Each year, the U.S. National Institutes of Health (NIH) invests substantial resources in core facilities that provide access to advanced cutting-edge technologies, expert consultation, and other services to scientific investigators. The facilities offer a number of services, ranging from systematic analysis and data processing, using specialized instrumentation, to access and expert advice on experimental design and evaluation needs, such as biostatistics, patient outreach, and clinical regulatory issues. The largest fraction of support for cores comes from the institutes and centers of the NIH, for example, through the National Cancer Institute (NCI) and National Center for Advancing Translational Sciences (NCATS). Significant NIH investment is spent on Center Core grants, particularly the NCI Cancer Centers, and the Clinical Translational Sciences Award Program supported by NCATS. The Office of the NIH Director’s Division of Program Coordination, Planning, and Strategic Initiatives (DPCPSI) also has a substantial investment in animal and biologic resource centers that provide models of human biology and disease for basic to clinical studies to researchers around the world. Many of these centers function like cores, as they provide the following: 1) high-quality, disease-free animals; 2) access to sophisticated technologies and facilities, as well as specialized animals; and 3) expert training by professional staff and consultation services. As an example, the NIH-supported National Primate Research Centers provide facilities, animals, and expertise for investigators who use nonhuman primates for biomedical research, facilitating >1000 individual research projects annually. The DPCPSI investment in core facilities also includes support through its Shared Instrument Grant program to purchase or upgrade expensive, specialized, commercially available instruments or integrated systems. This program promotes cost effectiveness; encourages optimal sharing among investigators, research groups, and departments; and fosters a collaborative, multidisciplinary environment. In many settings, the instrument is integrated in a centralized core facility. Through these investments and many other programs not listed, NIH’s annual support for research cores is estimated conservatively at $900 million. Given this large investment, it is critical that both NIH and the research institutions receiving support for these resources identify and implement approaches that enhance core resource efficiencies.
{"title":"Sharing Core Facilities and Research Resources--An Investment in Accelerating Scientific Discoveries.","authors":"Michael C Chang, F. Grieder","doi":"10.7171/jbt.16-2701-004","DOIUrl":"https://doi.org/10.7171/jbt.16-2701-004","url":null,"abstract":"Each year, the U.S. National Institutes of Health (NIH) invests substantial resources in core facilities that provide access to advanced cutting-edge technologies, expert consultation, and other services to scientific investigators. The facilities offer a number of services, ranging from systematic analysis and data processing, using specialized instrumentation, to access and expert advice on experimental design and evaluation needs, such as biostatistics, patient outreach, and clinical regulatory issues. The largest fraction of support for cores comes from the institutes and centers of the NIH, for example, through the National Cancer Institute (NCI) and National Center for Advancing Translational Sciences (NCATS). Significant NIH investment is spent on Center Core grants, particularly the NCI Cancer Centers, and the Clinical Translational Sciences Award Program supported by NCATS. The Office of the NIH Director’s Division of Program Coordination, Planning, and Strategic Initiatives (DPCPSI) also has a substantial investment in animal and biologic resource centers that provide models of human biology and disease for basic to clinical studies to researchers around the world. Many of these centers function like cores, as they provide the following: 1) high-quality, disease-free animals; 2) access to sophisticated technologies and facilities, as well as specialized animals; and 3) expert training by professional staff and consultation services. As an example, the NIH-supported National Primate Research Centers provide facilities, animals, and expertise for investigators who use nonhuman primates for biomedical research, facilitating >1000 individual research projects annually. The DPCPSI investment in core facilities also includes support through its Shared Instrument Grant program to purchase or upgrade expensive, specialized, commercially available instruments or integrated systems. This program promotes cost effectiveness; encourages optimal sharing among investigators, research groups, and departments; and fosters a collaborative, multidisciplinary environment. In many settings, the instrument is integrated in a centralized core facility. Through these investments and many other programs not listed, NIH’s annual support for research cores is estimated conservatively at $900 million. Given this large investment, it is critical that both NIH and the research institutions receiving support for these resources identify and implement approaches that enhance core resource efficiencies.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"37 1","pages":"2-3"},"PeriodicalIF":0.0,"publicationDate":"2016-01-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87737375","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Progress in biomedical research is largely driven by improvements, innovations, and breakthroughs in technology, an increasingly complex collaboration among basic, preclinical, and clinical science. Unfortunately, this increasing sophistication correlates with both growing research costs and a shrinking federal research budget. Taken together, it makes a strong argument for investing in core facilities. Cores are not new, having been a part of the research landscape for decades. Cores leverage expertise and state-of-the-art technology in centralized facilities and support institutional research in a cost-effective and efficient manner. There is a growing recognition that centralized administration of cores ensures best practices for institutional investment, financial and resource management, and core scientist professional and career development. Centralization maximizes institutional research capabilities by providing state-of-the-art instrumentation and multidisciplinary expertise for the entire research community, establishing a culture of collaboration integrated and aligned with institutional strategic goals and success in a highly competitive playground. However, to be successful, cores requires funding agencies and partners who identify opportunities and provide financial support, scientists who embrace team science, core scientists who personify the ethos of collaborative sharing, and committed institutions and policies that make it happen.
{"title":"JBT Special Issue on Core Management.","authors":"S. Mische","doi":"10.7171/jbt.16-2701-005","DOIUrl":"https://doi.org/10.7171/jbt.16-2701-005","url":null,"abstract":"Progress in biomedical research is largely driven by improvements, innovations, and breakthroughs in technology, an increasingly complex collaboration among basic, preclinical, and clinical science. Unfortunately, this increasing sophistication correlates with both growing research costs and a shrinking federal research budget. Taken together, it makes a strong argument for investing in core facilities. Cores are not new, having been a part of the research landscape for decades. Cores leverage expertise and state-of-the-art technology in centralized facilities and support institutional research in a cost-effective and efficient manner. There is a growing recognition that centralized administration of cores ensures best practices for institutional investment, financial and resource management, and core scientist professional and career development. Centralization maximizes institutional research capabilities by providing state-of-the-art instrumentation and multidisciplinary expertise for the entire research community, establishing a culture of collaboration integrated and aligned with institutional strategic goals and success in a highly competitive playground. However, to be successful, cores requires funding agencies and partners who identify opportunities and provide financial support, scientists who embrace team science, core scientists who personify the ethos of collaborative sharing, and committed institutions and policies that make it happen.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"5 1","pages":"1"},"PeriodicalIF":0.0,"publicationDate":"2016-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87481198","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to: Clive Slaughter, GRU-UGA Medical Partnership, 1425 Prince Ave., Athens, GA 30606, USA. Tel: (706) 713-2216; Fax: (706) 713-2221; E-mail: cslaught@uga.edu; or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the association.
本专栏重点介绍本出版物的读者感兴趣的最近发表的文章。我们鼓励ABRF成员将他们认为重要和有用的文章信息转发给:Clive Slaughter, GRU-UGA医疗合作伙伴，1425 Prince Ave.， Athens, GA 30606, USA。电话:(706)713-2216;传真:(706)713-2221;电子邮件:cslaught@uga.edu;或者给任何编辑委员会的成员。文章摘要反映的是审稿人的意见，而不一定是协会的意见。
{"title":"Article Watch: December 2015.","authors":"C. Slaughter","doi":"10.7171/jbt.15-2604-004","DOIUrl":"https://doi.org/10.7171/jbt.15-2604-004","url":null,"abstract":"This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to: Clive Slaughter, GRU-UGA Medical Partnership, 1425 Prince Ave., Athens, GA 30606, USA. Tel: (706) 713-2216; Fax: (706) 713-2221; E-mail: cslaught@uga.edu; or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the association.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"8 1","pages":"150-6"},"PeriodicalIF":0.0,"publicationDate":"2015-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91101606","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
A. Yamniuk, J. Newitt, M. Doyle, F. Arisaka, A. M. Giannetti, P. Hensley, D. Myszka, F. Schwarz, J. Thomson, E. Eisenstein
A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar. It is demonstrated that this system can serve as a benchmarking tool for the quantitative analysis of 2-site protein-protein interactions. The protein interaction pair enables determination of precise binding constants for the barstar protein binding to 2 distinct sites on the bivalent barnase binding partner (termed binase), where the 2 binding sites were engineered to possess affinities that differed by 2 orders of magnitude. Multiple MIRG laboratories characterized the interaction using isothermal titration calorimetry (ITC), AUC, and surface plasmon resonance (SPR) methods to evaluate the feasibility of the system as a benchmarking model. Although general agreement was seen for the binding constants measured using solution-based ITC and AUC approaches, weaker affinity was seen for surface-based method SPR, with protein immobilization likely affecting affinity. An analysis of the results from multiple MIRG laboratories suggests that the bivalent barnase-barstar system is a suitable model for benchmarking new approaches for the quantitative characterization of complex biomolecular interactions.
{"title":"Development of a Model Protein Interaction Pair as a Benchmarking Tool for the Quantitative Analysis of 2-Site Protein-Protein Interactions.","authors":"A. Yamniuk, J. Newitt, M. Doyle, F. Arisaka, A. M. Giannetti, P. Hensley, D. Myszka, F. Schwarz, J. Thomson, E. Eisenstein","doi":"10.7171/jbt.15-2604-001","DOIUrl":"https://doi.org/10.7171/jbt.15-2604-001","url":null,"abstract":"A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar. It is demonstrated that this system can serve as a benchmarking tool for the quantitative analysis of 2-site protein-protein interactions. The protein interaction pair enables determination of precise binding constants for the barstar protein binding to 2 distinct sites on the bivalent barnase binding partner (termed binase), where the 2 binding sites were engineered to possess affinities that differed by 2 orders of magnitude. Multiple MIRG laboratories characterized the interaction using isothermal titration calorimetry (ITC), AUC, and surface plasmon resonance (SPR) methods to evaluate the feasibility of the system as a benchmarking model. Although general agreement was seen for the binding constants measured using solution-based ITC and AUC approaches, weaker affinity was seen for surface-based method SPR, with protein immobilization likely affecting affinity. An analysis of the results from multiple MIRG laboratories suggests that the bivalent barnase-barstar system is a suitable model for benchmarking new approaches for the quantitative characterization of complex biomolecular interactions.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"44 1","pages":"125-41"},"PeriodicalIF":0.0,"publicationDate":"2015-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83021351","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Lauren N Kajiura, Scott D. Stewart, J. Dresios, C. Uyehara
Molecular detection of microbial pathogens in clinical samples requires the application of efficient sample lysis protocols and subsequent extraction and isolation of their nucleic acids. Here, we describe a simple and time-efficient method for simultaneous extraction of genomic DNA from gram-positive and -negative bacteria, as well as RNA from viral agents present in a sample. This method compared well with existing bacterial- and viral-specialized extraction protocols, worked reliably on clinical samples, and was not pathogen specific. This method may be used to extract DNA and RNA concurrently from viral and bacterial pathogens present in a sample and effectively detect coinfections in routine clinical diagnostics.
{"title":"Simultaneous Extraction of Viral and Bacterial Nucleic Acids for Molecular Diagnostic Applications.","authors":"Lauren N Kajiura, Scott D. Stewart, J. Dresios, C. Uyehara","doi":"10.7171/jbt.15-2604-002","DOIUrl":"https://doi.org/10.7171/jbt.15-2604-002","url":null,"abstract":"Molecular detection of microbial pathogens in clinical samples requires the application of efficient sample lysis protocols and subsequent extraction and isolation of their nucleic acids. Here, we describe a simple and time-efficient method for simultaneous extraction of genomic DNA from gram-positive and -negative bacteria, as well as RNA from viral agents present in a sample. This method compared well with existing bacterial- and viral-specialized extraction protocols, worked reliably on clinical samples, and was not pathogen specific. This method may be used to extract DNA and RNA concurrently from viral and bacterial pathogens present in a sample and effectively detect coinfections in routine clinical diagnostics.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"3 1","pages":"118-24"},"PeriodicalIF":0.0,"publicationDate":"2015-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76089075","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.
{"title":"An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.","authors":"Ryan Muller, M. C. Hammond, D. Rio, Yeon J Lee","doi":"10.7171/jbt.15-2604-003","DOIUrl":"https://doi.org/10.7171/jbt.15-2604-003","url":null,"abstract":"The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"46 1","pages":"142-9"},"PeriodicalIF":0.0,"publicationDate":"2015-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80466667","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
S. Collaud, T. Wiedl, E. Cattaneo, A. Soltermann, S. Hillinger, W. Weder, S. Arni
Laser-capture microdissection (LCM) enables the selection of a specific and pure cell population from a heterogenous tissue such as tumors. Activity-based protein profiling/profile (ABPP) is a chemical technology using enzyme-specific active site-directed probes to read out the functional state of many enzymes directly in any proteome. The aim of this work was to assess the compatibility of LCM with downstream ABPP for serine hydrolase (SH) in human lung adenocarcinoma. Fresh frozen lung adenocarcinoma tissue was stained with hematoxylin, toluidine blue, or methyl green (MG). Proteome from stained tissue was labeled further with SH-directed probes, and ABPPs were determined on a one-dimensional gel-based approach. This allowed us to assess the impact of staining procedures on their ABPPs. The effect of the LCM process on ABPPs was assessed furthermore using MG-stained lung adenocarcinoma tissue. The staining procedures led to strong changes in ABPPs. However, MG staining seemed the most compatible with downstream ABPP. MG-stained, laser-captured, microdissected tissue showed additional change in profiles as a result of the denaturing property of extraction buffer but not to the microdissection process itself. LCM staining procedures but not microdissection per se interfered with downstream ABPP and led to a strong change in ABPPs of SHs in human lung adenocarcinoma.
{"title":"Laser-capture microdissection impairs activity-based protein profiles for serine hydrolase in human lung adenocarcinoma.","authors":"S. Collaud, T. Wiedl, E. Cattaneo, A. Soltermann, S. Hillinger, W. Weder, S. Arni","doi":"10.5167/UZH-44238","DOIUrl":"https://doi.org/10.5167/UZH-44238","url":null,"abstract":"Laser-capture microdissection (LCM) enables the selection of a specific and pure cell population from a heterogenous tissue such as tumors. Activity-based protein profiling/profile (ABPP) is a chemical technology using enzyme-specific active site-directed probes to read out the functional state of many enzymes directly in any proteome. The aim of this work was to assess the compatibility of LCM with downstream ABPP for serine hydrolase (SH) in human lung adenocarcinoma. Fresh frozen lung adenocarcinoma tissue was stained with hematoxylin, toluidine blue, or methyl green (MG). Proteome from stained tissue was labeled further with SH-directed probes, and ABPPs were determined on a one-dimensional gel-based approach. This allowed us to assess the impact of staining procedures on their ABPPs. The effect of the LCM process on ABPPs was assessed furthermore using MG-stained lung adenocarcinoma tissue. The staining procedures led to strong changes in ABPPs. However, MG staining seemed the most compatible with downstream ABPP. MG-stained, laser-captured, microdissected tissue showed additional change in profiles as a result of the denaturing property of extraction buffer but not to the microdissection process itself. LCM staining procedures but not microdissection per se interfered with downstream ABPP and led to a strong change in ABPPs of SHs in human lung adenocarcinoma.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"21 1 1","pages":"25-8"},"PeriodicalIF":0.0,"publicationDate":"2010-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80254688","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
R. Thoma, J. Smith, W. Sandoval, J. Leone, P. Hunziker, B. Hampton, K. Linse, N. Denslow
The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield (R.Y.) information and the amount of lag were evaluated. Information about instrumentation and sample treatment was also collected as part of the study. For this study, the majority of participating laboratories successfully called the amino acid sequence for 17 cycles for all three test proteins. In general, laboratories found it more difficult to call the sequence containing the polyhistidine tag. Lag was observed earlier and more consistently with the polyhistidine-tagged protein than the polyalanine-tagged protein. Histidine yields were significantly less than the alanine yields in the tag portion of each analysis. The polyhistidine and polyalanine protein-R.Y. calculations were found to be equivalent. These calculations showed that the nontagged portion from each protein was equivalent. The terminal histidines from the tagged portion of the protein were demonstrated to be responsible for the high lag during N-terminal sequence analysis.
{"title":"The ABRF Edman Sequencing Research Group 2008 Study: investigation into homopolymeric amino acid N-terminal sequence tags and their effects on automated Edman degradation.","authors":"R. Thoma, J. Smith, W. Sandoval, J. Leone, P. Hunziker, B. Hampton, K. Linse, N. Denslow","doi":"10.5167/UZH-28072","DOIUrl":"https://doi.org/10.5167/UZH-28072","url":null,"abstract":"The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield (R.Y.) information and the amount of lag were evaluated. Information about instrumentation and sample treatment was also collected as part of the study. For this study, the majority of participating laboratories successfully called the amino acid sequence for 17 cycles for all three test proteins. In general, laboratories found it more difficult to call the sequence containing the polyhistidine tag. Lag was observed earlier and more consistently with the polyhistidine-tagged protein than the polyalanine-tagged protein. Histidine yields were significantly less than the alanine yields in the tag portion of each analysis. The polyhistidine and polyalanine protein-R.Y. calculations were found to be equivalent. These calculations showed that the nontagged portion from each protein was equivalent. The terminal histidines from the tagged portion of the protein were demonstrated to be responsible for the high lag during N-terminal sequence analysis.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"3 1","pages":"216-25"},"PeriodicalIF":0.0,"publicationDate":"2009-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84171420","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The analysis of intact and underivatised lipids in body fluids as well as in cell and tissue extracts is of utmost importance in the field of early diagnosis. Therefore, fast, reliable, and automated analytical methods are needed to detect known as well as unknown species. The combination of solid phase extraction, high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy are best at meeting this challenge. Herein, we show a workflow for the reliable analysis of individual components in phosphatidylethanolamine extracts. The limitations and advantages of the individual methods are discussed.
{"title":"Hyphenated tools for phospholipidomics.","authors":"J. Willmann, D. Leibfritz, H. Thiele","doi":"10.4172/jpb.s1000223","DOIUrl":"https://doi.org/10.4172/jpb.s1000223","url":null,"abstract":"The analysis of intact and underivatised lipids in body fluids as well as in cell and tissue extracts is of utmost importance in the field of early diagnosis. Therefore, fast, reliable, and automated analytical methods are needed to detect known as well as unknown species. The combination of solid phase extraction, high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy are best at meeting this challenge. Herein, we show a workflow for the reliable analysis of individual components in phosphatidylethanolamine extracts. The limitations and advantages of the individual methods are discussed.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"30 1","pages":"211-6"},"PeriodicalIF":0.0,"publicationDate":"2008-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84395365","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
James G Rohrbough, Linda A. Breci, Nirav C. Merchant, Susan Miller, P. Haynes
Data produced from the MudPIT analysis of yeast (S. cerevisiae) and rice (O. sativa) were used to develop a technique to validate single-peptide protein identifications using complementary database search algorithms. This results in a considerable reduction of overall false-positive rates for protein identifications; the overall false discovery rates in yeast are reduced from near 25% to less than 1%, and the false discovery rate of yeast single-peptide protein identifications becomes negligible. This technique can be employed by laboratories utilizing a SEQUEST-based proteomic analysis platform, incorporating the XTandem algorithm as a complementary tool for verification of single-peptide protein identifications. We have achieved this using open-source software, including several data-manipulation software tools developed in our laboratory, which are freely available to download.
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