods. It has been presumed but not proven that the clinical benefits of rapid testing to patients with infection offset the added cost. The intent of this study was to objectively define the clinical impact of rapid bacterial identification and antimicrobial susceptibility testing. A 1-year study was performed in which infected, hospitalized patients in a tertiary-care, teaching, medical center were randomly assigned to one of two groups: patients for whom identification and susceptibility testing was performed by using a semi-automated, rapid, same-day procedure and those for whom testing was accomplished by using traditional overnight techniques. The two groups were compared with respect to numerous demographic descriptors, and then patients were monitored prospectively through the end of their hospitalization with the aim of determining whether there existed objectively defineable differences in management and outcome between the two groups. The mean lengths of time to provision of susceptibility and identification test results in the rapid test group were 11.3 and 9.6 h, respectively. In the overnight test group, these values were 19.6 and 25.9 h, respectively (P < 0.0005). There were 273 evaluable patients in the first group and 300 in the second group. Other than the length of time required to provide susceptibility and identification test results, no significant differences were noted between the two groups with respect to > 100 demographic descriptors. With regard to measures of outcome, the mean lengths of hospitalization were also the same in both groups. Mortality rates were however, lower in the rapid test

  3. Growth medium for the rapid isolation and identification of anthrax

    NASA Astrophysics Data System (ADS)

    Kiel, Johnathan L.; Parker, Jill E.; Grubbs, Teri R.; Alls, John L.

    2000-07-01

    Anthrax has been recognized as a highly likely biological warfare or terrorist agent. The purpose of this work was to design a culture technique to rapidly isolate and identify `live' anthrax. In liquid or solid media form, 3AT medium (3-amino-L-tyrosine, the main ingredient) accelerated germination and growth of anthrax spores in 5 to 6 hours to a point expected at 18 to 24 hours with ordinary medium. During accelerated growth, standard definitive diagnostic tests such as sensitivity to lysis by penicillin or bacteriophage can be run. During this time, the bacteria synthesized a fluorescent and thermochemiluminescent polymer. Bacteria captured by specific antibody are, therefore, already labeled. Because living bacteria are required to generate the polymer, the test converts immunoassays for anthrax into viability assays. Furthermore, the polymer formation leads to the death of the vegetative form and non-viability of the spores produced in the medium. By altering the formulation of the medium, other microbes and even animal and human cells can be grown in it and labeled (including viruses grown in the animal or human cells).

  4. Rapid identification of antibiotic resistance using droplet microfluidics.

    PubMed

    Keays, Marie C; O'Brien, Mark; Hussain, Anam; Kiely, Patrick A; Dalton, Tara

    2016-04-01

    Culturing bacteria and monitoring bacterial cell growth is a critical issue when dealing with patients who present with bacterial infections. One of the main challenges that arises is the time taken to identify the particular strain of bacteria and consequently, decide the correct treatment. In the majority of cases, broad spectrum antibiotics are used to target infections when a narrow spectrum drug would be more appropriate. The efficient monitoring of bacterial growth and potential antibiotic resistance is necessary to identify the best treatment options for patients. Minturising the reactions into microfluidic droplets offers a novel method to rapidy analyze bacteria. Microfluidics facilitates low volume reactions that provide a unique system where each droplet reaction acts as an individual bioreactor. Here, we designed and built a novel platform that allowed us to create and monitor E.coli microfluidic droplet cultures. Optical capacity was built in and measurements of bacterial cultures were captured facilitating the continuous monitoring of individual reactions. The capacity of the instrument was demonstrated by the application of treatments to both bacteria and drug resistant strains of bacteria. We were able to detect responses within one hour in the droplet cultures, demonstrating the capacity of this workflow to the culture and rapid characterization of bacterial strains. PMID:26942773

  5. Identification of four plastid-localized protein kinases.

    PubMed

    Richter, Andreas S; Gartmann, Hans; Fechler, Mona; Rödiger, Anja; Baginsky, Sacha; Grimm, Bernhard

    2016-06-01

    In chloroplasts, protein phosphorylation regulates important processes, including metabolism, photosynthesis, gene expression, and signaling. Because the hitherto known plastid protein kinases represent only a fraction of existing kinases, we aimed at the identification of novel plastid-localized protein kinases that potentially phosphorylate enzymes of the tetrapyrrole biosynthesis (TBS) pathway. We screened publicly available databases for proteins annotated as putative protein kinase family proteins with predicted chloroplast localization. Additionally, we analyzed chloroplast fractions which were separated by sucrose density gradient centrifugation by mass spectrometry. We identified four new candidates for protein kinases, which were confirmed to be plastid localized by expression of GFP-fusion proteins in tobacco leaves. A phosphorylation assay with the purified kinases confirmed the protein kinase activity for two of them. PMID:27214872

  6. Rapid and Accurate Identification of Animal Species in Natural Leather Goods by Liquid Chromatography/Mass Spectrometry.

    PubMed

    Izuchi, Yukari; Takashima, Tsuneo; Hatano, Naoya

    2016-01-01

    The demand for leather goods has grown globally in recent years. Industry revenue is forecast to reach $91.2 billion by 2018. There is an ongoing labelling problem in the leather items market, in that it is currently impossible to identify the species that a given piece of leather is derived from. To address this issue, we developed a rapid and simple method for the specific identification of leather derived from cattle, horses, pigs, sheep, goats, and deer by analysing peptides produced by the trypsin-digestion of proteins contained in leather goods using liquid chromatography/mass spectrometry. We determined species-specific amino acid sequences by liquid chromatography/tandem mass spectrometry analysis using the Mascot software program and demonstrated that collagen α-1(I), collagen α-2(I), and collagen α-1(III) from the dermal layer of the skin are particularly useful in species identification. PMID:27313979

  7. Rapid and Accurate Identification of Animal Species in Natural Leather Goods by Liquid Chromatography/Mass Spectrometry

    PubMed Central

    Izuchi, Yukari; Takashima, Tsuneo; Hatano, Naoya

    2016-01-01

    The demand for leather goods has grown globally in recent years. Industry revenue is forecast to reach $91.2 billion by 2018. There is an ongoing labelling problem in the leather items market, in that it is currently impossible to identify the species that a given piece of leather is derived from. To address this issue, we developed a rapid and simple method for the specific identification of leather derived from cattle, horses, pigs, sheep, goats, and deer by analysing peptides produced by the trypsin-digestion of proteins contained in leather goods using liquid chromatography/mass spectrometry. We determined species-specific amino acid sequences by liquid chromatography/tandem mass spectrometry analysis using the Mascot software program and demonstrated that collagen α-1(I), collagen α-2(I), and collagen α-1(III) from the dermal layer of the skin are particularly useful in species identification. PMID:27313979

  8. Network understanding of herb medicine via rapid identification of ingredient-target interactions.

    PubMed

    Zhang, Hai-Ping; Pan, Jian-Bo; Zhang, Chi; Ji, Nan; Wang, Hao; Ji, Zhi-Liang

    2014-01-01

    Today, herb medicines have become the major source for discovery of novel agents in countermining diseases. However, many of them are largely under-explored in pharmacology due to the limitation of current experimental approaches. Therefore, we proposed a computational framework in this study for network understanding of herb pharmacology via rapid identification of putative ingredient-target interactions in human structural proteome level. A marketing anti-cancer herb medicine in China, Yadanzi (Brucea javanica), was chosen for mechanistic study. Total 7,119 ingredient-target interactions were identified for thirteen Yadanzi active ingredients. Among them, about 29.5% were estimated to have better binding affinity than their corresponding marketing drug-target interactions. Further Bioinformatics analyses suggest that simultaneous manipulation of multiple proteins in the MAPK signaling pathway and the phosphorylation process of anti-apoptosis may largely answer for Yadanzi against non-small cell lung cancers. In summary, our strategy provides an efficient however economic solution for systematic understanding of herbs' power. PMID:24429698

  9. Network Understanding of Herb Medicine via Rapid Identification of Ingredient-Target Interactions

    NASA Astrophysics Data System (ADS)

    Zhang, Hai-Ping; Pan, Jian-Bo; Zhang, Chi; Ji, Nan; Wang, Hao; Ji, Zhi-Liang

    2014-01-01

    Today, herb medicines have become the major source for discovery of novel agents in countermining diseases. However, many of them are largely under-explored in pharmacology due to the limitation of current experimental approaches. Therefore, we proposed a computational framework in this study for network understanding of herb pharmacology via rapid identification of putative ingredient-target interactions in human structural proteome level. A marketing anti-cancer herb medicine in China, Yadanzi (Brucea javanica), was chosen for mechanistic study. Total 7,119 ingredient-target interactions were identified for thirteen Yadanzi active ingredients. Among them, about 29.5% were estimated to have better binding affinity than their corresponding marketing drug-target interactions. Further Bioinformatics analyses suggest that simultaneous manipulation of multiple proteins in the MAPK signaling pathway and the phosphorylation process of anti-apoptosis may largely answer for Yadanzi against non-small cell lung cancers. In summary, our strategy provides an efficient however economic solution for systematic understanding of herbs' power.

  10. Rapid alteration of protein phosphorylation during postmortem: implication in the study of protein phosphorylation.

    PubMed

    Wang, Yifan; Zhang, Yanchong; Hu, Wen; Xie, Shutao; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

    2015-01-01

    Protein phosphorylation is an important post-translational modification of proteins. Postmortem tissues are widely being utilized in the biomedical studies, but the effects of postmortem on protein phosphorylation have not been received enough attention. In the present study, we found here that most proteins in mouse brain, heart, liver, and kidney were rapidly dephosphorylated to various degrees during 20 sec to 10 min postmortem. Phosphorylation of tau at Thr212 and glycogen synthase kinase 3β (GSK-3β) at Ser9 was reduced by 50% in the brain with 40 sec postmortem, a regular time for tissue processing. During postmortem, phosphorylation of cAMP-dependent protein kinase (PKA) and AMP activated kinase (AMPK) was increased in the brain, but not in other organs. Perfusion of the brain with cold or room temperature phosphate-buffered saline (PBS) also caused significant alteration of protein phosphorylation. Cooling down and maintaining mouse brains in the ice-cold buffer prevented the alteration effectively. This study suggests that phosphorylation of proteins is rapidly changed during postmortem. Thus, immediate processing of tissues followed by cooling down in ice-cold buffer is vitally important and perfusion has to be avoided when protein phosphorylation is to be studied. PMID:26511732

  11. Rapid alteration of protein phosphorylation during postmortem: implication in the study of protein phosphorylation

    PubMed Central

    Wang, Yifan; Zhang, Yanchong; Hu, Wen; Xie, Shutao; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

    2015-01-01

    Protein phosphorylation is an important post-translational modification of proteins. Postmortem tissues are widely being utilized in the biomedical studies, but the effects of postmortem on protein phosphorylation have not been received enough attention. In the present study, we found here that most proteins in mouse brain, heart, liver, and kidney were rapidly dephosphorylated to various degrees during 20 sec to 10 min postmortem. Phosphorylation of tau at Thr212 and glycogen synthase kinase 3β (GSK-3β) at Ser9 was reduced by 50% in the brain with 40 sec postmortem, a regular time for tissue processing. During postmortem, phosphorylation of cAMP-dependent protein kinase (PKA) and AMP activated kinase (AMPK) was increased in the brain, but not in other organs. Perfusion of the brain with cold or room temperature phosphate-buffered saline (PBS) also caused significant alteration of protein phosphorylation. Cooling down and maintaining mouse brains in the ice-cold buffer prevented the alteration effectively. This study suggests that phosphorylation of proteins is rapidly changed during postmortem. Thus, immediate processing of tissues followed by cooling down in ice-cold buffer is vitally important and perfusion has to be avoided when protein phosphorylation is to be studied. PMID:26511732

  12. Proteomics: Protein Identification Using Online Databases

    ERIC Educational Resources Information Center

    Eurich, Chris; Fields, Peter A.; Rice, Elizabeth

    2012-01-01

    Proteomics is an emerging area of systems biology that allows simultaneous study of thousands of proteins expressed in cells, tissues, or whole organisms. We have developed this activity to enable high school or college students to explore proteomic databases using mass spectrometry data files generated from yeast proteins in a college laboratory…

  13. Direct Maximization of Protein Identifications from Tandem Mass Spectra*

    PubMed Central

    Spivak, Marina; Weston, Jason; Tomazela, Daniela; MacCoss, Michael J.; Noble, William Stafford

    2012-01-01

    The goal of many shotgun proteomics experiments is to determine the protein complement of a complex biological mixture. For many mixtures, most methodological approaches fall significantly short of this goal. Existing solutions to this problem typically subdivide the task into two stages: first identifying a collection of peptides with a low false discovery rate and then inferring from the peptides a corresponding set of proteins. In contrast, we formulate the protein identification problem as a single optimization problem, which we solve using machine learning methods. This approach is motivated by the observation that the peptide and protein level tasks are cooperative, and the solution to each can be improved by using information about the solution to the other. The resulting algorithm directly controls the relevant error rate, can incorporate a wide variety of evidence and, for complex samples, provides 18–34% more protein identifications than the current state of the art approaches. PMID:22052992

  14. Simple Protein Complex Purification and Identification Method Suitable for High- throughput Mapping of Protein Interaction Networks

    SciTech Connect

    Markillie, Lye Meng; Lin, Chiann Tso; Adkins, Joshua N.; Auberry, Deanna L.; Hill, Eric A.; Hooker, Brian S.; Moore, Priscilla A.; Moore, Ronald J.; Shi, Liang; Wiley, H. S.; Kery, Vladimir

    2005-04-11

    Most of the current methods for purification and identification of protein complexes use endogenous expression of affinity tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, gel separation, in-gel digestion and mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pulldown assay with denaturing elution, trypsin digestion in organic solvent and LC ESI MS/MS protein identification using SEQUEST analysis. Our method is simple, easy to scale up and automate thus suitable for high throughput mapping of protein interaction networks and functional proteomics.

  15. Using Semantics, Grammar, Phonology, and Rapid Naming Tasks To Predict Word Identification.

    ERIC Educational Resources Information Center

    Hammill, Donald D.; Mather, Nancy; Allen, Elizabeth A.; Roberts, Rhia

    2002-01-01

    This study investigated the relative importance of semantic, grammatical, phonological, and rapid naming abilities in predicting word identification skills in 200 children (grades 1-6) using correlation, factor analysis, multiple regression, and predictive outcome analysis techniques. Composite measures of these abilities correlated significantly…

  16. A Perl procedure for protein identification by Peptide Mass Fingerprinting

    PubMed Central

    Tiengo, Alessandra; Barbarini, Nicola; Troiani, Sonia; Rusconi, Luisa; Magni, Paolo

    2009-01-01

    Background One of the topics of major interest in proteomics is protein identification. Protein identification can be achieved by analyzing the mass spectrum of a protein sample through different approaches. One of them, called Peptide Mass Fingerprinting (PMF), combines mass spectrometry (MS) data with searching strategies in a suitable database of known protein to provide a list of candidate proteins ranked by a score. To this aim, several algorithms and software tools have been proposed. However, the scoring methods and mainly the statistical evaluation of the results can be significantly improved. Results In this work, a Perl procedure for protein identification by PMF, called MsPI (Mass spectrometry Protein Identification), is presented. The implemented scoring methods were derived from the literature. MsPI implements a strategy to remove the contaminant masses present in the acquired spectra. Moreover, MsPI includes a statistical method to assign to each candidate protein, in addition to the scoring value, a p-value. Results obtained by MsPI on a dataset of 10 protein samples were compared with those achieved using two other software tools, i.e. Piums and Mascot. Piums implements one of the scoring methods available in MsPI, while Mascot is one of the most frequently used software tools in the protein identification field. MsPI scripts are available for downloading on the web site . Conclusion The performances of MsPI seem to be better than those of Piums and Mascot. In fact, on the considered dataset, MsPI includes in its candidate proteins list, the "true" proteins nine times over ten, whereas Piums includes in its list the "true" proteins only four time over ten. Even if Mascot also correctly includes in the candidates list the "true" proteins nine times over ten, it provides longer candidate lists, therefore increasing the number of false positives when the molecular weight of the proteins in the sample is approximatively known (e.g. by the 1-D/2-D

  17. Efficiency of a Multitest System (Enterotube) for Rapid Identification of Enterobacteriaceae

    PubMed Central

    Grunberg, E.; Titsworth, E.; Beskid, G.; Cleeland, R.; Delorenzo, W. F.

    1969-01-01

    Enterotube, a multiple-test system which combines nine biochemical tests useful in the identification of members of the family Enterobacteriaceae, was tested and compared with the PathoTec test system in the identification of gram-negative bacilli isolated from clinical specimens. It was found that both the Enterotube and the PathoTec systems rapidly and accurately defined the position of the organisms in the major groups of the family Enterobacteriaceae. Discrepancies were noted between the results of citrate tests on Simmons' citrate-agar and in the Enterotube, as well as between Simmons' citrate-agar and the PathoTec citrate test. It was concluded that the Enterotube system provides a simple, reliable, and rapid method for the presumptive identification of Enterobacteriaceae. The major advantage of the Enterotube is that all tests are done simultaneously by inoculation from a single isolated colony. PMID:4979941

  18. Evaluation of the rapid NFT system for identification of gram-negative, nonfermenting rods.

    PubMed

    Appelbaum, P C; Leathers, D J

    1984-10-01

    This study evaluated the ability of the Rapid NFT system (API System SA, Montalieu-Vercieu, France) to accurately identify 262 clinically isolated, gram-negative, nonfermentative rods without additional tests. Identifications were classified as correct; low discrimination, with a spectrum of two or more possibilities (additional tests necessary for accurate identification); and incorrect. Correct identification rates were analyzed in two categories: (i) correct to species or biotype for all organism groups except Alcaligenes faecalis-odorans, Moraxella, Pseudomonas testosteroni-alcaligenes-pseudoalcaligenes, and Acinetobacter calcoaceticus biotype haemolyticus-alcaligenes (in this category, the latter four genus-biotype group identifications were taken as correct) and (ii) correct to species or biotype in all cases, including the above four groups. In category i, 87.4% of the strains were correctly identified, with 4.2% low discrimination and 8.4% incorrect. When the criteria of category ii were used, 71.8% of the strains were correctly identified, with 19.9% low discrimination. The Rapid NFT system provided excellent species identification of Pseudomonas and Flavobacterium spp., Bordetella bronchiseptica, and Achromobacter xylosoxidans strains. Within Acinetobacter calcoaceticus, differentiation between biotypes anitratus and lwoffi was satisfactory, but the system did not differentiate between biotypes haemolyticus and alcaligenes. Species resolution within the genera Moraxella and Alcaligenes was incomplete. All Alcaligenes faecalis strains were misidentified and accounted for 50% of misidentifications with the Rapid NFT system; however, these results may reflect taxonomic differences rather than true misidentifications. The Rapid NFT system is easy to inoculate and interpret and represents a worthwhile advance in the identification of gram-negative, nonfermentative rods. PMID:6490857

  19. Identification of Ina proteins from Fusarium acuminatum

    NASA Astrophysics Data System (ADS)

    Scheel, Jan Frederik; Kunert, Anna Theresa; Pöschl, Ulrich; Fröhlich-Nowoisky, Janine

    2015-04-01

    Freezing of water above -36° C is based on ice nucleation activity (INA) mediated by ice nucleators (IN) which can be of various origins. Beside mineral IN, biological particles are a potentially important source of atmospheric IN. The best-known biological IN are common plant-associated bacteria. The IN activity of these bacteria is induced by a surface protein on the outer cell membrane, which is fully characterized. In contrast, much less is known about the nature of fungal IN. The fungal genus Fusarium is widely spread throughout the earth. It belongs to the Ascomycota and is one of the most severe fungal pathogens. It can affect a variety of organisms from plants to animals including humans. INA of Fusarium was already described about 30 years ago and INA of Fusarium as well as other fungal genera is assumed to be mediated by proteins or at least to contain a proteinaceous compound. Although many efforts were made the precise INA machinery of Fusarium and other fungal species including the proteins and their corresponding genes remain unidentified. In this study preparations from living fungal samples of F. acuminatum were fractionated by liquid chromatography and IN active fractions were identified by freezing assays. SDS-page and de novo sequencing by mass spectrometry were used to identify the primary structure of the protein. Preliminary results show that the INA protein of F. acuminatum is contained in the early size exclusion chromatography fractions indicating a high molecular size. Moreover we could identify a single protein band from IN active fractions at 130-145 kDa corresponding to sizes of IN proteins from bacterial species. To our knowledge this is for the first time an isolation of a single protein from in vivo samples, which can be assigned as IN active from Fusarium.

  20. Identification of kinetically hot residues in proteins.

    PubMed Central

    Demirel, M. C.; Atilgan, A. R.; Jernigan, R. L.; Erman, B.; Bahar, I.

    1998-01-01

    A number of recent studies called attention to the presence of kinetically important residues underlying the formation and stabilization of folding nuclei in proteins, and to the possible existence of a correlation between conserved residues and those participating in the folding nuclei. Here, we use the Gaussian network model (GNM), which recently proved useful in describing the dynamic characteristics of proteins for identifying the kinetically hot residues in folded structures. These are the residues involved in the highest frequency fluctuations near the native state coordinates. Their high frequency is a manifestation of the steepness of the energy landscape near their native state positions. The theory is applied to a series of proteins whose kinetically important residues have been extensively explored: chymotrypsin inhibitor 2, cytochrome c, and related C2 proteins. Most of the residues previously pointed out to underlie the folding process of these proteins, and to be critically important for the stabilization of the tertiary fold, are correctly identified, indicating a correlation between the kinetic hot spots and the early forming structural elements in proteins. Additionally, a strong correlation between kinetically hot residues and loci of conserved residues is observed. Finally, residues that may be important for the stability of the tertiary structure of CheY are proposed. PMID:9865946

  1. Rapid Identification of Candida Species and Other Clinically Important Yeast Species by Flow Cytometry†

    PubMed Central

    Page, Brent T.; Kurtzman, Cletus P.

    2005-01-01

    Two rapid diagnostic assays, utilizing two different Luminex flow cytometry methods, were developed for identification of clinically important ascomycetous yeast species. Direct hybridization and allele-specific primer extension methods were both successful in establishing a DNA-based assay that can rapidly and accurately identify Candida albicans, Candida krusei, Candida parapsilosis, Candida glabrata, and Candida tropicalis as well as other clinical species. The direct hybridization assay was designed to identify a total of 19 ascomycetous yeast species, and the allele-specific primer extension assay was designed to identify a total of 34 species. Probes were validated against 438 strains representing 303 species. From culture to identification, the allele-specific primer extension method takes 8 h and the direct hybridization method takes less than 5 h to complete. These assays represent comprehensive, rapid tests that are well suited for the clinical laboratory. PMID:16145099

  2. Rapid identification of Leishmania species by specific hybridization of kinetoplast DNA in cutaneous lesions.

    PubMed Central

    Wirth, D F; Pratt, D M

    1982-01-01

    Kinetoplast DNA (kDNA) was isolated from various species of the protozoic parasite Leishmania and analyzed by nucleic acid hybridization to detect species-related heterogeneity of kDNA. Purified DNA isolated from L. mexicana and L. braziliensis displayed no homology in nucleic acid hybridization studies. These results confirmed that rapid kDNA sequence change and evolution is occurring in New World species of Leishmania and suggested that such isolated kDNA could be used as a specific hybridization probe for the rapid identification of Leishmania species by using whole organisms. This work further demonstrates that such species-specific identification is feasible on isolated Leishmania promastigotes and, more important, directly on tissue touch blots derived from the cutaneous lesion. Thus, specific hybridization of isolated kDNA provides the basis for a rapid, accurate method for the diagnosis of human leishmaniasis directly from infected tissue. Images PMID:6960359

  3. Rapid identification of Leishmania species by specific hybridization of kinetoplast DNA in cutaneous lesions.

    PubMed

    Wirth, D F; Pratt, D M

    1982-11-01

    Kinetoplast DNA (kDNA) was isolated from various species of the protozoic parasite Leishmania and analyzed by nucleic acid hybridization to detect species-related heterogeneity of kDNA. Purified DNA isolated from L. mexicana and L. braziliensis displayed no homology in nucleic acid hybridization studies. These results confirmed that rapid kDNA sequence change and evolution is occurring in New World species of Leishmania and suggested that such isolated kDNA could be used as a specific hybridization probe for the rapid identification of Leishmania species by using whole organisms. This work further demonstrates that such species-specific identification is feasible on isolated Leishmania promastigotes and, more important, directly on tissue touch blots derived from the cutaneous lesion. Thus, specific hybridization of isolated kDNA provides the basis for a rapid, accurate method for the diagnosis of human leishmaniasis directly from infected tissue. PMID:6960359

  4. BioID Identification of Lamin-Associated Proteins.

    PubMed

    Mehus, Aaron A; Anderson, Ruthellen H; Roux, Kyle J

    2016-01-01

    A- and B-type lamins support the nuclear envelope, contribute to heterochromatin organization, and regulate a myriad of nuclear processes. The mechanisms by which lamins function in different cell types and the mechanisms by which lamin mutations cause over a dozen human diseases (laminopathies) remain unclear. The identification of proteins associated with lamins is likely to provide fundamental insight into these mechanisms. BioID (proximity-dependent biotin identification) is a unique and powerful method for identifying protein-protein and proximity-based interactions in living cells. BioID utilizes a mutant biotin ligase from bacteria that is fused to a protein of interest (bait). When expressed in living cells and stimulated with excess biotin, this BioID-fusion protein promiscuously biotinylates directly interacting and vicinal endogenous proteins. Following biotin-affinity capture, the biotinylated proteins can be identified using mass spectrometry. BioID thus enables screening for physiologically relevant protein associations that occur over time in living cells. BioID is applicable to insoluble proteins such as lamins that are often refractory to study by other methods and can identify weak and/or transient interactions. We discuss the use of BioID to elucidate novel lamin-interacting proteins and its applications in a broad range of biological systems, and provide detailed protocols to guide new applications. PMID:26778550

  5. Pyrosequencing as a tool for rapid fish species identification and commercial fraud detection.

    PubMed

    De Battisti, Cristian; Marciano, Sabrina; Magnabosco, Cristian; Busato, Sara; Arcangeli, Giuseppe; Cattoli, Giovanni

    2014-01-01

    The increased consumption of fish products, as well as the occurrence of exotic fish species in the Mediterranean Sea and in the fish market, has increased the risk of commercial fraud. Furthermore, the great amount of processed seafood products has greatly limited the application of classic identification systems. DNA-based identification allows a clear and unambiguous detection of polymorphisms between species, permitting differentiation and identification of both commercial fraud and introduction of species with potential toxic effects on humans. In this study, a novel DNA-based approach for differentiation of fish species based on pyrosequencing technology has been developed. Raw and processed fish products were tested, and up to 25 species of fish belonging to Clupeiformes and Pleuronectiformes groups were uniquely and rapidly identified. The proper identification based on short and unique genetic sequence signatures demonstrates that this approach is promising and cost-effective for large-scale surveys. PMID:24350776

  6. Rapid and Accurate Identification of Coagulase-Negative Staphylococci by Real-Time PCR

    PubMed Central

    Edwards, K. J.; Kaufmann, M. E.; Saunders, N. A.

    2001-01-01

    Biprobe identification assays based on real-time PCR were designed for 15 species of coagulase-negative staphylococci (CNS). Three sets of primers and four biprobes were designed from two variable regions of the 16S rRNA gene. An identification scheme was developed based on the pattern of melting peaks observed with the four biprobes that had been tested on 24 type strains. This scheme was then tested on 100 previously identified clinical isolates and 42 blindly tested isolates. For 125 of the 142 clinical isolates there was a perfect correlation between the biprobe identification and the result of the ID 32 Staph phenotypic tests and PCR. For 12 of the other isolates a 300-bp portion of the 16S rRNA gene was sequenced to determine identity. The remaining five isolates could not be fully identified. LightCycler real-time PCR allowed rapid and accurate identification of the important CNS implicated in infection. PMID:11526126

  7. Rapid Calculation of Protein pKa Values Using Rosetta

    PubMed Central

    Kilambi, Krishna Praneeth; Gray, Jeffrey J.

    2012-01-01

    We developed a Rosetta-based Monte Carlo method to calculate the pKa values of protein residues that commonly exhibit variable protonation states (Asp, Glu, Lys, His, and Tyr). We tested the technique by calculating pKa values for 264 residues from 34 proteins. The standard Rosetta score function, which is independent of any environmental conditions, failed to capture pKa shifts. After incorporating a Coulomb electrostatic potential and optimizing the solvation reference energies for pKa calculations, we employed a method that allowed side-chain flexibility and achieved a root mean-square deviation (RMSD) of 0.83 from experimental values (0.68 after discounting 11 predictions with an error over 2 pH units). Additional degrees of side-chain conformational freedom for the proximal residues facilitated the capture of charge-charge interactions in a few cases, resulting in an overall RMSD of 0.85 pH units. The addition of backbone flexibility increased the overall RMSD to 0.93 pH units but improved relative pKa predictions for proximal catalytic residues. The method also captures large pKa shifts of lysine and some glutamate point mutations in staphylococcal nuclease. Thus, a simple and fast method based on the Rosetta score function and limited conformational sampling produces pKa values that will be useful when rapid estimation is essential, such as in docking, design, and folding. PMID:22947875

  8. Peroxymonosulfate Rapidly Inactivates the Disease-Associated Prion Protein.

    PubMed

    Chesney, Alexandra R; Booth, Clarissa J; Lietz, Christopher B; Li, Lingjun; Pedersen, Joel A

    2016-07-01

    Prions, the etiological agents in transmissible spongiform encephalopathies, exhibit remarkable resistance to most methods of inactivation that are effective against conventional pathogens. Prions are composed of pathogenic conformers of the prion protein (PrP(TSE)). Some prion diseases are transmitted, in part, through environmental routes. The recalcitrance of prions to inactivation may lead to a persistent reservoir of infectivity that contributes to the environmental maintenance of epizootics. At present, few methods exist to remediate prion-contaminated land surfaces. Here we conducted a proof-of-principle study to examine the ability of peroxymonosulfate to degrade PrP(TSE). We find that peroxymonosulfate rapidly degrades PrP(TSE) from two species. Transition-metal-catalyzed decomposition of peroxymonosulfate to produce sulfate radicals appears to enhance degradation. We further demonstrate that exposure to peroxymonosulfate significantly reduced PrP(C) to PrP(TSE) converting ability as measured by protein misfolding cyclic amplification, used as a proxy for infectivity. Liquid chromatography-tandem mass spectrometry revealed that exposure to peroxymonosulfate results in oxidative modifications to methionine and tryptophan residues. This study indicates that peroxymonosulfate may hold promise for decontamination of prion-contaminated surfaces. PMID:27247993

  9. Multicolor Quantum Dot-Based Chemical Nose for Rapid and Array-Free Differentiation of Multiple Proteins.

    PubMed

    Xu, Qinfeng; Zhang, Yihong; Tang, Bo; Zhang, Chun-yang

    2016-02-16

    Nanomaterial-based differential sensors (e.g., chemical nose) have shown great potential for identification of multiple proteins because of their modulatable recognition and transduction capability but with the limitation of array separation, single-channel read-out, and long incubation time. Here, we develop a multicolor quantum dot (QD)-based multichannel sensing platform for rapid identification of multiple proteins in an array-free format within 1 min. A protein-binding dye of bromophenol blue (BPB) is explored as an efficient reversible quencher of QDs, and the mixture of BPB with multicolor QDs may generate the quenched QD-BPB complexes. The addition of proteins will disrupt the QD-BPB complexes as a result of the competitive protein-BPB binding, inducing the separation of BPB from the QDs and the generation of distinct fluorescence patterns. The multicolor patterns may be collected at a single-wavelength excitation and differentiated by a linear discriminant analysis (LDA). This multichannel sensing platform allows for the discrimination of ten proteins and seven cell lines with the fastest response rate reported to date, holding great promise for rapid and high-throughput medical diagnostics. PMID:26759896

  10. Detection and identification of protein citrullination in complex biological systems.

    PubMed

    Clancy, Kathleen W; Weerapana, Eranthie; Thompson, Paul R

    2016-02-01

    Protein citrullination is a post-translational modification of arginine that is catalyzed by the Protein Arginine Deiminase (PAD) family of enzymes. Aberrantly increased citrullination is associated with a host of inflammatory diseases and cancer and PAD inhibitors have shown remarkable efficacy in a range of diseases including rheumatoid arthritis, lupus, atherosclerosis, and ulcerative colitis. In rheumatoid arthritis, citrullinated proteins serve as key antigens for rheumatoid arthritis-associated autoantibodies. These data suggest that citrullinated proteins may serve more generally as biomarkers of specific disease states, however, the identification of citrullinated residues remains challenging due to the small 1Da mass change that occurs upon citrullination. Herein, we highlight the available techniques to identify citrullinated proteins/residues focusing on advanced MS techniques as well as chemical derivatization strategies that are currently being employed to identify citrullinated proteins as well as the specific residues modified within those proteins. PMID:26517730

  11. Rapid identification of haloarchaea and methanoarchaea using the matrix assisted laser desorption/ionization time-of-flight mass spectrometry

    PubMed Central

    Shih, Chao-Jen; Chen, Sheng-Chung; Weng, Chieh-Yin; Lai, Mei-Chin; Yang, Yu-Liang

    2015-01-01

    The aim of this study was to classify certain environmental haloarchaea and methanoarchaea using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and to expand the archaeal mass spectral database. A total of 69 archaea were collected including type strains and samples isolated locally from different environments. For extraction of the haloarchaeal total cell peptides/proteins, a simple method of acetonitrile extraction was developed. Cluster analysis conducted with the MALDI-TOF MS data overcame the high divergence in intragenomic 16S rRNA sequences in haloarchaea and clearly distinguished Methanohalophilus mahii from M. portucalensis. Putative biomarkers that can distinguish several particular archaeal genera were also assigned. In conclusion, this study expands the mass spectral database of peptide/protein fingerprints from bacteria and fungi to the archaea domain and provides a rapid identification platform for environmental archaeal samples. PMID:26541644

  12. Rapid Genus- and Species-Specific Identification of Cronobacter spp. by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry▿

    PubMed Central

    Stephan, Roger; Ziegler, Dominik; Pflüger, Valentin; Vogel, Guido; Lehner, Angelika

    2010-01-01

    Cronobacter spp. are Gram-negative opportunistic food-borne pathogens and are known as rare but important causes of life-threatening neonatal infections. Rapid and reliable identification of Cronobacter species and their differentiation from phenotypically similar, nonpathogenic Enterobacter turicensis, Enterobacter helveticus, and Enterobacter pulveris have become increasingly important. We evaluated here the application of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid genus and species identification of the six Cronobacter species recognized so far. To this end, we developed a reference MS database library that includes 54 Cronobacter target strains as well as 17 nontarget strains. The strains provided reproducible and unique mass spectra profiles covering a wide molecular mass range (2,000 to 30,000 Da). Genus- and species-specific biomarker protein mass patterns were determined. The defined biomarker mass patterns (Spectral Archive and Microbial Identification System [SARAMIS] SuperSpectrum) were validated using 36 strains from various Cronobacter species as well as eight nontarget strains. For all strains the mass spectrometry-based identification scheme yielded identical results as with a PCR-based identification system. All strains were correctly identified, and no nontarget strain was misidentified as Cronobacter. Our study demonstrates that MALDI-TOF MS is a reliable and powerful tool for the rapid identification of Cronobacter strains to the genus and species level. PMID:20554814

  13. Recovery and identification of mature enamel proteins in ancient teeth.

    PubMed

    Porto, Isabel M; Laure, Helen J; Tykot, Robert H; de Sousa, Frederico B; Rosa, Jose C; Gerlach, Raquel F

    2011-12-01

    Proteins in mineralized tissues provide a window to the past, and dental enamel is peculiar in being highly resistant to diagenesis and providing information on a very narrow window of time, such as the developing period; however, to date, complete proteins have not been extracted successfully from ancient teeth. In this work we tested the ability of a whole-crown micro-etch technique to obtain enamel protein samples from mature enamel of recently extracted (n = 2) and ancient (n = 2; ad 800 to 1100) third molars. Samples were analyzed using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry, and the resulting spectra were searched against the Swiss-Prot protein database using the Mascot software for protein identification. In our protocol, the separation of proteins in gel is not necessary. Successful identification of specific enamel proteins was obtained after whole-crown superficial enamel etching with 10% HCl. Most protein fragments recovered from dry teeth and mummy teeth contained amino-terminal amelogenin peptides. Only one peptide specific for the amelogenin X-isoform was identified. In conclusion, the reported techniques allowed the successful recovery of proteins specific to dental enamel from samples obtained in a very conservative manner, which may also be important in forensic and/or archeological science. PMID:22243232

  14. Imaging metals in proteins by combining electrophoresis with rapid x-ray fluorescence mapping.

    PubMed

    Finney, Lydia; Chishti, Yasmin; Khare, Tripti; Giometti, Carol; Levina, Aviva; Lay, Peter A; Vogt, Stefan

    2010-06-18

    Growing evidence points toward a very dynamic role for metals in biology. This suggests that physiological circumstance may mandate metal ion redistribution among ligands. This work addresses a critical need for technology that detects, identifies, and measures the metal-containing components of complex biological matrixes. We describe a direct, user-friendly approach for identifying and quantifying metal-protein adducts in complex samples using native- or SDS-PAGE, blotting, and rapid synchrotron X-ray fluorescence mapping with micro-XANES (X-ray absorption near-edge structure) of entire blots. The identification and quantification of each metal bound to a protein spot has been demonstrated, and the technique has been applied in two exemplary cases. In the first, the speciation of the in vitro binding of exogenous chromium to blood serum proteins was influenced markedly by both the oxidation state of chromium exposed to the serum proteins and the treatment conditions, which is of relevance to the biochemistry of Cr dietary supplements. In the second case, in vivo changes in endogenous metal speciation were examined to probe the influence of oxygen depletion on iron speciation in Shewanella oneidensis. PMID:20392082

  15. Imaging metals in proteins by combining electrophoresis with rapid x-ray fluorescence mapping.

    SciTech Connect

    Finney, L.; Chishti, Y.; Khare, T.; Giometti, C.; Levina, A.; Lay, P. A.; Vogt, S.; Univ. of Sydney; Northwestern Univ.

    2010-01-01

    Growing evidence points toward a very dynamic role for metals in biology. This suggests that physiological circumstance may mandate metal ion redistribution among ligands. This work addresses a critical need for technology that detects, identifies, and measures the metal-containing components of complex biological matrixes. We describe a direct, user-friendly approach for identifying and quantifying metal?protein adducts in complex samples using native- or SDS-PAGE, blotting, and rapid synchrotron X-ray fluorescence mapping with micro-XANES (X-ray absorption near-edge structure) of entire blots. The identification and quantification of each metal bound to a protein spot has been demonstrated, and the technique has been applied in two exemplary cases. In the first, the speciation of the in vitro binding of exogenous chromium to blood serum proteins was influenced markedly by both the oxidation state of chromium exposed to the serum proteins and the treatment conditions, which is of relevance to the biochemistry of Cr dietary supplements. In the second case, in vivo changes in endogenous metal speciation were examined to probe the influence of oxygen depletion on iron speciation in Shewanella oneidensis.

  16. The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.

    PubMed

    Xu, Xiaoli; Song, Yuan; Li, Yuhua; Chang, Jianfeng; Zhang, Hua; An, Lizhe

    2010-08-01

    Isolation and identification of protein partners in multi-protein complexes are important in gaining further insights into the cellular roles of proteins and determining the possible mechanisms by which proteins have an effect in the molecular environment. The tandem affinity purification (TAP) method was originally developed in yeast for the purification of protein complexes and identification of protein-protein interactions. With modifications to this method and many variations in the original tag made over the past few years, the TAP system could be applied in mammalian, plant, bacteria and other systems for protein complex analysis. In this review, we describe the application of the TAP method in various organisms, the modification in the tag, the disadvantages, the developments and the future prospects of the TAP method. PMID:20399864

  17. Identification of Chemical-Adducted Proteins in Urine by Multi-dimensional Protein Identification Technology (LC/LC–MS/MS)

    PubMed Central

    Labenski, Matthew T.; Fisher, Ashley A.; Monks, Terrence J.; Lau, Serrine S.

    2014-01-01

    Recent technological advancements in mass spectrometry facilitate the detection of chemical-induced posttranslational modifications (PTMs) that may alter cell signaling pathways or alter the structure and function of the modified proteins. To identify such protein adducts (Kleiner et al., Chem Res Toxicol 11:1283–1290, 1998), multi-dimensional protein identification technology (MuDPIT) has been utilized. MuDPIT was first described by Link et al. as a new technique useful for protein identification from a complex mixture of proteins (Link et al., Nat Biotechnol 17:676–682, 1999). MuDPIT utilizes two different HPLC columns to further enhance peptide separation, increasing the number of peptide hits and protein coverage. The technology is extremely useful for proteomes, such as the urine proteome, samples from immunoprecipitations, and 1D gel bands resolved from a tissue homogenate or lysate. In particular, MuDPIT has enhanced the field of adduct hunting for adducted peptides, since it is more capable of identifying lesser abundant peptides, such as those that are adducted, than the more standard LC–MS/MS. The site-specific identification of covalently adducted proteins is a prerequisite for understanding the biological significance of chemical-induced PTMs and the subsequent toxicological response they elicit. PMID:20972764

  18. Methods and Approaches to Mass Spectroscopy Based Protein Identification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This book chapter is a review of current mass spectrometers and the role in the field of proteomics. Various instruments are discussed and their strengths and weaknesses are highlighted. In addition, the methods of protein identification using a mass spectrometer are explained as well as data vali...

  19. Rapid whole protein quantitation of staphylococcal enterotoxins A and B by liquid chromatography/mass spectrometry.

    PubMed

    Sospedra, Isabel; Soler, Carla; Mañes, Jordi; Soriano, José Miguel

    2012-05-18

    Staphylococcus aureus is an important pathogen and has been indicated as the fifth causative agent of food-borne human illness throughout the world. Staphylococcal enterotoxins (SEs) are toxic compounds excreted mainly by strains of S. aureus. Among these toxins, enterotoxins A (SEA) and B (SEB) are both of the most prevalent compounds in staphylococcal food poisoning. In this work, reverse phase liquid chromatography coupled to ESI mass spectrometry (LC-ESI/MS) has been applied for its rapid identification and quantification. Limit of detection (LOD) values were 0.5 and 0.2 ng for SEA and SEB, respectively and limit of quantification (LOQ) value was 1 ng for both enterotoxins. SEA and SEB have been analyzed as intact proteins in milk and fruit juices. Analytical methods are essential for routine monitoring purposes and safeguard public health and the proposed technique can detect and quantify successfully SEA and SEB in food samples. PMID:22498351

  20. Rapid identification of Yersinia pestis and Brucella melitensis by chip-based continuous flow PCR

    NASA Astrophysics Data System (ADS)

    Dietzsch, Michael; Hlawatsch, Nadine; Melzer, Falk; Tomaso, Herbert; Gärtner, Claudia; Neubauer, Heinrich

    2012-06-01

    To combat the threat of biological agents like Yersinia pestis and Brucella melitensis in bioterroristic scenarios requires fast, easy-to-use and safe identification systems. In this study we describe a system for rapid amplification of specific genetic markers for the identification of Yersinia pestis and Brucella melitensis. Using chip based PCR and continuous flow technology we were able to amplify the targets simultaneously with a 2-step reaction profile within 20 minutes. The subsequent analysis of amplified fragments by standard gel electrophoresis requires another 45 minutes. We were able to detect both pathogens within 75 minutes being much faster than most other nucleic acid amplification technologies.

  1. Simple and rapid multiplex PCR for identification of the main human diarrheagenic Escherichia coli.

    PubMed

    Tobias, Joshua; Vutukuru, Sreekanth-Reddy

    2012-10-12

    Establishment of a simple and rapid multiplex PCR system for identification of the main diarrheagenic E. coli categories, including enteroaggregative E. coli, enterotoxigenic E. coli, enteropathogenic E. coli, and enterohemorrhagic E. coli, is described. This two-step multiplex PCR system allows the identification by targeting CVD432, LT, STh, STp, Eae, Bfp, Stx1, and Stx2. By applying the developed multiplex PCR system, categorization of E. coli isolates isolated from stool samples of infants with diarrhea into the main diarrheagenic E. coli categories is also shown. PMID:22192837

  2. Identification & Characterization of Fungal Ice Nucleation Proteins

    NASA Astrophysics Data System (ADS)

    Scheel, Jan Frederik; Kunert, Anna Theresa; Kampf, Christopher Johannes; Mauri, Sergio; Weidner, Tobias; Pöschl, Ulrich; Fröhlich-Nowoisky, Janine

    2016-04-01

    Freezing of water at relatively warm subfreezing temperatures is dependent on ice nucleation catalysis facilitated by ice nuclei (IN). These IN can be of various origins and although extensive research was done and progress was achieved, the nature and mechanisms leading to an effective IN are to date still poorly understood. Some of the most important processes of our geosphere like the water cycle are highly dependent on effective ice nucleation at temperatures between -2°C - -8°C, a temperature range which is almost exclusively covered by biological IN (BioIN). BioIN are usually macromolecular structures of biological polymers. Sugars as well as proteins have been reported to serve as IN and the best characterized BioIN are ice nucleation proteins (IN-P) from gram negative bacteria. Fungal strains from Fusarium spp. were described to be effective IN at subfreezing temperatures up to -2°C already 25 years ago and more and more fungal species are described to serve as efficient IN. Fungal IN are also thought to be proteins or at least contain a proteinaceous compound, but to date the fungal IN-P primary structure as well as their coding genetic elements of all IN active fungi are unknown. The aim of this study is a.) to identify the proteins and their coding genetic elements from IN active fungi (F. acuminatum, F. avenaceum, M. alpina) and b.) to characterize the mechanisms by which fungal IN serve as effective IN. We designed an interdisciplinary approach using biological, analytical and physical methods to identify fungal IN-P and describe their biological, chemical, and physical properties.

  3. Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory

    PubMed Central

    Suarez, Stéphanie; Ferroni, Agnès; Lotz, Aurélie; Jolley, Keith A.; Guérin, Philippe; Leto, Julie; Dauphin, Brunhilde; Jamet, Anne; Maiden, Martin C.J.; Nassif, Xavier; Armengaud, Jean

    2014-01-01

    Whole-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640–12000 m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates. PMID:23916798

  4. Tuber borchii fruit body: 2-dimensional profile and protein identification.

    PubMed

    Pierleoni, Raffaella; Buffalini, Michele; Vallorani, Luciana; Guidi, Chiara; Zeppa, Sabrina; Sacconi, Cinzia; Pucci, Piero; Amoresano, Angela; Casbarra, Annarita; Stocchi, Vilberto

    2004-04-01

    The formation of the fruit body represents the final phase of the ectomycorrhizal fungus T. borchii life cycle. Very little is known concerning the molecular and biochemical processes involved in the fructification phase. 2-DE maps of unripe and ripe ascocarps revealed different protein expression levels and the comparison of the electropherograms led to the identification of specific proteins for each developmental phase. Associating micropreparative 2-DE to microchemical approaches, such as N-terminal sequencing and 2-D gel-electrophoresis mass-spectrometry, proteins playing pivotal roles in truffle physiology were identified. PMID:15081280

  5. Bioinformatics pipeline for functional identification and characterization of proteins

    NASA Astrophysics Data System (ADS)

    Skarzyńska, Agnieszka; Pawełkowicz, Magdalena; Krzywkowski, Tomasz; Świerkula, Katarzyna; PlÄ der, Wojciech; Przybecki, Zbigniew

    2015-09-01

    The new sequencing methods, called Next Generation Sequencing gives an opportunity to possess a vast amount of data in short time. This data requires structural and functional annotation. Functional identification and characterization of predicted proteins could be done by in silico approches, thanks to a numerous computational tools available nowadays. However, there is a need to confirm the results of proteins function prediction using different programs and comparing the results or confirm experimentally. Here we present a bioinformatics pipeline for structural and functional annotation of proteins.

  6. Potential use of microarray technology for rapid identification of central nervous system pathogens.

    PubMed

    Hanson, Eric H; Niemeyer, Debra M; Folio, Les; Agan, Brian K; Rowley, Robb K

    2004-08-01

    Outbreaks of central nervous system (CNS) diseases result in significant productivity and financial losses, threatening peace and wartime readiness capabilities. To meet this threat, rapid clinical diagnostic tools for detecting and identifying CNS pathogens are needed. Current tools and techniques cannot efficiently deal with CNS pathogen diversity; they cannot provide real-time identification of pathogen serogroups and strains, and they require days, sometimes weeks, for examination of tissue culture. Rapid and precise CNS pathogen diagnostics are needed to provide the opportunity for tailored therapeutic regimens and focused preventive efforts to decrease morbidity and mortality. Such diagnostics are available through genetic and genomic technologies, which have the potential for reducing the time required in serogroup or strain identification from 500+ hours for some viral cultures to less than 3 hours for all pathogens. In the near future, microarray diagnostics and future derivations of these technologies will change the paradigm used for outbreak investigations and will improve health care for all. PMID:15379069

  7. Identification and analysis of multi-protein complexes in placenta.

    PubMed

    Wang, Fuqiang; Wang, Ling; Xu, Zhiyang; Liang, Gaolin

    2013-01-01

    Placental malfunction induces pregnancy disorders which contribute to life-threatening complications for both the mother and the fetus. Identification and characterization of placental multi-protein complexes is an important step to integratedly understand the protein-protein interaction networks in placenta which determine placental function. In this study, blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (BN/SDS-PAGE) and Liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to screen the multi-protein complexes in placenta. 733 unique proteins and 34 known and novel heterooligomeric multi-protein complexes including mitochondrial respiratory chain complexes, integrin complexes, proteasome complexes, histone complex, and heat shock protein complexes were identified. A novel protein complex, which involves clathrin and small conductance calcium-activated potassium (SK) channel protein 2, was identified and validated by antibody based gel shift assay, co-immunoprecipitation and immunofluorescence staining. These results suggest that BN/SDS-PAGE, when integrated with LC-MS/MS, is a very powerful and versatile tool for the investigation of placental protein complexes. This work paves the way for deeper functional characterization of the placental protein complexes associated with pregnancy disorders. PMID:23638173

  8. Rapid Identification of Black Grain Eumycetoma Causative Agents Using Rolling Circle Amplification

    PubMed Central

    Ahmed, Sarah A.; van den Ende, Bert H. G. Gerrits; Fahal, Ahmed H.; van de Sande, Wendy W. J.; de Hoog, G. S.

    2014-01-01

    Accurate identification of mycetoma causative agent is a priority for treatment. However, current identification tools are far from being satisfactory for both reliable diagnosis and epidemiological investigations. A rapid, simple, and highly efficient molecular based method for identification of agents of black grain eumycetoma is introduced, aiming to improve diagnostic in endemic areas. Rolling Circle Amplification (RCA) uses species-specific padlock probes and isothermal DNA amplification. The tests were based on ITS sequences and developed for Falciformispora senegalensis, F. tompkinsii, Madurella fahalii, M. mycetomatis, M. pseudomycetomatis, M. tropicana, Medicopsis romeroi, and Trematosphaeria grisea. With the isothermal RCA assay, 62 isolates were successfully identified with 100% specificity and no cross reactivity or false results. The main advantage of this technique is the low-cost, high specificity, and simplicity. In addition, it is highly reproducible and can be performed within a single day. PMID:25474355

  9. Rapid chemical test for the identification of chromium-molybdenum steel

    NASA Technical Reports Server (NTRS)

    Redmond, John C

    1932-01-01

    This note describes a simple, rapid, qualitative test which can be applied to solutions of drilling or chips for the identification of chromium-molybdenum steel. The test is based on the orange-red compound which is formed when thiocyanate and inequivalent molybdenum react. This test is much more reliable than the potassium ethylxanthate test which has been recommended for a like purpose. A list of the apparatus and reagents which are required, and a description of the procedure follows.

  10. Rapid Bacterial Identification, Resistance, Virulence and Type Profiling using Selected Reaction Monitoring Mass Spectrometry.

    PubMed

    Charretier, Yannick; Dauwalder, Olivier; Franceschi, Christine; Degout-Charmette, Elodie; Zambardi, Gilles; Cecchini, Tiphaine; Bardet, Chloe; Lacoux, Xavier; Dufour, Philippe; Veron, Laurent; Rostaing, Hervé; Lanet, Veronique; Fortin, Tanguy; Beaulieu, Corinne; Perrot, Nadine; Dechaume, Dominique; Pons, Sylvie; Girard, Victoria; Salvador, Arnaud; Durand, Géraldine; Mallard, Frédéric; Theretz, Alain; Broyer, Patrick; Chatellier, Sonia; Gervasi, Gaspard; Van Nuenen, Marc; Roitsch, Carolyn Ann; Van Belkum, Alex; Lemoine, Jérôme; Vandenesch, François; Charrier, Jean-Philippe

    2015-01-01

    Mass spectrometry (MS) in Selected Reaction Monitoring (SRM) mode is proposed for in-depth characterisation of microorganisms in a multiplexed analysis. Within 60-80 minutes, the SRM method performs microbial identification (I), antibiotic-resistance detection (R), virulence assessment (V) and it provides epidemiological typing information (T). This SRM application is illustrated by the analysis of the human pathogen Staphylococcus aureus, demonstrating its promise for rapid characterisation of bacteria from positive blood cultures of sepsis patients. PMID:26350205

  11. Rapid Bacterial Identification, Resistance, Virulence and Type Profiling using Selected Reaction Monitoring Mass Spectrometry

    PubMed Central

    Charretier, Yannick; Dauwalder, Olivier; Franceschi, Christine; Degout-Charmette, Elodie; Zambardi, Gilles; Cecchini, Tiphaine; Bardet, Chloe; Lacoux, Xavier; Dufour, Philippe; Veron, Laurent; Rostaing, Hervé; Lanet, Veronique; Fortin, Tanguy; Beaulieu, Corinne; Perrot, Nadine; Dechaume, Dominique; Pons, Sylvie; Girard, Victoria; Salvador, Arnaud; Durand, Géraldine; Mallard, Frédéric; Theretz, Alain; Broyer, Patrick; Chatellier, Sonia; Gervasi, Gaspard; Van Nuenen, Marc; Ann Roitsch, Carolyn; Van Belkum, Alex; Lemoine, Jérôme; Vandenesch, François; Charrier, Jean-Philippe

    2015-01-01

    Mass spectrometry (MS) in Selected Reaction Monitoring (SRM) mode is proposed for in-depth characterisation of microorganisms in a multiplexed analysis. Within 60–80 minutes, the SRM method performs microbial identification (I), antibiotic-resistance detection (R), virulence assessment (V) and it provides epidemiological typing information (T). This SRM application is illustrated by the analysis of the human pathogen Staphylococcus aureus, demonstrating its promise for rapid characterisation of bacteria from positive blood cultures of sepsis patients. PMID:26350205

  12. Rapid Identification of the Genus Fonsecaea by PCR with Specific Oligonucleotide Primers

    PubMed Central

    Abliz, Paride; Fukushima, Kazutaka; Takizawa, Kayoko; Nieda, Norikazu; Miyaji, Makoto; Nishimura, Kazuko

    2003-01-01

    An oligonucleotide primer set based on internal transcribed spacer regions of ribosomal DNA for PCR which gives the amplicon for only the DNA from Fonsecaea species was designed. This set yielded an amplicon with 333 bp for all strains of Fonsecaea pedrosoi and Fonsecaea compacta examined but no amplicons for related dematiaceous fungi and pathogenic yeasts. PCR using this primer set was considered to be a useful method for the rapid identification of the genus Fonsecaea. PMID:12574304

  13. Evaluation of an Immunochromatographic Assay Kit for Rapid Identification of Mycobacterium tuberculosis Complex in Clinical Isolates▿

    PubMed Central

    Park, Mi Young; Kim, Young Jin; Hwang, Sang Hyun; Kim, Hyoung Hoi; Lee, Eun Yup; Jeong, Seok Hoon; Chang, Chulhun L.

    2009-01-01

    We evaluated a new immunochromatographic assay (ICA) using mouse monoclonal anti-MPT64 antibody for rapid discrimination between Mycobacterium tuberculosis and nontuberculous mycobacteria in clinical isolates. A study with mycobacteria and other organisms showed excellent sensitivity (≅99%) and specificity (100%) and an appropriate detection limit (105 CFU/ml) when tested with M. tuberculosis H37Rv. This ICA can simplify the identification of M. tuberculosis in clinical laboratories. PMID:19052177

  14. An integral strategy toward the rapid identification of analogous nontarget compounds from complex mixtures.

    PubMed

    Wu, Liang; Gong, Ping; Wu, Yuzheng; Liao, Ke; Shen, Hanyuan; Qi, Qu; Liu, Huiying; Wang, Guangji; Hao, Haiping

    2013-08-16

    Identification of nontarget compounds in complex mixtures is of significant importance in various scientific fields. On the basis of the universal property that the compounds in complex mixtures can be classified to various analogous families, this study presents a general strategy for the rapid identification of nontarget compounds from complex matrixes using herbal medicine as an example. The proposed strategy consists of three sequential steps. First, a blank control sample is prepared for the purpose of removing interferences in the complex matrixes via automatic chromatographic and mass spectrometric data comparisons. Second, the diagnostic ions guided bridging network strategy is developed for the rapid classification of analogous compounds and structural characterizations. Finally, a quantitative structure retention relationship (QSRR) is built to validate the identifications and to differentiate isomers. Using this strategy, we have successfully identified a total of 45 organic acids from Mai-Luo-Ning and Flos Lonicerae injection, and 46 ginsenosides from Shen-Mai injection samples. The QSRR approach enabled a successful differentiation of most isomers. The proposed strategy will be expected to be applicable to the identification of nontarget compounds in complex mixtures. PMID:23838303

  15. Rapid Identification of Candida Species with Species-Specific DNA Probes

    PubMed Central

    Elie, Cheryl M.; Lott, Timothy J.; Reiss, Errol; Morrison, Christine J.

    1998-01-01

    Rapid identification of Candida species has become more important because of an increase in infections caused by species other than Candida albicans, including species innately resistant to azole antifungal drugs. We previously developed a PCR assay with an enzyme immunoassay (EIA) format to detect amplicons from the five most common Candida species by using universal fungal primers and species-specific probes directed to the ITS2 region of the gene for rRNA. We designed probes to detect seven additional Candida species (C. guilliermondii, C. kefyr, C. lambica, C. lusitaniae, C. pelliculosa, C. rugosa, and C. zeylanoides) included in the API 20C sugar assimilation panel, five probes for species not identified by API 20C (C. haemulonii, C. norvegica, C. norvegensis, C. utilis, and C. viswanathii), and a probe for the newly described species C. dubliniensis, creating a panel of 18 Candida species probes. The PCR-EIA correctly identified multiple strains of each species tested, including five identified as C. albicans by the currently available API 20C database but determined to be C. dubliniensis by genotypic and nonroutine phenotypic characteristics. Species identification time was reduced from a mean of 3.5 days by conventional identification methods to 7 h by the PCR-EIA. This method is simple, rapid, and feasible for identifying Candida species in clinical laboratories that utilize molecular identification techniques and provides a novel method to differentiate the new species, C. dubliniensis, from C. albicans. PMID:9774576

  16. Vibratory reaction unit for the rapid analysis of proteins and glycochains.

    PubMed

    Sasakura, Yukie; Nogami, Makoto; Kobayashi, Noriko; Kanda, Katsuhiro

    2007-01-01

    A protein digestion system using immobilized enzymes for protein identification and glycochain analyses has been developed, and a vibration reaction unit for micro-scale sample convection on an enzyme-immobilized solid surface was constructed. BSA as a model substrate was digested by this unit, and was successfully identified by mass spectrometry (MS) analyses. Compared to the conventional liquid-phase digestion, the reaction unit increased the number of matched peptides from 9 to 26, protein score from 455 to 1247, and sequence coverage from 21% to 48%. Glycopeptidase F (NGF), an enzyme that cleaves N-glycans from glycoproteins, was also immobilized and used to remove the glycochains from human immunoglobulin G (IgG). Trypsin and NGF were immobilized on the same solid surface and used to remove glycochains from IgG in single-step. Glycochains were labeled with fluorescent reagent and analyzed by HPLC. Several peaks corresponding to the glycochains of IgG were detected. These results suggested that the single-step digestion system, by immobilized multiple enzymes (trypsin and NGF) would be effective for the rapid structural analysis of glycoproteins. PMID:19662179

  17. Rapid and Efficient Protein Digestion using Trypsin Coated Magnetic Nanoparticles under Pressure Cycles

    SciTech Connect

    Lee, Byoungsoo; Lopez-Ferrer, Daniel; Kim, Byoung Chan; Na, Hyon Bin; Park, Yong Il; Weitz, Karl K.; Warner, Marvin G.; Hyeon, Taeghwan; Lee, Sang-Won; Smith, Richard D.; Kim, Jungbae

    2011-01-01

    Trypsin-coated magnetic nanoparticles (EC-TR/NPs), prepared via a simple crosslinking of the enzyme to magnetic nanoparticles, were highly stable and could be easily captured using a magnet after the digestion was complete. EC-TR/NPs showed a negligible loss of trypsin activity after multiple uses and continuous shaking, while a control sample of covalently-attached trypsin on NPs resulted in a rapid inactivation under the same conditions due to the denaturation and autolysis of trypsin. Digestions were carried out on a single model protein, a five protein mixture, and a whole mouse brain proteome, and also compared for digestion at atmospheric pressure and 37 ºC for 12 h, and in combination with pressure cycling technology (PCT) at room temperature for 1 min. In all cases, the EC-TR/NPs performed equally as well or better than free trypsin in terms of the number of peptide/protein identifications and reproducibility across technical replicates. However, the concomitant use of EC-TR/NPs and PCT resulted in very fast (~1 min) and more reproducible digestions.

  18. Identification and Validation of ISG15 Target Proteins.

    PubMed

    Durfee, Larissa A; Huibregtse, Jon M

    2010-01-01

    ISG15 is an interferon-induced ubiquitin-like protein (Ubl) that has antiviral properties. The core E1, E2 and E3 enzymes for conjugation of human ISG15 are Ube1L, UbcH8 and Herc5, all of which are induced at the transcriptional level by Type 1 interferon signaling. Several proteomics studies have, together, identified over 300 cellular proteins as ISG15 targets. These targets include a broad range of constitutively expressed proteins and approximately 15 interferon-induced proteins. This chapter provides an overview of the target identification process and the validation of these targets. We also discuss the limited number of examples where the biochemical effect of ISG15 conjugation on target proteins has been characterized. PMID:21222286

  19. Identification of Secreted Candida Proteins Using Mass Spectrometry.

    PubMed

    Gómez-Molero, Emilia; Dekker, Henk L; de Boer, Albert D; de Groot, Piet W J

    2016-01-01

    Analysis of fungal secretomes using mass spectrometry is a useful technique in cell biology. Knowledge of the secretome of a human fungal pathogen may yield important information of host-pathogen interactions and may be useful for identifying vaccines candidates or diagnostic markers for antifungal strategies. In this chapter, with a main focus on sample preparation aspects, we describe the methodology that we apply for gel-independent batch identification and quantification of proteins that are secreted during growth in liquid cultures. Using these techniques with Candida and other yeast species, the majority of the identified proteins are classical secretory proteins and cell wall proteins containing N-terminal signal peptides for secretion, although dependent on sample preparation quality and the mass spectrometric analysis also usually, a number of nonsecretory proteins are identified. PMID:26519067

  20. Identification of PDC-109-like protein(s) in buffalo seminal plasma.

    PubMed

    Harshan, Hiron M; Sankar, Surya; Singh, L P; Singh, Manish Kumar; Sudharani, S; Ansari, M R; Singh, S K; Majumdar, A C; Joshi, P

    2009-10-01

    The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma. PMID:19117702

  1. Proteomics technologies for the global identification and quantification of proteins.

    PubMed

    Brewis, Ian A; Brennan, P

    2010-01-01

    This review provides an introduction for the nonspecialist to proteomics and in particular the major approaches available for global protein identification and quantification. Proteomics technologies offer considerable opportunities for improved biological understanding and biomarker discovery. The central platform for proteomics is tandem mass spectrometry (MS) but a number of other technologies, resources, and expertise are absolutely required to perform meaningful experiments. These include protein separation science (and protein biochemistry in general), genomics, and bioinformatics. There are a range of workflows available for protein (or peptide) separation prior to tandem MS and subsequent bioinformatics analysis to achieve protein identifications. The predominant approaches are 2D electrophoresis (2DE) and subsequent MS, liquid chromatography-MS (LC-MS), and GeLC-MS. Beyond protein identification, there are a number of well-established options available for protein quantification. Difference gel electrophoresis (DIGE) following 2DE is one option but MS-based methods (most commonly iTRAQ-Isobaric Tags for Relative and Absolute Quantification or SILAC-Stable Isotope Labeling by Amino Acids) are now the preferred options. Sample preparation is critical to performing good experiments and subcellular fractionation can additionally provide protein localization information compared with whole cell lysates. Differential detergent solubilization is another valid option. With biological fluids, it is possible to remove the most abundant proteins by immunodepletion. Sample enrichment is also used extensively in certain analyses and most commonly in phosphoproteomics with the initial purification of phosphopeptides. Proteomics produces considerable datasets and resources to facilitate the necessary extended analysis of this data are improving all the time. Beyond the opportunities afforded by proteomics there are definite challenges to achieving full proteomic coverage

  2. Identification of differential protein interactors of lamin A and progerin.

    PubMed

    Kubben, Nard; Voncken, Jan Willem; Demmers, Jeroen; Calis, Chantal; van Almen, Geert; Pinto, Yigal; Misteli, Tom

    2010-01-01

    The nuclear lamina is an interconnected meshwork of intermediate filament proteins underlying the nuclear envelope. The lamina is an important regulator of nuclear structural integrity as well as nuclear processes, including transcription, DNA replication and chromatin remodeling. The major components of the lamina are A- and B-type lamins. Mutations in lamins impair lamina functions and cause a set of highly tissue-specific diseases collectively referred to as laminopathies. The phenotypic diversity amongst laminopathies is hypothesized to be caused by mutations affecting specific protein interactions, possibly in a tissue-specific manner. Current technologies to identify interaction partners of lamin A and its mutants are hampered by the insoluble nature of lamina components. To overcome the limitations of current technologies, we developed and applied a novel, unbiased approach to identify lamin A-interacting proteins. This approach involves expression of the high-affinity OneSTrEP-tag, precipitation of lamin-protein complexes after reversible protein cross-linking and subsequent protein identification by mass spectrometry. We used this approach to identify in mouse embryonic fibroblasts and cardiac myocyte NklTAg cell lines proteins that interact with lamin A and its mutant isoform progerin, which causes the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS). We identified a total of 313 lamina-interacting proteins, including several novel lamin A interactors, and we characterize a set of 35 proteins which preferentially interact with lamin A or progerin. PMID:21327095

  3. Rapid identification of Candida species in blood cultures by a clinically useful PCR method.

    PubMed Central

    Shin, J H; Nolte, F S; Morrison, C J

    1997-01-01

    Widespread use of fluconazole for the prophylaxis and treatment of candidiasis has led to a reduction in the number of cases of candidemia caused by Candida albicans but has also resulted in the emergence of candidemias caused by innately fluconazole-resistant, non-C. albicans Candida species. Given the fulminant and rapidly fatal outcome of acute disseminated candidiasis, rapid identification of newly emerging Candida species in blood culture is critical for the implementation of appropriately targeted antifungal drug therapy. Therefore, we used a PCR-based assay to rapidly identify Candida species from positive blood culture bottles. This assay used fungus-specific, universal primers for DNA amplification and species-specific probes to identify C. albicans, C. krusei, C. parapsilosis, C. tropicalis, or C. glabrata amplicons. It also used a simpler and more rapid (1.5-h) sample preparation technique than those described previously and used detergent, heat, and mechanical breakage to recover Candida species DNA from blood cultures. A simple and rapid (3.5-h) enzyme immunosorbent assay (EIA)-based format was then used for amplicon detection. One hundred fifty blood culture bottles, including 73 positive blood culture bottle sets (aerobic and anaerobic) from 31 patients with candidemia, were tested. The combined PCR and EIA methods (PCR-EIA) correctly identified all Candida species in 73 blood culture bottle sets, including bottles containing bacteria coisolated with yeasts and 3 cultures of samples from patients with mixed candidemias originally identified as single-species infections by routine phenotypic identification methods. Species identification time was reduced from a mean of 3.5 days by routine phenotypic methods to 7 h by the PCR-EIA method. No false-positive results were obtained for patients with bacteremias (n = 18), artificially produced non-Candida fungemias (n = 3), or bottles with no growth (n = 20). Analytical sensitivity was 1 cell per 2-microl

  4. Separation and identification of the light harvesting proteins contained in grana and stroma thylakoid membrane fractions.

    PubMed

    Timperio, Anna Maria; Huber, Christian G; Zolla, Lello

    2004-06-18

    This paper presents the results of a study performed to develop a rapid and straightforward method to resolve and simultaneously identify the light-harvesting proteins of photosystem I (LHCI) and photosystem II (LHCII) present in the grana and stroma of the thylakoid membranes of higher plants. These hydrophobic proteins are embedded in the phospholipid membrane, and their extraction usually requires detergent and time consuming manipulations that may introduce artifacts. The method presented here makes use of digitonin, a detergent which causes rapid (within less than 3 min) cleavage of the thylakoid membrane into two subfractions: appressed (grana) and non-appressed (stroma) membranes, the former enriched in photosystem II and the latter containing mainly photosystem I. From these two fractions identification of the protein components was performed by separating them by reversed-phase high-performance liquid chromatography (RP-HPLC) and determining the intact molecular mass by electrospray ionization mass spectrometry (ESI-MS). By this strategy the ion suppression during ESI-MS that normally occurs in the presence of membrane phospholipids was avoided, since RP-HPLC removed most phospholipids from the analytes. Consequently, high quality mass spectra were extracted from the reconstructed ion chromatograms. The specific cleavage of thylakoid membranes by digitonin, as well as the rapid identification and quantification of the antenna composition of the two complexes facilitate future studies of the lateral migration of the chlorophyll-protein complexes along thylakoid membranes, which is well known to be induced by high intensity light or other environmental stresses. Such investigations could not be performed by sodium dodecylsulfate-polyacrylamide gel electrophoresis because of insufficient resolution of the proteins having molecular masses between 22,000 and 25,000. PMID:15248427

  5. Systematic identification of protein combinations mediating chromatin looping.

    PubMed

    Zhang, Kai; Li, Nan; Ainsworth, Richard I; Wang, Wei

    2016-01-01

    Chromatin looping plays a pivotal role in gene expression and other biological processes through bringing distal regulatory elements into spatial proximity. The formation of chromatin loops is mainly mediated by DNA-binding proteins (DBPs) that bind to the interacting sites and form complexes in three-dimensional (3D) space. Previously, identification of DBP cooperation has been limited to those binding to neighbouring regions in the proximal linear genome (1D cooperation). Here we present the first study that integrates protein ChIP-seq and Hi-C data to systematically identify both the 1D- and 3D-cooperation between DBPs. We develop a new network model that allows identification of cooperation between multiple DBPs and reveals cell-type-specific and -independent regulations. Using this framework, we retrieve many known and previously unknown 3D-cooperations between DBPs in chromosomal loops that may be a key factor in influencing the 3D organization of chromatin. PMID:27461729

  6. Identification of short peptide sequences in complex milk protein hydrolysates.

    PubMed

    O'Keeffe, Martina B; FitzGerald, Richard J

    2015-10-01

    Numerous low molecular mass bioactive peptides (BAPs) can be generated during the hydrolysis of bovine milk proteins. Low molecular mass BAP sequences are less likely to be broken down by digestive enzymes and are thus more likely to be active in vivo. However, the identification of short peptides remains a challenge during mass spectrometry (MS) analysis due to issues with the transfer and over-fragmentation of low molecular mass ions. A method is described herein using time-of-flight ESI-MS/MS to effectively fragment and identify short peptides. This includes (a) short synthetic peptides, (b) short peptides within a defined hydrolysate sample, i.e. a prolyl endoproteinase hydrolysate of β-casein and (c) short peptides within a complex hydrolysate, i.e. a Corolase PP digest of sodium caseinate. The methodology may find widespread utilisation in the efficient identification of low molecular mass peptide sequences in food protein hydrolysates. PMID:25872436

  7. Systematic identification of protein combinations mediating chromatin looping

    PubMed Central

    Zhang, Kai; Li, Nan; Ainsworth, Richard I.; Wang, Wei

    2016-01-01

    Chromatin looping plays a pivotal role in gene expression and other biological processes through bringing distal regulatory elements into spatial proximity. The formation of chromatin loops is mainly mediated by DNA-binding proteins (DBPs) that bind to the interacting sites and form complexes in three-dimensional (3D) space. Previously, identification of DBP cooperation has been limited to those binding to neighbouring regions in the proximal linear genome (1D cooperation). Here we present the first study that integrates protein ChIP-seq and Hi-C data to systematically identify both the 1D- and 3D-cooperation between DBPs. We develop a new network model that allows identification of cooperation between multiple DBPs and reveals cell-type-specific and -independent regulations. Using this framework, we retrieve many known and previously unknown 3D-cooperations between DBPs in chromosomal loops that may be a key factor in influencing the 3D organization of chromatin. PMID:27461729

  8. Rapid identification of oral Actinomyces species cultivated from subgingival biofilm by MALDI-TOF-MS

    PubMed Central

    Stingu, Catalina S.; Borgmann, Toralf; Rodloff, Arne C.; Vielkind, Paul; Jentsch, Holger; Schellenberger, Wolfgang; Eschrich, Klaus

    2015-01-01

    Background Actinomyces are a common part of the residential flora of the human intestinal tract, genitourinary system and skin. Isolation and identification of Actinomyces by conventional methods is often difficult and time consuming. In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has become a rapid and simple method to identify bacteria. Objective The present study evaluated a new in-house algorithm using MALDI-TOF-MS for rapid identification of different species of oral Actinomyces cultivated from subgingival biofilm. Design Eleven reference strains and 674 clinical strains were used in this study. All the strains were preliminarily identified using biochemical methods and then subjected to MALDI-TOF-MS analysis using both similarity-based analysis and classification methods (support vector machine [SVM]). The genotype of the reference strains and of 232 clinical strains was identified by sequence analysis of the 16S ribosomal RNA (rRNA). Results The sequence analysis of the 16S rRNA gene of all references strains confirmed their previous identification. The MALDI-TOF-MS spectra obtained from the reference strains and the other clinical strains undoubtedly identified as Actinomyces by 16S rRNA sequencing were used to create the mass spectra reference database. Already a visual inspection of the mass spectra of different species reveals both similarities and differences. However, the differences between them are not large enough to allow a reliable differentiation by similarity analysis. Therefore, classification methods were applied as an alternative approach for differentiation and identification of Actinomyces at the species level. A cross-validation of the reference database representing 14 Actinomyces species yielded correct results for all species which were represented by more than two strains in the database. Conclusions Our results suggest that a combination of MALDI-TOF-MS with powerful

  9. Functional module identification in protein interaction networks by interaction patterns

    PubMed Central

    Wang, Yijie; Qian, Xiaoning

    2014-01-01

    Motivation: Identifying functional modules in protein–protein interaction (PPI) networks may shed light on cellular functional organization and thereafter underlying cellular mechanisms. Many existing module identification algorithms aim to detect densely connected groups of proteins as potential modules. However, based on this simple topological criterion of ‘higher than expected connectivity’, those algorithms may miss biologically meaningful modules of functional significance, in which proteins have similar interaction patterns to other proteins in networks but may not be densely connected to each other. A few blockmodel module identification algorithms have been proposed to address the problem but the lack of global optimum guarantee and the prohibitive computational complexity have been the bottleneck of their applications in real-world large-scale PPI networks. Results: In this article, we propose a novel optimization formulation LCP2 (low two-hop conductance sets) using the concept of Markov random walk on graphs, which enables simultaneous identification of both dense and sparse modules based on protein interaction patterns in given networks through searching for LCP2 by random walk. A spectral approximate algorithm SLCP2 is derived to identify non-overlapping functional modules. Based on a bottom-up greedy strategy, we further extend LCP2 to a new algorithm (greedy algorithm for LCP2) GLCP2 to identify overlapping functional modules. We compare SLCP2 and GLCP2 with a range of state-of-the-art algorithms on synthetic networks and real-world PPI networks. The performance evaluation based on several criteria with respect to protein complex prediction, high level Gene Ontology term prediction and especially sparse module detection, has demonstrated that our algorithms based on searching for LCP2 outperform all other compared algorithms. Availability and implementation: All data and code are available at http://www.cse.usf.edu/∼xqian/fmi/slcp2hop

  10. Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering

    PubMed Central

    Blackburn, Matthew C.; Petrova, Ekaterina; Correia, Bruno E.; Maerkl, Sebastian J.

    2016-01-01

    The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3–4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF–DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering. PMID:26704969

  11. POLYPHENOLS AND MECHANICAL MACERATION SHIFT PROTEIN FRACTIONS IN LEGUME HAYS FROM RAPIDLY TO SLOWLY DEGRADED FORMS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid proteolysis of forage protein during rumen fermentation can impair protein use by dairy cattle. The severity of conditioning at harvest may influence protein degradability in forages, particularly if protein-binding polyphenols are present. In 2002 and 2003, first and second cuttings of alfalf...

  12. Identification of novel CBP interacting proteins in embryonic orofacial tissue

    SciTech Connect

    Yin Xiaolong; Warner, Dennis R.; Roberts, Emily A.; Pisano, M. Michele; Greene, Robert M. . E-mail: greene@louisville.edu

    2005-04-15

    cAMP response element-binding protein (CREB)-binding protein (CBP) plays an important role as a general co-integrator of multiple signaling pathways and interacts with a large number of transcription factors and co-factors, through its numerous protein-binding domains. To identify nuclear factors associated with CBP in developing orofacial tissue, a yeast two-hybrid screen of a cDNA library derived from orofacial tissue from gestational day 11 to 13 mouse embryos was conducted. Using the carboxy terminus (amino acid residues 1676-2441) of CBP as bait, several novel proteins that bind CBP were identified, including an Msx-interacting-zinc finger protein, CDC42 interaction protein 4/thyroid hormone receptor interactor 10, SH3-domain GRB2-like 1, CCR4-NOT transcription complex subunit 3, adaptor protein complex AP-1 {beta}1 subunit, eukaryotic translation initiation factor 2B subunit 1 ({alpha}), and cyclin G-associated kinase. Results of the yeast two-hybrid screen were confirmed by glutathione S-transferase pull-down assays. The identification of these proteins as novel CBP-binding partners allows exploration of new mechanisms by which CBP regulates and integrates diverse cell signaling pathways.

  13. Identification of Topping Responsive Proteins in Tobacco Roots

    PubMed Central

    Li, Fei; Zhang, Huizhen; Wang, Shaoxin; Xiao, Wanfu; Ding, Chao; Liu, Weiqun; Guo, Hongxiang

    2016-01-01

    The process of topping elicits many responses in the tobacco plant, including an increase in nicotine biosynthesis, and the secondary growth of roots. Some topping responsive miRNAs and genes have been identified in our previous study, but the mechanism of the tobacco response to topping has not yet been fully elucidated. In this study, topping responsive proteins isolated from tobacco roots were screened using two-dimensional electrophoresis. Of the proteins identified, calreticulin and auxin-responsive protein indole acetic acid (IAA9) were involved in the secondary growth of roots; leucine-rich repeat disease resistance, heat shock protein 70, and farnesyl pyrophosphate synthase 1 were involved in the wounding stress response; and F-box protein played an important role in promoting the ability of nicotine synthesis after topping. In addition, we identified five tobacco bHLH proteins (NtbHLH, NtMYC1a, NtMYC1b, NtMYC2a, and NtMYC2b) related to nicotine biosynthesis. NtMYC2 was suggested to be the main positive transcription factor, with NtbHLH protein being a negative regulator in the jasmonic acid (JA)-mediated activation of nicotine biosynthesis after topping. Tobacco topping activates a comprehensive range of biological processes involving the IAA and JA signaling pathways, and the identification of proteins involved in these processes will improve our understanding of the topping response. PMID:27200055

  14. Identification of Topping Responsive Proteins in Tobacco Roots.

    PubMed

    Li, Fei; Zhang, Huizhen; Wang, Shaoxin; Xiao, Wanfu; Ding, Chao; Liu, Weiqun; Guo, Hongxiang

    2016-01-01

    The process of topping elicits many responses in the tobacco plant, including an increase in nicotine biosynthesis, and the secondary growth of roots. Some topping responsive miRNAs and genes have been identified in our previous study, but the mechanism of the tobacco response to topping has not yet been fully elucidated. In this study, topping responsive proteins isolated from tobacco roots were screened using two-dimensional electrophoresis. Of the proteins identified, calreticulin and auxin-responsive protein indole acetic acid (IAA9) were involved in the secondary growth of roots; leucine-rich repeat disease resistance, heat shock protein 70, and farnesyl pyrophosphate synthase 1 were involved in the wounding stress response; and F-box protein played an important role in promoting the ability of nicotine synthesis after topping. In addition, we identified five tobacco bHLH proteins (NtbHLH, NtMYC1a, NtMYC1b, NtMYC2a, and NtMYC2b) related to nicotine biosynthesis. NtMYC2 was suggested to be the main positive transcription factor, with NtbHLH protein being a negative regulator in the jasmonic acid (JA)-mediated activation of nicotine biosynthesis after topping. Tobacco topping activates a comprehensive range of biological processes involving the IAA and JA signaling pathways, and the identification of proteins involved in these processes will improve our understanding of the topping response. PMID:27200055

  15. Identification of AOSC-binding proteins in neurons

    NASA Astrophysics Data System (ADS)

    Liu, Ming; Nie, Qin; Xin, Xianliang; Geng, Meiyu

    2008-11-01

    Acidic oligosaccharide sugar chain (AOSC), a D-mannuronic acid oligosaccharide, derived from brown algae polysaccharide, has been completed Phase I clinical trial in China as an anti-Alzheimer’s Disease (AD) drug candidate. The identification of AOSC-binding protein(s) in neurons is very important for understanding its action mechanism. To determine the binding protein(s) of AOSC in neurons mediating its anti-AD activities, confocal microscopy, affinity chromatography, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were used. Confocal microscopy analysis shows that AOSC binds to SH-SY5Y cells in concentration-, time-, and temperature-dependent fashions. The AOSC binding proteins were purified by affinity chromatography and identified by LC-MS/MS analysis. The results showed that there are 349 proteins binding AOSC, including clathrin, adaptor protein-2 (AP-2) and amyloid precursor protein (APP). These results suggest that the binding/entrance of AOSC to neurons is probably responsible for anti-AD activities.

  16. Separating the wheat from the chaff: unbiased filtering of background tandem mass spectra improves protein identification.

    PubMed

    Junqueira, Magno; Spirin, Victor; Santana Balbuena, Tiago; Waridel, Patrice; Surendranath, Vineeth; Kryukov, Grigoriy; Adzhubei, Ivan; Thomas, Henrik; Sunyaev, Shamil; Shevchenko, Andrej

    2008-08-01

    Only a small fraction of spectra acquired in LC-MS/MS runs matches peptides from target proteins upon database searches. The remaining, operationally termed background, spectra originate from a variety of poorly controlled sources and affect the throughput and confidence of database searches. Here, we report an algorithm and its software implementation that rapidly removes background spectra, regardless of their precise origin. The method estimates the dissimilarity distance between screened MS/MS spectra and unannotated spectra from a partially redundant background library compiled from several control and blank runs. Filtering MS/MS queries enhanced the protein identification capacity when searches lacked spectrum to sequence matching specificity. In sequence-similarity searches it reduced by, on average, 30-fold the number of orphan hits, which were not explicitly related to background protein contaminants and required manual validation. Removing high quality background MS/MS spectra, while preserving in the data set the genuine spectra from target proteins, decreased the false positive rate of stringent database searches and improved the identification of low-abundance proteins. PMID:18558732

  17. A novel multiplex isothermal amplification method for rapid detection and identification of viruses

    PubMed Central

    Nyan, Dougbeh-Chris; Swinson, Kevin L.

    2015-01-01

    A rapid multiplex isothermal amplification assay has been developed for detection and identification of multiple blood-borne viruses that infect millions of people world-wide. These infections may lead to chronic diseases or death if not diagnosed and treated in a timely manner. Sets of virus-specific oligonucleotides and oligofluorophores were designed and used in a reverse-transcription loop-mediated multiplexed isothermal amplification reaction for detection and gel electrophoretic identification of human Immunodeficiency virus (HIV), hepatitis-B virus (HBV), hepatitis-C virus (HCV), hepatitis-E virus (HEV), dengue virus (DENV), and West Nile (WNV) virus infection in blood plasma. Amplification was catalyzed with two thermostable enzymes for 30–60 minutes under isothermal condition, utilizing a simple digital heat source. Electrophoretic analysis of amplified products demonstrated simultaneous detection of 6 viruses that were distinctly identified by unique ladder-like banding patterns. Naked-eye fluorescent visualization of amplicons revealed intensely fluorescing products that indicated positive detection. The test demonstrated a 97% sensitivity and a 100% specificity, with no cross-reaction with other viruses observed. This portable detection tool may have clinical and field utility in the developing and developed world settings. This may enable rapid diagnosis and identification of viruses for targeted therapeutic intervention and prevention of disease transmission. PMID:26643761

  18. Rapid and accurate identification of microorganisms contaminating cosmetic products based on DNA sequence homology.

    PubMed

    Fujita, Y; Shibayama, H; Suzuki, Y; Karita, S; Takamatsu, S

    2005-12-01

    The aim of this study was to develop rapid and accurate procedures to identify microorganisms contaminating cosmetic products, based on the identity of the nucleotide sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA coding DNA (rDNA). Five types of microorganisms were isolated from the inner portion of lotion bottle caps, skin care lotions, and cleansing gels. The rDNA ITS region of microorganisms was amplified through the use of colony-direct PCR or ordinal PCR using DNA extracts as templates. The nucleotide sequences of the amplified DNA were determined and subjected to homology search of a publicly available DNA database. Thereby, we obtained DNA sequences possessing high similarity with the query sequences from the databases of all the five organisms analyzed. The traditional identification procedure requires expert skills, and a time period of approximately 1 month to identify the microorganisms. On the contrary, 3-7 days were sufficient to complete all the procedures employed in the current method, including isolation and cultivation of organisms, DNA sequencing, and the database homology search. Moreover, it was possible to develop the skills necessary to perform the molecular techniques required for the identification procedures within 1 week. Consequently, the current method is useful for rapid and accurate identification of microorganisms, contaminating cosmetics. PMID:18492168

  19. Rapid Multi-Damage Identification for Health Monitoring of Laminated Composites Using Piezoelectric Wafer Sensor Arrays

    PubMed Central

    Si, Liang; Wang, Qian

    2016-01-01

    Through the use of the wave reflection from any damage in a structure, a Hilbert spectral analysis-based rapid multi-damage identification (HSA-RMDI) technique with piezoelectric wafer sensor arrays (PWSA) is developed to monitor and identify the presence, location and severity of damage in carbon fiber composite structures. The capability of the rapid multi-damage identification technique to extract and estimate hidden significant information from the collected data and to provide a high-resolution energy-time spectrum can be employed to successfully interpret the Lamb waves interactions with single/multiple damage. Nevertheless, to accomplish the precise positioning and effective quantification of multiple damage in a composite structure, two functional metrics from the RMDI technique are proposed and used in damage identification, which are the energy density metric and the energy time-phase shift metric. In the designed damage experimental tests, invisible damage to the naked eyes, especially delaminations, were detected in the leftward propagating waves as well as in the selected sensor responses, where the time-phase shift spectra could locate the multiple damage whereas the energy density spectra were used to quantify the multiple damage. The increasing damage was shown to follow a linear trend calculated by the RMDI technique. All damage cases considered showed completely the developed RMDI technique potential as an effective online damage inspection and assessment tool. PMID:27153070

  20. Rapid Multi-Damage Identification for Health Monitoring of Laminated Composites Using Piezoelectric Wafer Sensor Arrays.

    PubMed

    Si, Liang; Wang, Qian

    2016-01-01

    Through the use of the wave reflection from any damage in a structure, a Hilbert spectral analysis-based rapid multi-damage identification (HSA-RMDI) technique with piezoelectric wafer sensor arrays (PWSA) is developed to monitor and identify the presence, location and severity of damage in carbon fiber composite structures. The capability of the rapid multi-damage identification technique to extract and estimate hidden significant information from the collected data and to provide a high-resolution energy-time spectrum can be employed to successfully interpret the Lamb waves interactions with single/multiple damage. Nevertheless, to accomplish the precise positioning and effective quantification of multiple damage in a composite structure, two functional metrics from the RMDI technique are proposed and used in damage identification, which are the energy density metric and the energy time-phase shift metric. In the designed damage experimental tests, invisible damage to the naked eyes, especially delaminations, were detected in the leftward propagating waves as well as in the selected sensor responses, where the time-phase shift spectra could locate the multiple damage whereas the energy density spectra were used to quantify the multiple damage. The increasing damage was shown to follow a linear trend calculated by the RMDI technique. All damage cases considered showed completely the developed RMDI technique potential as an effective online damage inspection and assessment tool. PMID:27153070

  1. Purify First: rapid expression and purification of proteins from XMRV.

    PubMed

    Gillette, William K; Esposito, Dominic; Taylor, Troy E; Hopkins, Ralph F; Bagni, Rachel K; Hartley, James L

    2011-04-01

    Purifying proteins from recombinant sources is often difficult, time-consuming, and costly. We have recently instituted a series of improvements in our protein purification pipeline that allows much more accurate choice of expression host and conditions and purification protocols. The key elements are parallel cloning, small scale parallel expression and lysate preparation, and small scale parallel protein purification. Compared to analyzing expression data only, results from multiple small scale protein purifications predict success at scale-up with greatly improved reliability. Using these new procedures we purified eight of nine proteins from xenotropic murine leukemia virus-related virus (XMRV) on the first attempt at large scale. PMID:21146612

  2. Etiological Analysis of Fungal Keratitis and Rapid Identification of Predominant Fungal Pathogens.

    PubMed

    He, Dan; Hao, Jilong; Gao, Song; Wan, Xue; Wang, Wanting; Shan, Qiushi; Wang, Li

    2016-02-01

    Fungal keratitis is a worldwide-distributed refractory and potentially blinding ocular infection caused by various fungi. It is necessary to investigate the etiological and epidemiological characteristics of this disease and establish a rapid and specific pathogenic identification method. Here, we isolated and identified fungal pathogens of 275 patients with presumed fungal keratitis from Jilin Province, China, and conducted statistical analyses of epidemiological information. The positive rate of fungal culture was 72.0 %. Fusarium sp. was the most common genus among 210 fungal isolates. The predominant species were Fusarium solani, Aspergillus fumigatus, and Candida glabrata, which accounted for over 50 % of the isolated organisms. Corneal trauma and previous use of drugs were the most important predisposing factors. In addition, a multiplex polymerase chain reaction (PCR) was designed with species-specific primers of the three species that could identify them with amplicons of approximately 330 bp from F. solani, 275 bp from A. fumigatus, and 230 bp from C. glabrata. Additionally, PCR with fungal universal primers and multiplex PCR were performed using DNA prepared by an improved DNA extraction method from corneal scrapings. With this method, fungal pathogens from corneal scrapings could be specifically and rapidly identified within 8 h. The culture-independent rapid identification of corneal scrapings may have great significance for the early diagnosis and treatment of fungal keratitis. PMID:26446032

  3. Evaluation of Verigene Blood Culture Test Systems for Rapid Identification of Positive Blood Cultures

    PubMed Central

    Kim, Jae-Seok; Kang, Go-Eun; Kim, Han-Sung; Song, Wonkeun; Lee, Kyu Man

    2016-01-01

    The performance of molecular tests using the Verigene Gram-Positive and Gram-Negative Blood Culture nucleic acid tests (BC-GP and BC-GN, resp.; Naosphere, Northbrook, IL, USA) was evaluated for the identification of microorganisms detected from blood cultures. Ninety-nine blood cultures containing Gram-positive bacteria and 150 containing Gram-negative bacteria were analyzed using the BC-GP and BC-GN assays, respectively. Blood cultures were performed using the Bactec blood culture system (BD Diagnostic Systems, Franklin Lakes, NJ, USA) and conventional identification and antibiotic-susceptibility tests were performed using a MicroScan system (Siemens, West Sacramento, CA, USA). When a single strain of bacteria was isolated from the blood culture, Verigene assays correctly identified 97.9% (94/96) of Gram-positive bacteria and 93.8% (137/146) of Gram-negative bacteria. Resistance genes mecA and vanA were correctly detected by the BC-GP assay, while the extended-spectrum β-lactamase CTX-M and the carbapenemase OXA resistance gene were detected from 30 cases cultures by the BC-GN assay. The BC-GP and BC-GN assays showed high agreement with conventional identification and susceptibility tests. These tests are useful for rapid identification of microorganisms and the detection of clinically important resistance genes from positive Bactec blood cultures. PMID:26904669

  4. Rapid urine preparation prior to identification of uropathogens by MALDI-TOF MS.

    PubMed

    Veron, L; Mailler, S; Girard, V; Muller, B H; L'Hostis, G; Ducruix, C; Lesenne, A; Richez, A; Rostaing, H; Lanet, V; Ghirardi, S; van Belkum, A; Mallard, F

    2015-09-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS) has been introduced in clinical routine microbiology laboratories. For the rapid diagnosis of urinary tract infections, culture-independent methods prior MALDI-mediated identification have been described. Here, we describe a comparison of three of these methods based on their performance of bacterial identification and their potential as a routine tool for microbiology labs : (i) differential centrifugation, (ii) urine filtration and (iii) a 5-h bacterial cultivation on solid culture media. For 19 urine samples, all methods were directly compared and correct bacterial species identification by MALDI was used as performance indicator. A higher percentage of correct MALDI identification was obtained after filtration (78.9 %) and the growth-based method (84.2 %) as compared to differential centrifugation (68.4 %). Additional testing of 76 mono-microbial specimens (bacteriuria > 10(5) CFU/mL) confirmed the good performance of short growth with a 90.8 % correct MALDI score, with a potentially better fit to the routine workflow of microbiology labs. PMID:26054715

  5. Final Progress Report: Isotope Identification Algorithm for Rapid and Accurate Determination of Radioisotopes Feasibility Study

    SciTech Connect

    Rawool-Sullivan, Mohini; Bounds, John Alan; Brumby, Steven P.; Prasad, Lakshman; Sullivan, John P.

    2012-04-30

    This is the final report of the project titled, 'Isotope Identification Algorithm for Rapid and Accurate Determination of Radioisotopes,' PMIS project number LA10-HUMANID-PD03. The goal of the work was to demonstrate principles of emulating a human analysis approach towards the data collected using radiation isotope identification devices (RIIDs). It summarizes work performed over the FY10 time period. The goal of the work was to demonstrate principles of emulating a human analysis approach towards the data collected using radiation isotope identification devices (RIIDs). Human analysts begin analyzing a spectrum based on features in the spectrum - lines and shapes that are present in a given spectrum. The proposed work was to carry out a feasibility study that will pick out all gamma ray peaks and other features such as Compton edges, bremsstrahlung, presence/absence of shielding and presence of neutrons and escape peaks. Ultimately success of this feasibility study will allow us to collectively explain identified features and form a realistic scenario that produced a given spectrum in the future. We wanted to develop and demonstrate machine learning algorithms that will qualitatively enhance the automated identification capabilities of portable radiological sensors that are currently being used in the field.

  6. Imaging Beads-Retained Prey Assay for Rapid and Quantitative Protein-Protein Interaction

    PubMed Central

    Zhou, Yan; Hong, Wanjin; Lu, Lei

    2013-01-01

    Conventional Western blot based pull-down methods involve lengthy and laborious work and the results are generally not quantitative. Here, we report the imaging beads-retained prey (IBRP) assay that is rapid and quantitative in studying protein-protein interactions. In this assay, the bait is immobilized onto beads and the prey is fused with a fluorescence protein. The assay takes advantage of the fluorescence of prey and directly quantifies the amount of prey binding to the immobilized bait under a microscope. We validated the assay using previously well studied interactions and found that the amount of prey retained on beads could have a relative linear relationship to both the inputs of bait and prey. IBRP assay provides a universal, fast, quantitative and economical method to study protein interactions and it could be developed to a medium- or high-throughput compatible method. With the availability of fluorescence tagged whole genome ORFs in several organisms, we predict IBRP assay should have wide applications. PMID:23555762

  7. Fluorescence in situ hybridization for rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis recovered from cystic fibrosis patients.

    PubMed

    Wellinghausen, Nele; Wirths, Beate; Poppert, Sven

    2006-09-01

    Achromobacter xylosoxidans is frequently isolated from the respiratory secretions of cystic fibrosis (CF) patients, but identification with biochemical tests is unreliable. We describe fluorescence in situ hybridization assays for the rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis. Both assays showed high sensitivities and high specificities with a collection of 155 nonfermenters from CF patients. PMID:16954289

  8. Rapid identification of acetic acid bacteria using MALDI-TOF mass spectrometry fingerprinting.

    PubMed

    Andrés-Barrao, Cristina; Benagli, Cinzia; Chappuis, Malou; Ortega Pérez, Ruben; Tonolla, Mauro; Barja, François

    2013-03-01

    Acetic acid bacteria (AAB) are widespread microorganisms characterized by their ability to transform alcohols and sugar-alcohols into their corresponding organic acids. The suitability of matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) for the identification of cultured AAB involved in the industrial production of vinegar was evaluated on 64 reference strains from the genera Acetobacter, Gluconacetobacter and Gluconobacter. Analysis of MS spectra obtained from single colonies of these strains confirmed their basic classification based on comparative 16S rRNA gene sequence analysis. MALDI-TOF analyses of isolates from vinegar cross-checked by comparative sequence analysis of 16S rRNA gene fragments allowed AAB to be identified, and it was possible to differentiate them from mixed cultures and non-AAB. The results showed that MALDI-TOF MS analysis was a rapid and reliable method for the clustering and identification of AAB species. PMID:23182036

  9. Evaluation of the MS-2 system for rapid identification of Enterobacteriaceae.

    PubMed Central

    McCracken, A W; Martin, W J; McCarthy, L R; Schwab, D A; Cooper, B H; Helgeson, N G; Prowant, S; Robson, J

    1980-01-01

    The precision, accuracy, and other performance characteristics of the MS-2 (Abbott Laboratories, Diagnostic Division, Dallas, Tex.) system for the identification of Enterobacteriaceae were evaluated in a collaborative study involving three clinical laboratories. When identifying 150 unknown, coded organisms, the MS-2 system was 97%, 98%, and 93% accurate, respectively, in three laboratories. The system showed an overall accuracy of 94% when compared with conventional manual tube methods in identifying 1,154 clinical isolates of 26 species of Enterobacteriaceae. Discrepancies between automated and conventional methods were chiefly caused by biochemical variants, especially among Enterobacter species. The MS-2 system was rapid and simple to operate and produced printed results of bacterial identification in 5 h. The cost of disposable components compared favorably with commercial, visually read systems for identifying Enterobacteriaceae. PMID:7024299

  10. Clinical evaluation of a simple, rapid procedure for the presumptive identification of anaerobic bacteria.

    PubMed Central

    Holland, J W; Gagnet, S M; Lewis, S A; Stauffer, L R

    1977-01-01

    A simple, rapid procedure for the presumptive identification of anaerobic bacteria has been evaluated. Two hundred and thirty-five clinical isolates were identified using gas-liquid chromatography and 3-ml volumes of a few selected test media. These test media were stored aerobically and incubated in GasPak anaerobic jars. The average incubation time was 39 h. This procedure, when compared to the results of our standard identification procedure, correctly identified 98% of the isolates to the genus level, 83% to the species level, and 83% of Bacteroides fragilis and Bacteroides melaninogenicus to the subspecies level. Fifty-three of the isolates were also identified by using 0.5-ml volumes of test media stored, inoculated, and incubated in an anaerobic glove box. The 3-ml-and the 0.5-ml-volume procedures correctly identified comparable percentages of the 53 isolates. PMID:323283

  11. Identification of Protein Interactions Involved in Cellular Signaling

    PubMed Central

    Westermarck, Jukka; Ivaska, Johanna; Corthals, Garry L.

    2013-01-01

    Protein-protein interactions drive biological processes. They are critical for all intra- and extracellular functions, and the technologies to analyze them are widely applied throughout the various fields of biological sciences. This study takes an in-depth view of some common principles of cellular regulation and provides a detailed account of approaches required to comprehensively map signaling protein-protein interactions in any particular cellular system or condition. We provide a critical review of the benefits and disadvantages of the yeast two-hybrid method and affinity purification coupled with mass spectrometric procedures for identification of signaling protein-protein interactions. In particular, we emphasize the quantitative and qualitative differences between tandem affinity and one-step purification (such as FLAG and Strep tag) methods. Although applicable to all types of interaction studies, a special section is devoted in this review to aspects that should be considered when attempting to identify signaling protein interactions that often are transient and weak by nature. Finally, we discuss shotgun and quantitative information that can be gleaned by MS-coupled methods for analysis of multiprotein complexes. PMID:23481661

  12. RBRIdent: An algorithm for improved identification of RNA-binding residues in proteins from primary sequences.

    PubMed

    Xiong, Dapeng; Zeng, Jianyang; Gong, Haipeng

    2015-06-01

    Rapid and correct identification of RNA-binding residues based on the protein primary sequences is of great importance. In most prevalent machine-learning-based identification methods; however, either some features are inefficiently represented, or the redundancy between features is not effectively removed. Both problems may weaken the performance of a classifier system and raise its computational complexity. Here, we addressed the above problems and developed a better classifier (RBRIdent) to identify the RNA-binding residues. In an independent benchmark test, RBRIdent achieved an accuracy of 76.79%, Matthews correlation coefficient of 0.3819 and F-measure of 75.58%, remarkably outperforming all prevalent methods. These results suggest the necessity of proper feature description and the essential role of feature selection in this project. All source data and codes are freely available at http://166.111.152.91/RBRIdent. PMID:25846271

  13. Identification of a Non-Pentapeptide Region Associated with Rapid Mycobacterial Evolution

    PubMed Central

    Warholm, Per; Light, Sara

    2016-01-01

    A large portion of the coding capacity of Mycobacterium tuberculosis is devoted to the production of proteins containing several copies of the pentapeptide-2 repeat, namely the PE/PPE_MPTR proteins. Protein domain repeats have a variety of binding properties and are involved in protein-protein interactions as well as binding to other ligands such as DNA and RNA. They are not as common in prokaryotes, compared to eukaryotes, but the enrichment of pentapeptide-2 repeats in Mycobacteria constitutes an exception to that rule. The genes encoding the PE/PPE_MPTR proteins have undergone many rearrangements and here we have identified the expansion patterns across the Mycobacteria. We have performed a reclassification of the PE/PPE_MPTR proteins using cohesive regions rather than sparse domain architectures. It is clear that these proteins have undergone large insertions of several pentapeptide-2 domains appearing adjacent to one another in a repetitive pattern. Further, we have identified a non-pentapeptide motif associated with rapid mycobacterial evolution. The sequence composition of this region suggests a different structure compared to pentapeptide-2 repeats. By studying the evolution of the PE/PPE_MPTR proteins, we have distinguished features pertaining to tuberculosis-inducing species. Further studies of the non-pentapeptide region associated with repeat expansions promises to shed light on the pathogenicity of Mycobacterium tuberculosis. PMID:27149271

  14. Selective, rapid and optically switchable regulation of protein function in live mammalian cells

    NASA Astrophysics Data System (ADS)

    Tsai, Yu-Hsuan; Essig, Sebastian; James, John R.; Lang, Kathrin; Chin, Jason W.

    2015-07-01

    The rapid and selective regulation of a target protein within living cells that contain closely related family members is an outstanding challenge. Here we introduce genetically directed bioorthogonal ligand tethering (BOLT) and demonstrate selective inhibition (iBOLT) of protein function. In iBOLT, inhibitor-conjugate/target protein pairs are created where the target protein contains a genetically encoded unnatural amino acid with bioorthogonal reactivity and the inhibitor conjugate contains a complementary bioorthogonal group. iBOLT enables the first rapid and specific inhibition of MEK isozymes, and introducing photoisomerizable linkers in the inhibitor conjugate enables reversible, optical regulation of protein activity (photo-BOLT) in live mammalian cells. We demonstrate that a pan kinase inhibitor conjugate allows selective and rapid inhibition of the lymphocyte specific kinase, indicating the modularity and scalability of BOLT. We anticipate that BOLT will enable the rapid and selective regulation of diverse proteins for which no selective small-molecule ligands exist.

  15. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays

    PubMed Central

    Yu, Xiaobo; LaBaer, Joshua

    2015-01-01

    Summary AMPylation (adenylylation) has been recognized as an important post translational modification employed by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes and is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method to identify new substrates using protein microarrays, which can significantly expand the list of potential substrates. Here, we describe procedures to detect AMPylated and auto-AMPylated proteins in a sensitive, high throughput, and non-radioactive manner. The approach employs high-density protein microarrays fabricated using NAPPA (Nucleic Acid Programmable Protein Arrays) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide–alkyne cycloaddition. The assay can be accomplished within 11 hours. PMID:25881200

  16. Identification of contractile vacuole proteins in Trypanosoma cruzi.

    PubMed

    Ulrich, Paul N; Jimenez, Veronica; Park, Miyoung; Martins, Vicente P; Atwood, James; Moles, Kristen; Collins, Dalis; Rohloff, Peter; Tarleton, Rick; Moreno, Silvia N J; Orlando, Ron; Docampo, Roberto

    2011-01-01

    Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism

  17. Retinal proteins modified by 4-hydroxynonenal: identification of molecular targets.

    PubMed

    Kapphahn, Rebecca J; Giwa, Babatomiwa M; Berg, Kristin M; Roehrich, Heidi; Feng, Xiao; Olsen, Timothy W; Ferrington, Deborah A

    2006-07-01

    The reactive aldehyde, 4-hydroxynonenal (HNE), is a product of lipid peroxidation that can covalently modify and inactivate proteins. Previously, we reported increased HNE modification of select retinal proteins resolved by one-dimensional gel electrophoresis in aged Fisher 344 x Brown Norway rats (Louie, J.L., Kapphahn, R.J., Ferrington, D.A., 2002. Proteasome function and protein oxidation in the aged retina. Exp. Eye Res. 75, 271-284). In the current study, quantitative assessment of HNE molar content using slot blot immunoassays showed HNE content is increased 30% in aged rat retina. In contrast, there was no age-related difference in HNE content in individual spots resolved by 2D gel electrophoresis suggesting the increased modification is likely on membrane proteins that are missing on 2D gels. The HNE-immunoreactive proteins resolved by 2D gel electrophoresis were identified by MALDI-TOF mass spectrometry. These proteins are involved in metabolism, chaperone function, and fatty acid transport. Proteins that were frequently modified and had the highest molar content of HNE included triosephosphate isomerase, alpha enolase, heat shock cognate 70 and betaB2 crystallin. Immunochemical detection of HNE adducts on retinal sections showed greater immune reaction in ganglion cells, photoreceptor inner segment, and the inner plexiform layer. Identification of HNE modified proteins in two alternative model systems, human retinal pigment epithelial cells in culture (ARPE19) and human donor eyes, indicated that triosephosphate isomerase and alpha enolase are generally modified. These results identify a common subset of proteins that contain HNE adducts and suggest that select retinal proteins are molecular targets for HNE modification. PMID:16530755

  18. Rapid Identification and Characterization of Francisella by Molecular Biology and Other Techniques

    PubMed Central

    Lai, Xin-He; Zhao, Long-Fei; Chen, Xiao-Ming; Ren, Yi

    2016-01-01

    Francisella tularensis is the causative pathogen of tularemia and a Tier 1 bioterror agent on the CDC list. Considering the fact that some subpopulation of the F. tularensis strains is more virulent, more significantly associated with mortality, and therefore poses more threat to humans, rapid identification and characterization of this subpopulation strains is of invaluable importance. This review summarizes the up-to-date developments of assays for mainly detecting and characterizing F. tularensis and a touch of caveats of some of the assays. PMID:27335619

  19. Identification of Sequences Encoding Symbiodinium minutum Mitochondrial Proteins

    PubMed Central

    Butterfield, Erin R.; Howe, Christopher J.; Nisbet, R. Ellen R.

    2016-01-01

    The dinoflagellates are an extremely diverse group of algae closely related to the Apicomplexa and the ciliates. Much work has previously been undertaken to determine the presence of various biochemical pathways within dinoflagellate mitochondria. However, these studies were unable to identify several key transcripts including those encoding proteins involved in the pyruvate dehydrogenase complex, iron–sulfur cluster biosynthesis, and protein import. Here, we analyze the draft nuclear genome of the dinoflagellate Symbiodinium minutum, as well as RNAseq data to identify nuclear genes encoding mitochondrial proteins. The results confirm the presence of a complete tricarboxylic acid cycle in the dinoflagellates. Results also demonstrate the difficulties in using the genome sequence for the identification of genes due to the large number of introns, but show that it is highly useful for the determination of gene duplication events. PMID:26798115

  20. Identification of Sequences Encoding Symbiodinium minutum Mitochondrial Proteins.

    PubMed

    Butterfield, Erin R; Howe, Christopher J; Nisbet, R Ellen R

    2016-02-01

    The dinoflagellates are an extremely diverse group of algae closely related to the Apicomplexa and the ciliates. Much work has previously been undertaken to determine the presence of various biochemical pathways within dinoflagellate mitochondria. However, these studies were unable to identify several key transcripts including those encoding proteins involved in the pyruvate dehydrogenase complex, iron-sulfur cluster biosynthesis, and protein import. Here, we analyze the draft nuclear genome of the dinoflagellate Symbiodinium minutum, as well as RNAseq data to identify nuclear genes encoding mitochondrial proteins. The results confirm the presence of a complete tricarboxylic acid cycle in the dinoflagellates. Results also demonstrate the difficulties in using the genome sequence for the identification of genes due to the large number of introns, but show that it is highly useful for the determination of gene duplication events. PMID:26798115

  1. Application of Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Rapid Identification and Clustering Analysis of Pantoea Species ▿ †

    PubMed Central

    Rezzonico, Fabio; Vogel, Guido; Duffy, Brion; Tonolla, Mauro

    2010-01-01

    Pantoea agglomerans is an ecologically diverse taxon that includes commercially important plant-beneficial strains and opportunistic clinical isolates. Standard biochemical identification methods in diagnostic laboratories were repeatedly shown to run into false-positive identifications of P. agglomerans, a fact which is also reflected by the high number of 16S rRNA gene sequences in public databases that are incorrectly assigned to this species. More reliable methods for rapid identification are required to ascertain the prevalence of this species in clinical samples and to evaluate the biosafety of beneficial isolates. Whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) methods and reference spectra (SuperSpectrum) were developed for accurate identification of P. agglomerans and related bacteria and used to detect differences in the protein profile within variants of the same strain, including a ribosomal point mutation conferring streptomycin resistance. MALDI-TOF MS-based clustering was shown to generally agree with classification based on gyrB sequencing, allowing rapid and reliable identification at the species level. PMID:20453125

  2. An Inducible System for Rapid Degradation of Specific Cellular Proteins Using Proteasome Adaptors

    PubMed Central

    Wilmington, Shameika R.; Matouschek, Andreas

    2016-01-01

    A common way to study protein function is to deplete the protein of interest from cells and observe the response. Traditional methods involve disrupting gene expression but these techniques are only effective against newly synthesized proteins and leave previously existing and stable proteins untouched. Here, we introduce a technique that induces the rapid degradation of specific proteins in mammalian cells by shuttling the proteins to the proteasome for degradation in a ubiquitin-independent manner. We present two implementations of the system in human culture cells that can be used individually to control protein concentration. Our study presents a simple, robust, and flexible technology platform for manipulating intracellular protein levels. PMID:27043013

  3. Identification of GI cancers utilising rapid mid-infrared spectral imaging

    NASA Astrophysics Data System (ADS)

    Nallala, Jayakrupakar; Lloyd, Gavin R.; Kendall, Catherine; Barr, Hugh; Shepherd, Neil; Stone, Nick

    2016-03-01

    Pathologists find it notoriously difficult to provide both inter- and intra-observer agreement on a diagnosis of early gastrointestinal cancers. Vibrational spectroscopic approaches have shown their value in providing molecular compositional data from tissue samples and therefore enabling the identification of disease specific changes, when combined with multivariate techniques. Mid-infrared microscopic imaging is undergoing rapid developments in sources, detectors and spectrometers. Here we explore the use of high magnification FTIR for GI cancers and consider how the MINERVA (MId- to NEaR infrared spectroscopy for improVed medical diAgnostics) project, which is developing discrete frequency IR imaging tools will enable histopathologists to obtain rapid molecular images form unstained tissue sections.

  4. Identification of the Species of Origin for Meat Products by Rapid Evaporative Ionization Mass Spectrometry.

    PubMed

    Balog, Julia; Perenyi, Dora; Guallar-Hoyas, Cristina; Egri, Attila; Pringle, Steven D; Stead, Sara; Chevallier, Olivier P; Elliott, Chris T; Takats, Zoltan

    2016-06-15

    Increasingly abundant food fraud cases have brought food authenticity and safety into major focus. This study presents a fast and effective way to identify meat products using rapid evaporative ionization mass spectrometry (REIMS). The experimental setup was demonstrated to be able to record a mass spectrometric profile of meat specimens in a time frame of <5 s. A multivariate statistical algorithm was developed and successfully tested for the identification of animal tissue with different anatomical origin, breed, and species with 100% accuracy at species and 97% accuracy at breed level. Detection of the presence of meat originating from a different species (horse, cattle, and venison) has also been demonstrated with high accuracy using mixed patties with a 5% detection limit. REIMS technology was found to be a promising tool in food safety applications providing a reliable and simple method for the rapid characterization of food products. PMID:27167240

  5. ERP and Adaptive Autoregressive identification with spectral power decomposition to study rapid auditory processing in infants.

    PubMed

    Piazza, C; Cantiani, C; Tacchino, G; Molteni, M; Reni, G; Bianchi, A M

    2014-01-01

    The ability to process rapidly-occurring auditory stimuli plays an important role in the mechanisms of language acquisition. For this reason, the research community has begun to investigate infant auditory processing, particularly using the Event Related Potentials (ERP) technique. In this paper we approach this issue by means of time domain and time-frequency domain analysis. For the latter, we propose the use of Adaptive Autoregressive (AAR) identification with spectral power decomposition. Results show EEG delta-theta oscillation enhancement related to the processing of acoustic frequency and duration changes, suggesting that, as expected, power modulation encodes rapid auditory processing (RAP) in infants and that the time-frequency analysis method proposed is able to identify this modulation. PMID:25571014

  6. Rapid Generation of Amyloid from Native Proteins In vitro

    PubMed Central

    Dorta-Estremera, Stephanie M; Li, Jingjing; Cao, Wei

    2013-01-01

    Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion. PMID:24335677

  7. Evaluation of the VITEK 2 system for rapid identification of yeasts and yeast-like organisms.

    PubMed

    Graf, B; Adam, T; Zill, E; Göbel, U B

    2000-05-01

    The new VITEK 2 system is a fully automated system dedicated to the identification and susceptibility testing of microorganisms. In conjunction with the VITEK ID-YST card the VITEK 2 system allows the identification of clinically important yeasts and yeast-like organisms in 15 h due to a sensitive fluorescence-based technology. The ID-YST card consists of 47 biochemical reactions. The database comprises 51 taxa, including newly described species. In this study we evaluated the reliability of the VITEK ID-YST card for the identification of yeasts and yeast-like organisms encountered in a clinical microbiology laboratory. A total of 241 strains representing 21 species were studied. The strains were isolated from clinical samples within a period of 60 days prior to the identification. The tests were performed using 24-h to 55-h subcultures on Sabouraud-gentamicin-chloramphenicol agar. Each strain was tested in parallel using the ID 32C strip as a comparison method combined with microscopic morphology and an agglutination test for C. krusei. Overall, 222 strains (92.1%) were unequivocally identified including 11 isolates (4.6%) identified with low discrimination resolved by simple additional tests. Ten strains (4. 1%) for which results were given with low discrimination could not be unequivocally identified with supplemental tests, 4 strains (1. 7%) were misidentified and 5 strains (2.1%) could not be identified. In conclusion, we found that the VITEK 2 system is a rapid and accurate method for the identification of medically important yeasts and yeast-like organisms. PMID:10790099

  8. Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins

    PubMed Central

    Saritas-Yildirim, Banu; Pliner, Hannah A.; Ochoa, Angelica; Silva, Elena M.

    2015-01-01

    Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development. PMID:26327321

  9. Identification of major immunogenic proteins of Mycoplasma synoviae isolates.

    PubMed

    Bercic, Rebeka Lucijana; Slavec, Brigita; Lavric, Miha; Narat, Mojca; Bidovec, Andrej; Dovc, Peter; Bencina, Dusan

    2008-02-01

    Mycoplasma synoviae isolates differ in patterns of immunogenic proteins, but most of them have not been identified yet. The main aim of this study was their identification in two closely related M. synoviae isolates, ULB 02/P4 and ULB 02/OV6, recovered recently from chickens in Slovenia. N-terminal sequencing identified 17 M. synoviae proteins. Amongst them were 14 major, highly expressed but previously unidentified proteins, including enzymes, chaperones and putative lipoproteins. ULB 02/P4 proteins with increasing molecular weight (M(w)) in the region above the lipoprotein MSPB (approximately 40 kDa) were elongation factor EF-Tu, enolase, NADH oxidase, haemagglutinin MSPA, ATP synthase beta chain, trigger factor, pyruvate kinase and chaperone DnaK. Enolase (approximately 47 kDa) seemed to be immunogenic for chickens infected with M. synoviae, whereas EF-Tu, which might cross-react with antibodies to the P1 adhesin of Mycoplasma pneumoniae, was not. ULB 02/OV6 synthesized several immunogenic proteins and those with M(w) of approximately 70, 78, 82, 90, 110 and 160 kDa, cross-reacted with antibodies to Mycoplasma gallisepticum. They remain to be identified, because besides putative lipoproteins, protein bands of 78, 82, 85 and 110 kDa contained also dehydrogenase PdhD, elongation factor EF-G, enzyme PtsG and putative neuraminidase, respectively. PMID:17720337

  10. Identification of 24 h Ixodes scapularis immunogenic tick saliva proteins

    PubMed Central

    Lewis, Lauren A.; Radulović, Željko M.; Kim, Tae K.; Porter, Lindsay M.; Mulenga, Albert

    2015-01-01

    Ixodes scapularis is arguably the most medically important tick species in the United States. This tick transmits 5 of the 14 human tick-borne disease (TBD) agents in the USA: Borrelia burgdorferi, Anaplasma phagocytophilum, B. miyamotoi, Babesia microti, and Powassan virus disease. Except for the Powassan virus disease, I. scapularis-vectored TBD agents require more than 24 h post attachment to be transmitted. This study describes identification of 24 h immunogenic I. scapularis tick saliva proteins, which could provide opportunities to develop strategies to stop tick feeding before transmission of the majority of pathogens. A 24 h fed female I. scapularis phage display cDNA expression library was biopanned using rabbit antibodies to 24 h fed I. scapularis female tick saliva proteins, subjected to next generation sequencing, de novo assembly, and bioinformatic analyses. A total of 182 contigs were assembled, of which ~19% (35/182) are novel and did not show identity to any known proteins in GenBank. The remaining ~81% (147/182) of contigs were provisionally identified based on matches in GenBank including ~18% (27/147) that matched protein sequences previously annotated as hypothetical and putative tick saliva proteins. Others include proteases and protease inhibitors (~3%, 5/147), transporters and/or ligand binding proteins (~6%, 9/147), immunogenic tick saliva housekeeping enzyme-like (17%, 25/147), ribosomal protein-like (~31%, 46/147), and those classified as miscellaneous (~24%, 35/147). Notable among the miscellaneous class include antimicrobial peptides (microplusin and ricinusin), myosin-like proteins that have been previously found in tick saliva, and heat shock tick saliva protein. Data in this study provides the foundation for in-depth analysis of I. scapularis feeding during the first 24 h, before the majority of TBD agents can be transmitted. PMID:25825233

  11. SIMULTANEOUS AND RAPID IDENTIFICATION OF ESCHERICHIA COLI, LISTERIA MONOCYTOGENES, AND SALMONELLA TYPHIMONIUM BY SURFACE-ENHANCED RAMAN SCATTERING SPECTROSCOPY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The development of rapid and routine identification methods for foodborne bacteria is of considerable importance due to concerns regarding bio-/agro-terrorism, public health, and economic loss. The traditional techniques are time consuming and are not sufficiently rapid to assure the safety of ready...

  12. Rapid Detection and Identification of Yersinia pestis from Food Using Immunomagnetic Separation and Pyrosequencing.

    PubMed

    Amoako, Kingsley K; Shields, Michael J; Goji, Noriko; Paquet, Chantal; Thomas, Matthew C; Janzen, Timothy W; Bin Kingombe, Cesar I; Kell, Arnold J; Hahn, Kristen R

    2012-01-01

    Interest has recently been renewed in the possible use of Y. pestis, the causative agent of plague, as a biological weapon by terrorists. The vulnerability of food to intentional contamination coupled with reports of humans having acquired plague through eating infected animals that were not adequately cooked or handling of meat from infected animals makes the possible use of Y. pestis in a foodborne bioterrorism attack a reality. Rapid, efficient food sample preparation and detection systems that will help overcome the problem associated with the complexity of the different matrices and also remove any ambiguity in results will enable rapid informed decisions to be made regarding contamination of food with biothreat agents. We have developed a rapid detection assay that combines the use of immunomagnetic separation and pyrosequencing in generating results for the unambiguous identification of Y. pestis from milk (0.9 CFU/mL), bagged salad (1.6 CFU/g), and processed meat (10 CFU/g). The low detection limits demonstrated in this assay provide a novel tool for the rapid detection and confirmation of Y. pestis in food without the need for enrichment. The combined use of the iCropTheBug system and pyrosequencing for efficient capture and detection of Y. pestis is novel and has potential applications in food biodefence. PMID:23091729

  13. Rapid protein immobilization for thin film continuous flow biocatalysis.

    PubMed

    Britton, Joshua; Raston, Colin L; Weiss, Gregory A

    2016-08-01

    A versatile enzyme immobilization strategy for thin film continuous flow processing is reported. Here, non-covalent and glutaraldehyde bioconjugation are used to immobilize enzymes on the surfaces of borosilicate reactors. This approach requires only ng of protein per reactor tube, with the stock protein solution readily recycled to sequentially coat >10 reactors. Confining reagents to thin films during immobilization reduced the amount of protein, piranha-cleaning solution, and other reagents by ∼96%. Through this technique, there was no loss of catalytic activity over 10 h processing. The results reported here combines the benefits of thin film flow processing with the mild conditions of biocatalysis. PMID:27461146

  14. Rapid evolution of 6-phenylpurine inhibitors of protein kinase B through structure-based design.

    PubMed

    Donald, Alastair; McHardy, Tatiana; Rowlands, Martin G; Hunter, Lisa-Jane K; Davies, Thomas G; Berdini, Valerio; Boyle, Robert G; Aherne, G Wynne; Garrett, Michelle D; Collins, Ian

    2007-05-17

    6-phenylpurines were identified as novel, ATP-competitive inhibitors of protein kinase B (PKB/Akt) from a fragment-based screen and were rapidly progressed to potent compounds using iterative protein-ligand crystallography with a PKA-PKB chimeric protein. An elaborated lead compound showed cell growth inhibition and effects on cellular signaling pathways characteristic of PKB inhibition. PMID:17451235

  15. A rapid method to improve protein detection by indirect ELISA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enzyme-linked immunosorbant assay (ELISA) is a rapid, high-throughput, quantitative immunoassay for the selective detection of target antigens. The general principle behind an ELISA is antibody mediated capture and detection of an antigen with a measureable substrate. Numerous incarnations of th...

  16. Crux: rapid open source protein tandem mass spectrometry analysis.

    PubMed

    McIlwain, Sean; Tamura, Kaipo; Kertesz-Farkas, Attila; Grant, Charles E; Diament, Benjamin; Frewen, Barbara; Howbert, J Jeffry; Hoopmann, Michael R; Käll, Lukas; Eng, Jimmy K; MacCoss, Michael J; Noble, William Stafford

    2014-10-01

    Efficiently and accurately analyzing big protein tandem mass spectrometry data sets requires robust software that incorporates state-of-the-art computational, machine learning, and statistical methods. The Crux mass spectrometry analysis software toolkit ( http://cruxtoolkit.sourceforge.net ) is an open source project that aims to provide users with a cross-platform suite of analysis tools for interpreting protein mass spectrometry data. PMID:25182276

  17. Ribosomal protein biomarkers provide root nodule bacterial identification by MALDI-TOF MS.

    PubMed

    Ziegler, Dominik; Pothier, Joël F; Ardley, Julie; Fossou, Romain Kouakou; Pflüger, Valentin; de Meyer, Sofie; Vogel, Guido; Tonolla, Mauro; Howieson, John; Reeve, Wayne; Perret, Xavier

    2015-07-01

    Accurate identification of soil bacteria that form nitrogen-fixing associations with legume crops is challenging given the phylogenetic diversity of root nodule bacteria (RNB). The labor-intensive and time-consuming 16S ribosomal RNA (rRNA) sequencing and/or multilocus sequence analysis (MLSA) of conserved genes so far remain the favored molecular tools to characterize symbiotic bacteria. With the development of mass spectrometry (MS) as an alternative method to rapidly identify bacterial isolates, we recently showed that matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) can accurately characterize RNB found inside plant nodules or grown in cultures. Here, we report on the development of a MALDI-TOF RNB-specific spectral database built on whole cell MS fingerprints of 116 strains representing the major rhizobial genera. In addition to this RNB-specific module, which was successfully tested on unknown field isolates, a subset of 13 ribosomal proteins extracted from genome data was found to be sufficient for the reliable identification of nodule isolates to rhizobial species as shown in the putatively ascribed ribosomal protein masses (PARPM) database. These results reveal that data gathered from genome sequences can be used to expand spectral libraries to aid the accurate identification of bacterial species by MALDI-TOF MS. PMID:25776061

  18. Rapid Identification of Pseudallescheria and Scedosporium Strains by Using Rolling Circle Amplification

    PubMed Central

    Lackner, Michaela; Najafzadeh, Mohammad Javad; Sun, Jiufeng; Lu, Qiaoyun

    2012-01-01

    The Pseudallescheria boydii complex, comprising environmental pathogens with Scedosporium anamorphs, has recently been subdivided into five main species: Scedosporium dehoogii, S. aurantiacum, Pseudallescheria minutispora, P. apiosperma, and P. boydii, while the validity of some other taxa is being debated. Several Pseudallescheria and Scedosporium species are indicator organisms of pollution in soil and water. Scedosporium dehoogii in particular is enriched in soils contaminated by aliphatic hydrocarbons. In addition, the fungi may cause life-threatening infections involving the central nervous system in severely impaired patients. For screening purposes, rapid and economic tools for species recognition are needed. Our aim is to establish rolling circle amplification (RCA) as a screening tool for species-specific identification of Pseudallescheria and Scedosporium. With this aim, a set of padlock probes was designed on the basis of the internal transcribed spacer (ITS) region, differing by up to 13 fixed mutations. Padlock probes were unique as judged from sequence comparison by BLAST search in GenBank and in dedicated research databases at CBS (Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre). RCA was applied as an in vitro tool, tested with pure DNA amplified from cultures. The species-specific padlock probes designed in this study yielded 100% specificity. The method presented here was found to be an attractive alternative to identification by restriction fragment length polymorphism (RFLP) or sequencing. The rapidity (<1 day), specificity, and low costs make RCA a promising screening tool for environmentally and clinically relevant fungi. PMID:22057865

  19. LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Trangoni, Marcos D; Gioffré, Andrea K; Cerón Cucchi, María E; Caimi, Karina C; Ruybal, Paula; Zumárraga, Martín J; Cravero, Silvio L

    2015-06-01

    In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR Green(TM) allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries. PMID:26273282

  20. Fluorescent In Situ Hybridization Allows Rapid Identification of Microorganisms in Blood Cultures

    PubMed Central

    Kempf, Volkhard A. J.; Trebesius, Karlheinz; Autenrieth, Ingo B.

    2000-01-01

    Using fluorescent in situ hybridization (FISH) with rRNA-targeted fluorescently labelled oligonucleotide probes, pathogens were rapidly detected and identified in positive blood culture bottles without cultivation and biotyping. In this study, 115 blood cultures with a positive growth index as determined by a continuous-reading automated blood culture system were examined by both conventional laboratory methods and FISH. For this purpose, oligonucleotide probes that allowed identification of approximately 95% of those pathogens typically associated with bacteremia were produced. The sensitivity and specificity of these probes were 100%. From all 115 blood cultures, microorganisms were grown after 1 day and identification to the family, genus, or species level was achieved after 1 to 3 days while 111 samples (96.5%) were similarly identified by FISH within 2.5 h. Staphylococci were identified in 62 of 62 samples, streptococci and enterococci were identified in 19 of 20 samples, gram-negative rods were identified in 28 of 30 samples, and fungi were identified in two of two samples. Thus, FISH is an appropriate method for identification of pathogens grown in blood cultures from septicemic patients. PMID:10655393

  1. Immunoprecipitation of Plasma Membrane Receptor-Like Kinases for Identification of Phosphorylation Sites and Associated Proteins.

    PubMed

    Kadota, Yasuhiro; Macho, Alberto P; Zipfel, Cyril

    2016-01-01

    Membrane proteins are difficult to study for numerous reasons. The surface of membrane proteins is relatively hydrophobic and sometimes very unstable, additionally requiring detergents for their extraction from the membrane. This leads to challenges at all levels, including expression, solubilization, purification, identification of associated proteins, and the identification of post-translational modifications. However, recent advances in immunoprecipitation technology allow to isolate membrane proteins efficiently, facilitating the study of protein-protein interactions, the identification of novel associated proteins, and to identify post-translational modifications, such as phosphorylation. Here, we describe an optimized immunoprecipitation protocol for plant plasma membrane receptor-like kinases. PMID:26577786

  2. Rapid Electrochemical Detection and Identification of Microbiological and Chemical Contaminants for Manned Spaceflight Project

    NASA Technical Reports Server (NTRS)

    Pierson, Duane; Botkin, Douglas; Gazda, Daniel

    2014-01-01

    Microbial control in the spacecraft environment is a daunting task, especially in the presence of human crew members. Currently, assessing the potential crew health risk associated with a microbial contamination event requires return of representative environmental samples that are analyzed in a ground-based laboratory. It is therefore not currently possible to quickly identify microbes during spaceflight. This project addresses the unmet need for spaceflight-compatible microbial identification technology. The electrochemical detection and identification platform is expected to provide a sensitive, specific, and rapid sample-to-answer capability for in-flight microbial monitoring that can distinguish between related microorganisms (pathogens and non-pathogens) as well as chemical contaminants. This will dramatically enhance our ability to monitor the spacecraft environment and the health risk to the crew. Further, the project is expected to eliminate the need for sample return while significantly reducing crew time required for detection of multiple targets. Initial work will focus on the optimization of bacterial detection and identification. The platform is designed to release nucleic acids (DNA and RNA) from microorganisms without the use of harmful chemicals. Bacterial DNA or RNA is captured by bacteria-specific probe molecules that are bound to a microelectrode, and that capture event can generate a small change in the electrical current (Lam, et al. 2012. Anal. Chem. 84(1): 21-5.). This current is measured, and a determination is made whether a given microbe is present in the sample analyzed. Chemical detection can be accomplished by directly applying a sample to the microelectrode and measuring the resulting current change. This rapid microbial and chemical detection device is designed to be a low-cost, low-power platform anticipated to be operated independently of an external power source, characteristics optimal for manned spaceflight and areas where power

  3. Identification of Proteins that Modify Cataract of the Eye Lens

    PubMed Central

    Hoehenwarter, Wolfgang; Tang, Yajun; Ackermann, Renate; Pleissner, Klaus-Peter; Schmid, Monika; Stein, Robert; Zimny-Arndt, Ursula; Kumar, Nalin M.; Jungblut, Peter R.

    2010-01-01

    The occurrence of a nuclear cataract in the eye lens due to disruption of theα3Cx46 connexin gene, Gja3, is dependent on strain background in a mouse model, implicating factors that modify the pathology. The differences upon cataractogenesis in the urea soluble proteins of the lens of two mouse strains, C57BL/6J and 129/SvJ, were analyzed by a comparative proteomics approach. Determination of the complete proteome of an organ offers the opportunity to characterize at a molecular level, differences in gene expression and post-translational modifications occurring during pathology and between individuals. The abundance of 63 protein species was altered between the strains. A unique aspect of this study is the identification of chaperonin subunit 6A, mortalin, ERp29 and syntaxin binding protein 6 in the eye lens. DNA polymorphisms resulting in non-conservative amino acid changes that led to altered physicochemical properties of the proteins were detected for mortalin, chaperonin subunit 6A, annexin A1 and possibly gamma N crystallin. The results show HSP27/25 and/or ERp29 are the likely major modifying factors for cataractogenesis. Extension of the results suggests that small heat shock proteins have a major role for influencing cataract formation in humans. PMID:19003866

  4. Identification of Inhibitors of Biological Interactions Involving Intrinsically Disordered Proteins

    PubMed Central

    Marasco, Daniela; Scognamiglio, Pasqualina Liana

    2015-01-01

    Protein–protein interactions involving disordered partners have unique features and represent prominent targets in drug discovery processes. Intrinsically Disordered Proteins (IDPs) are involved in cellular regulation, signaling and control: they bind to multiple partners and these high-specificity/low-affinity interactions play crucial roles in many human diseases. Disordered regions, terminal tails and flexible linkers are particularly abundant in DNA-binding proteins and play crucial roles in the affinity and specificity of DNA recognizing processes. Protein complexes involving IDPs are short-lived and typically involve short amino acid stretches bearing few “hot spots”, thus the identification of molecules able to modulate them can produce important lead compounds: in this scenario peptides and/or peptidomimetics, deriving from structure-based, combinatorial or protein dissection approaches, can play a key role as hit compounds. Here, we propose a panoramic review of the structural features of IDPs and how they regulate molecular recognition mechanisms focusing attention on recently reported drug-design strategies in the field of IDPs. PMID:25849651

  5. Identification of specific protein markers in microdissected hepatocellular carcinoma.

    PubMed

    Melle, Christian; Ernst, Günther; Scheibner, Olaf; Kaufmann, Roland; Schimmel, Bettina; Bleul, Annett; Settmacher, Utz; Hommann, Merten; Claussen, Uwe; von Eggeling, Ferdinand

    2007-01-01

    At present, the molecular mechanisms of hepatocellular carcinogenesis are not well-understood, and hepatocellular carcinoma (HCC) stays one of the most frequent and high-risk metastatic visceral neoplasms worldwide. For the identification of tumor-relevant proteins, we analyzed microdissected cells from nontumorous liver tissue (n = 28) and tissue derived from hepatic tumor center (n = 25), as well as tumor margin (n = 23). We unequivocally identified 53 proteins from hepatic tumor tissues by peptide fingerprint mapping and SELDI mass spectrometry that were separated using two-dimensional gel electrophoresis. Among a number of signals that were detected as significantly different in the protein profiling analysis, we identified for the first time ferritin light subunit (FLS) and adenylate kinase 3 alpha-like 1 (AK3), showing decreased expressions in hepatic tumor, as well as biliverdin reductase B (BVRB) that was upregulated in HCC. The use of ProteinChip technology in combination with tissue microdissection gives insight of the complex changes occurring at the protein level in hepatocellular cancer associated with tumor development and progression and resulted in three new potential diagnostically useful markers. PMID:17203974

  6. Identification of differentially expressed serum proteins in gastric adenocarcinoma☆

    PubMed Central

    Subbannayya, Yashwanth; Mir, Sartaj Ahmad; Renuse, Santosh; Manda, Srikanth S.; Pinto, Sneha M.; Puttamallesh, Vinuth N.; Solanki, Hitendra Singh; Manju, H.C.; Syed, Nazia; Sharma, Rakesh; Christopher, Rita; Vijayakumar, M.; Kumar, K.V. Veerendra; Prasad, T.S. Keshava; Ramaswamy, Girija; Kumar, Rekha V.; Chatterjee, Aditi; Pandey, Akhilesh; Gowda, Harsha

    2015-01-01

    Gastric adenocarcinoma is an aggressive cancer with poor prognosis. Blood based biomarkers of gastric cancer have the potential to improve diagnosis and monitoring of these tumors. Proteins that show altered levels in the circulation of gastric cancer patients could prove useful as putative biomarkers. We used an iTRAQ-based quantitative proteomic approach to identify proteins that show altered levels in the sera of patients with gastric cancer. Our study resulted in identification of 643 proteins, of which 48 proteins showed increased levels and 11 proteins showed decreased levels in serum from gastric cancer patients compared to age and sex matched healthy controls. Proteins that showed increased expression in gastric cancer included inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Mannose-binding protein C (MBL2), sex hormone-binding globulin (SHBG), insulin-like growth factor-binding protein 2 (IGFBP2), serum amyloid A protein (SAA1), Orosomucoid 1 (ORM1) and extracellular superoxide dismutase [Cu–Zn] (SOD3). We used multiple reaction monitoring assays and validated elevated levels of ITIH4 and SAA1 proteins in serum from gastric cancer patients. Biological significance Gastric cancer is a highly aggressive cancer associated with high mortality. Serum-based biomarkers are of considerable interest in diagnosis and monitoring of various diseases including cancers. Gastric cancer is often diagnosed at advanced stages resulting in poor prognosis and high mortality. Pathological diagnosis using biopsy specimens remains the gold standard for diagnosis of gastric cancer. Serum-based biomarkers are of considerable importance as they are minimally invasive. In this study, we carried out quantitative proteomic profiling of serum from gastric cancer patients to identify proteins that show altered levels in gastric cancer patients. We identified more than 50 proteins that showed altered levels in gastric cancer patient sera. Validation in a large cohort of well

  7. Rapid visualization of hydrogen positions in neutron protein crystallography structures

    SciTech Connect

    Blakeley, Matthew P.; Meilleur, Flora; Myles, Dean A A; Weiss, Kevin L; Munshi, Parthapratim; Shang-Lin, Chung

    2012-01-01

    Neutron crystallography is a powerful technique to visualize experimentally the position of light atoms, including hydrogen and its isotope deuterium. Over the last several years, structural biologists have shown an increasing interest for the technique as it uniquely complements X-ray crystallographic data by revealing the position of hydrogen/deuterium atoms in macromolecules. With this regained interest, access to macromolecule neutron crystallography beam lines is becoming a limiting step. In this report we show that rapid data collection could be a valuable alternative to long data collection time when appropriate. Comparison of perdeuterated Rubredoxin structures refined against neutron data sets collected over hours and up to five days shows that rapid neutron data collection in just 14 hours is sufficient to provide the position of 262 hydrogen positions atoms without ambiguity.

  8. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.

    PubMed

    Natsume, Toyoaki; Kiyomitsu, Tomomi; Saga, Yumiko; Kanemaki, Masato T

    2016-04-01

    Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID) technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES) cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function. PMID:27052166

  9. A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification

    PubMed Central

    Kouzaki, Yuji; Maeda, Takuya; Sasaki, Hiroaki; Tamura, Shinsuke; Hamamoto, Takaaki; Yuki, Atsushi; Sato, Akinori; Miyahira, Yasushi; Kawana, Akihiko

    2015-01-01

    Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64°C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 103 cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 103 cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation

  10. Rapid Detection and Identification of Human Hookworm Infections through High Resolution Melting (HRM) Analysis

    PubMed Central

    Ngui, Romano; Lim, Yvonne A. L.; Chua, Kek Heng

    2012-01-01

    Background Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species. Methods Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method's sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples. Conclusion The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species. PMID:22844538