The Experts below are selected from a list of 2328 Experts worldwide ranked by ideXlab platform
Ruedi Aebersold - One of the best experts on this subject based on the ideXlab platform.
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Quantitative Protein Profiling Via Isotope-Coded Affinity Tag (ICAT) and Tandem Mass Spectrometry I. STATISTICALLY ANNOTATED DATASETS FOR PEPTIDE SEQUENCES AND PROTEINS IDENTIFIED VIA THE
2016Co-Authors: Alexey I. Nesvizhskii, David R Goodlett, Ruedi Aebersold, Jimmy Eng, Julian D. WattsAbstract:Lipid rafts were prepared according to standard protocols from Jurkat T cells stimulated via T cell receptor/CD28 cross-linking and from control (unstimulated) cells. Co-isolating proteins from the control and stimulated cell preparations were labeled with isotopically normal (d0) and heavy (d8) versions of the same Isotope-Coded Affinity Tag (ICAT) reagent, respectively. Samples were combined, proteolyzed, and resultant peptides fractionated via cat-ion exchange chromatography. Cysteine-containing (ICAT-labeled) peptides were recovered via the biotin Tag component of the ICAT reagents by avidin-Affinity chro-matography. On-line micro-capillary liquid chromatogra-phy tandem mass spectrometry was performed on both avidin-Affinity (ICAT-labeled) and flow-through (unlabeled) fractions. Initial peptide sequence identification was b
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FOCUS: PROTEOMICS Proteomic Analysis of Pseudomonas aeruginosa Grown Under Magnesium Limitation
2014Co-Authors: Tina Guina, Ruedi Aebersold, David R Goodlett, Jimmy Eng, Samuel I. Miller, Samuel O. Purvine, Robert K. Ernst, Kimberly A. LeeAbstract:In this study, large-scale qualitative and quantitative proteomic technology was applied to the analysis of the opportunistic bacterial pathogen Pseudomonas aeruginosa grown under magne-sium limitation, an environmental condition previously shown to induce expression of various virulence factors. For quantitative analysis, whole cell and membrane proteins were differen-tially labeled with Isotope-Coded Affinity Tag (ICAT) reagents and ICAT reagent-labeled peptides were separated by two-dimensional chromatography prior to analysis by electros-pray ionization-tandem mass spectrometry (ESI-MS/MS) in an ion trap mass spectrometer (ITMS). To increase the number of protein identifications, gas-phase fractionation (GPF) in the m/z dimension was employed for analysis of ICAT peptides derived from whole cell extracts. The experiments confirmed expression of 1331 P. aeruginosa proteins of which 145 were differentially expressed upon limitation of magnesium. A number of conserved Gram-negative magnesium stress-response proteins involved in bacterial virulence were among the most abundant proteins induced in low magnesium. Comparative ICAT analysis of membrane versus whole cell protein indicated that growth of P. aeruginosa in low magnesium resulted in altered subcellular compartmentalization of large enzyme complexes such as ribosomes. Thi
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quantitative proteomic analysis of myc induced apoptosis a direct role for myc induction of the mitochondrial chloride ion channel mtclic clic4
Journal of Biological Chemistry, 2006Co-Authors: Hookeun Lee, Yuzuru Shiio, Kwang S Suh, Stuart H Yuspa, Robert N Eisenman, Ruedi AebersoldAbstract:Myc is a key regulatory protein in higher eukaryotes controlling important cellular functions such as proliferation, differentiation, and apoptosis. Myc is profoundly involved in the genesis of many human and animal cancers, and the abrogation of Myc-induced apoptosis is a critical event in cancer progression. Because the mechanisms that mediate Myc-induced apoptosis are largely unknown, we analyzed protein expression during Myc-induced apoptosis using an Isotope-Coded Affinity Tag quantitative proteomics approach and identified that a proapoptotic mitochondrial chloride ion channel, mtCLIC/CLIC4, is induced by Myc. Myc binds to the mtCLIC gene promoter and activates its transcription. Suppression of mtCLIC expression by RNA interference inhibited Myc-induced apoptosis in response to different stress conditions and abolished the cooperative induction of apoptosis by Myc and Bax. We also found that Myc reduces the expression of Bcl-2 and Bcl-xL and that the apoptosis-inducing stimuli up-regulate Bax expression. These results suggest that up-regulation of mtCLIC, together with a reduction in Bcl-2 and Bcl-xL, sensitizes Myc-expressing cells to the proapoptotic action of Bax.
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eLS - Quantitative Proteomics (ICAT
Encyclopedia of Life Sciences, 2006Co-Authors: Timothy J Griffin, Jamie Sherman, Ruedi AebersoldAbstract:An essential aspect of proteomic analysis is the identification and quantification of each protein present in two or more complex mixtures. The use of Isotope-Coded Affinity Tag (ICAT™) reagents and mass spectrometry forms part of quantitative proteomic analysis technology. Keywords: quantitative proteomics; mass spectrometry; liquid chromatography; two-dimensional gel electrophoresis
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Quantitative proteomic analysis of the budding yeast cell cycle using acid-cleavable Isotope-Coded Affinity Tag reagents.
Proteomics, 2006Co-Authors: Mark R. Flory, Hookeun Lee, Richard Bonneau, Parag Mallick, Kyle A. Serikawa, David R. Morris, Ruedi AebersoldAbstract:Quantitative profiling of proteins, the direct effectors of nearly all biological functions, will undoubtedly complement technologies for the measurement of mRNA. Systematic proteomic measurement of the cell cycle is now possible by using stable isotopic labeling with Isotope-Coded Affinity Tag reagents and software tools for high-throughput analysis of LC-MS/MS data. We provide here the first such study achieving quantitative, global proteomic measurement of a time-course gene expression experiment in a model eukaryote, the budding yeast Saccharomyces cerevisiae, during the cell cycle. We sampled 48% of all predicted ORFs, and provide the data, including identifications, quantitations, and statistical measures of certainty, to the community in a sortable matrix. We do not detect significant concordance in the dynamics of the system over the time-course tested between our proteomic measurements and microarray measures collected from similarly treated yeast cultures. Our proteomic dataset therefore provides a necessary and complementary measure of eukaryotic gene expression, establishes a rich database for the functional analysis of S. cerevisiae proteins, and will enable further development of technologies for global proteomic analysis of higher eukaryotes.
David R Goodlett - One of the best experts on this subject based on the ideXlab platform.
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Quantitative Protein Profiling Via Isotope-Coded Affinity Tag (ICAT) and Tandem Mass Spectrometry I. STATISTICALLY ANNOTATED DATASETS FOR PEPTIDE SEQUENCES AND PROTEINS IDENTIFIED VIA THE
2016Co-Authors: Alexey I. Nesvizhskii, David R Goodlett, Ruedi Aebersold, Jimmy Eng, Julian D. WattsAbstract:Lipid rafts were prepared according to standard protocols from Jurkat T cells stimulated via T cell receptor/CD28 cross-linking and from control (unstimulated) cells. Co-isolating proteins from the control and stimulated cell preparations were labeled with isotopically normal (d0) and heavy (d8) versions of the same Isotope-Coded Affinity Tag (ICAT) reagent, respectively. Samples were combined, proteolyzed, and resultant peptides fractionated via cat-ion exchange chromatography. Cysteine-containing (ICAT-labeled) peptides were recovered via the biotin Tag component of the ICAT reagents by avidin-Affinity chro-matography. On-line micro-capillary liquid chromatogra-phy tandem mass spectrometry was performed on both avidin-Affinity (ICAT-labeled) and flow-through (unlabeled) fractions. Initial peptide sequence identification was b
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FOCUS: PROTEOMICS Proteomic Analysis of Pseudomonas aeruginosa Grown Under Magnesium Limitation
2014Co-Authors: Tina Guina, Ruedi Aebersold, David R Goodlett, Jimmy Eng, Samuel I. Miller, Samuel O. Purvine, Robert K. Ernst, Kimberly A. LeeAbstract:In this study, large-scale qualitative and quantitative proteomic technology was applied to the analysis of the opportunistic bacterial pathogen Pseudomonas aeruginosa grown under magne-sium limitation, an environmental condition previously shown to induce expression of various virulence factors. For quantitative analysis, whole cell and membrane proteins were differen-tially labeled with Isotope-Coded Affinity Tag (ICAT) reagents and ICAT reagent-labeled peptides were separated by two-dimensional chromatography prior to analysis by electros-pray ionization-tandem mass spectrometry (ESI-MS/MS) in an ion trap mass spectrometer (ITMS). To increase the number of protein identifications, gas-phase fractionation (GPF) in the m/z dimension was employed for analysis of ICAT peptides derived from whole cell extracts. The experiments confirmed expression of 1331 P. aeruginosa proteins of which 145 were differentially expressed upon limitation of magnesium. A number of conserved Gram-negative magnesium stress-response proteins involved in bacterial virulence were among the most abundant proteins induced in low magnesium. Comparative ICAT analysis of membrane versus whole cell protein indicated that growth of P. aeruginosa in low magnesium resulted in altered subcellular compartmentalization of large enzyme complexes such as ribosomes. Thi
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METHODOLOGY Comparison of a Label-Free Quantitative Proteomic Method Based on Peptide Ion Current Area to the Isotope Coded Affi nity Tag Method
2013Co-Authors: Soyoung Ryu, Byron Gallis, Young Ah Goo, Scott A. Shaffer, Dragan Radulovic, David R GoodlettAbstract:Abstract: Recently, several research groups have published methods for the determination of proteomic expression profiling by mass spectrometry without the use of exogenously added stable isotopes or stable isotope dilution theory. These so-called label-free, methods have the advanTage of allowing data on each sample to be acquired independently from all other samples to which they can later be compared in silico for the purpose of measuring changes in protein expression between various biological states. We developed label free software based on direct measurement of peptide ion current area (PICA) and compared it to two other methods, a simpler label free method known as spectral counting and the isotope coded Affinity Tag (ICAT) method. Data analysis by these methods of a standard mixture containing proteins of known, but varying, concentrations showed that they performed similarly with a mean squared error of 0.09. Additionally, complex bacterial protein mixtures spiked with known concentrations of standard proteins were analyzed using the PICA label-free method. These results indicated that the PICA method detected all levels of standard spiked proteins at the 90 % confidence level in this complex biological sample. This finding confirms that label-free methods, based on direct measurement of the area under a single ion current trace, performed as well as the standard ICAT method. Given the fact that the label-free methods provide ease in experimental design well beyond pair-wise comparison, label-free methods such as our PICA method are well suited for proteomic expression profiling of large numbers of samples as is needed in clinical analysis. Keywords: label-free quantification, peptide ion current area (PICA), isotope coded Affinity Tag (ICAT), spectral coun
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Comparison of a Label-Free Quantitative Proteomic Method Based on Peptide Ion Current Area to the Isotope Coded Affinity Tag Method
Cancer informatics, 2008Co-Authors: Soyoung Ryu, Byron Gallis, Young Ah Goo, Scott A. Shaffer, Dragan Radulovic, David R GoodlettAbstract:Recently, several research groups have published methods for the determination of proteomic expression profiling by mass spectrometry without the use of exogenously added stable isotopes or stable isotope dilution theory. These so-called label-free, methods have the advanTage of allowing data on each sample to be acquired independently from all other samples to which they can later be compared in silico for the purpose of measuring changes in protein expression between various biological states. We developed label free software based on direct measurement of peptide ion current area (PICA) and compared it to two other methods, a simpler label free method known as spectral counting and the isotope coded Affinity Tag (ICAT) method. Data analysis by these methods of a standard mixture containing proteins of known, but varying, concentrations showed that they performed similarly with a mean squared error of 0.09. Additionally, complex bacterial protein mixtures spiked with known concentrations of standard proteins were analyzed using the PICA label-free method. These results indicated that the PICA method detected all levels of standard spiked proteins at the 90% confidence level in this complex biological sample. This finding confirms that label-free methods, based on direct measurement of the area under a single ion current trace, performed as well as the standard ICAT method. Given the fact that the label-free methods provide ease in experimental design well beyond pair-wise comparison, label-free methods such as our PICA method are well suited for proteomic expression profiling of large numbers of samples as is needed in clinical analysis.
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Increased quantitative proteome coverage with 13C/12C‐based, acid‐cleavable isotope‐coded Affinity Tag reagent and modified data acquisition scheme
Proteomics, 2005Co-Authors: Kelly Cooke, David R Goodlett, Hookeun Lee, Brian Raught, Andrew Page, Victoria Aneliunas, Phil Hieter, Ruedi AebersoldAbstract:Quantitative protein profiling using the Isotope-Coded Affinity Tag (ICAT) method and tandem mass spectrometry (MS) enables the pair-wise comparison of protein expression levels in biological samples. A new version of the ICAT reagent with an acid-cleavable bond, which allows removal of the biotin moiety prior to MS and which utilizes 13 C substitution for 12 C in the heavy-ICATreagent rather than 2 H (for 1 H) as in the original reagent (Gygi, S. P., Rist, B., Gerber, S. A., Frantisek, T., Gelb, M. H., Aebersold, R., Quantitative analysis of complex protein mixtures using Isotope-Coded Affinity Tags. Nat. Biotechnol. 1999, 17, 994-999), was investigated. We developed and validated an MS data acquisition strategy using this new reagent that results in an increased number of protein identifications per experiment, without losing the accuracy of protein quantification. This was achieved by following a single survey (precursor) ion scan and serial collision-induced dissociations (CIDs) of four different precursor ions observed in the prior survey scan. This strategy is common to many high-performance liquid chromatography-electrospray ionization (HPLC-ESI)-MS shotgun proteomic strategies, but heretofore not to ICAT experiments. This advance is possible because the new ICAT reagent uses 13 C as the heavy element rather than 2 H, thus, eliminating the slight delay in retention time of ICAT-labeled light peptides on a C18-based HPLC separation that occurs with 2 H and 1 H. Analyses using this new scheme of an ICAT-labeled trypsin-digested six protein mixture as well as a tryptic digest of a total yeast lysate, indicated that about two times more proteins were identified in a single analysis, and that there was no loss in accuracy of quantification.
Jimmy K. Eng - One of the best experts on this subject based on the ideXlab platform.
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The Pseudomonas aeruginosa Proteome during Anaerobic Growth
Journal of bacteriology, 2005Co-Authors: Tina Guina, Jimmy K. Eng, Mitchell J. Brittnacher, Hai Nguyen, Samuel I. MillerAbstract:Isotope-Coded Affinity Tag analysis and two-dimensional gel electrophoresis followed by tandem mass spectrometry were used to identify Pseudomonas aeruginosa proteins expressed during anaerobic growth. Out of the 617 proteins identified, 158 were changed in abundance during anaerobic growth compared to during aerobic growth, including proteins whose increased expression was expected based on their role in anaerobic metabolism. These results form the basis for future analyses of alterations in bacterial protein content during growth in various environments, including the cystic fibrosis airway.
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Pancreatic Cancer Proteome: The Proteins That Underlie Invasion, Metastasis, and Immunologic Escape
Gastroenterology, 2005Co-Authors: Ru Chen, David R Goodlett, Kelly Cooke, Samuel Donohoe, Jimmy K. Eng, Sheng Pan, David A. Crispin, Zhaoli Lane, Mary P. BronnerAbstract:Pancreatic cancer is a highly lethal disease that has seen little headway in diagnosis and treatment for the past few decades. The effective treatment of pancreatic cancer is critically relying on the diagnosis of the disease at an early sTage, which still remains challenging. New experimental approaches, such as quantitative proteomics, have shown great potential for the study of cancer and have opened new opportunities to investigate crucial events underlying pancreatic tumorigenesis and to exploit this knowledge for early detection and better intervention. To systematically study protein expression in pancreatic cancer, we used Isotope-Coded Affinity Tag technology and tandem mass spectrometry to perform quantitative proteomic profiling of pancreatic cancer tissues and normal pancreas. A total of 656 proteins were identified and quantified in 2 pancreatic cancer samples, of which 151 were differentially expressed in cancer by at least 2-fold. This study revealed numerous proteins that are newly discovered to be associated with pancreatic cancer, providing candidates for future early diagnosis biomarkers and targets for therapy. Several differentially expressed proteins were further validated by tissue microarray immunohistochemistry. Many of the differentially expressed proteins identified are involved in protein-driven interactions between the ductal epithelium and the extracellular matrix that orchestrate tumor growth, migration, angiogenesis, invasion, metastasis, and immunologic escape. Our study is the first application of Isotope-Coded Affinity Tag technology for proteomic analysis of human cancer tissue and has shown the value of this technology in identifying differentially expressed proteins in cancer.
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Proteomic analysis of synaptosomes using Isotope-Coded Affinity Tags and mass spectrometry.
Proteomics, 2005Co-Authors: Sabine P. Schrimpf, Ruedi Aebersold, Jimmy K. Eng, Virginia Meskenaite, Erich Brunner, Dorothea Rutishauser, Pascal Walther, Peter SondereggerAbstract:Synaptosomes are isolated synapses produced by subcellular fractionation of brain tissue. They contain the complete presynaptic terminal, including mitochondria and synaptic vesicles, and portions of the postsynaptic side, including the postsynaptic membrane and the postsynaptic density (PSyD). A proteomic characterisation of synaptosomes isolated from mouse brain was performed employing the Isotope-Coded Affinity Tag (ICAT) method and tandem mass spectrometry (MS/MS). After isotopic labelling and tryptic digestion, peptides were fractionated by cation exchange chromatography and cysteine-containing peptides were isolated by Affinity chromatography. The peptides were identified by microcapillary liquid chromatography-electrospray ionisation MS/MS (muLC-ESI MS/MS). In two experiments, peptides representing a total of 1131 database entries were identified. They are involved in different presynaptic and postsynaptic functions, including synaptic vesicle exocytosis for neurotransmitter release, vesicle endocytosis for synaptic vesicle recycling, as well as postsynaptic receptors and proteins constituting the PSyD. Moreover, a large number of soluble and membrane-bound molecules serving functions in synaptic signal transduction and metabolism were detected. The results provide an inventory of the synaptic proteome and confirm the suitability of the ICAT method for the assessment of synaptic structure, function and plasticity.
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The Application of New Software Tools to Quantitative Protein Profiling Via Isotope-Coded Affinity Tag (ICAT) and Tandem Mass Spectrometry I. Statistically Annotated Datasets for Peptide Sequences and Proteins Identified via the Application of ICAT a
Molecular & cellular proteomics : MCP, 2003Co-Authors: Priska D. Von Haller, David R Goodlett, Alexey I. Nesvizhskii, Samuel Donohoe, Kelly Vaughn, Andrew Keller, Jimmy K. Eng, Ruedi AebersoldAbstract:Lipid rafts were prepared according to standard protocols from Jurkat T cells stimulated via T cell receptor/CD28 cross-linking and from control (unstimulated) cells. Co-isolating proteins from the control and stimulated cell preparations were labeled with isotopically normal (d0) and heavy (d8) versions of the same Isotope-Coded Affinity Tag (ICAT) reagent, respectively. Samples were combined, proteolyzed, and resultant peptides fractionated via cation exchange chromatography. Cysteine-containing (ICAT-labeled) peptides were recovered via the biotin Tag component of the ICAT reagents by avidin-Affinity chromatography. On-line micro-capillary liquid chromatography tandem mass spectrometry was performed on both avidin-Affinity (ICAT-labeled) and flow-through (unlabeled) fractions. Initial peptide sequence identification was by searching recorded tandem mass spectrometry spectra against a human sequence data base using SEQUEST software. New statistical data modeling algorithms were then applied to the SEQUEST search results. These allowed for discrimination between likely "correct" and "incorrect" peptide assignments, and from these the inferred proteins that they collectively represented, by calculating estimated probabilities that each peptide assignment and subsequent protein identification was a member of the "correct" population. For convenience, the resultant lists of peptide sequences assigned and the proteins to which they corresponded were filtered at an arbitrarily set cut-off of 0.5 (i.e. 50% likely to be "correct") and above and compiled into two separate datasets. In total, these data sets contained 7667 individual peptide identifications, which represented 2669 unique peptide sequences, corresponding to 685 proteins and related protein groups.
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The Application of New Software Tools to Quantitative Protein Profiling Via Isotope-Coded Affinity Tag (ICAT) and Tandem Mass Spectrometry II. Evaluation of Tandem Mass Spectrometry Methodologies for Large-Scale Protein Analysis, and the Application
Molecular & cellular proteomics : MCP, 2003Co-Authors: Priska D. Von Haller, David R Goodlett, Alexey I. Nesvizhskii, Samuel Donohoe, Kelly Vaughn, Andrew Keller, Jimmy K. Eng, Ruedi AebersoldAbstract:Proteomic approaches to biological research that will prove the most useful and productive require robust, sensitive, and reproducible technologies for both the qualitative and quantitative analysis of complex protein mixtures. Here we applied the Isotope-Coded Affinity Tag (ICAT) approach to quantitative protein profiling, in this case proteins that copurified with lipid raft plasma membrane domains isolated from control and stimulated Jurkat human T cells. With the ICAT approach, cysteine residues of the two related protein isolates were covalently labeled with isotopically normal and heavy versions of the same reagent, respectively. Following proteolytic cleavage of combined labeled proteins, peptides were fractionated by multidimensional chromatography and subsequently analyzed via automated tandem mass spectrometry. Individual tandem mass spectrometry spectra were searched against a human sequence database, and a variety of recently developed, publicly available software applications were used to sort, filter, analyze, and compare the results of two repetitions of the same experiment. In particular, robust statistical modeling algorithms were used to assign measures of confidence to both peptide sequences and the proteins from which they were likely derived, identified via the database searches. We show that by applying such statistical tools to the identification of T cell lipid raft-associated proteins, we were able to estimate the accuracy of peptide and protein identifications made. These tools also allow for determination of the false positive rate as a function of user-defined data filtering parameters, thus giving the user significant control over and information about the final output of large-scale proteomic experiments. With the ability to assign probabilities to all identifications, the need for manual verification of results is substantially reduced, thus making the rapid evaluation of large proteomic datasets possible. Finally, by repeating the experiment, information relating to the general reproducibility and validity of this approach to large-scale proteomic analyses was also obtained.
Hookeun Lee - One of the best experts on this subject based on the ideXlab platform.
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quantitative proteomic analysis of myc induced apoptosis a direct role for myc induction of the mitochondrial chloride ion channel mtclic clic4
Journal of Biological Chemistry, 2006Co-Authors: Hookeun Lee, Yuzuru Shiio, Kwang S Suh, Stuart H Yuspa, Robert N Eisenman, Ruedi AebersoldAbstract:Myc is a key regulatory protein in higher eukaryotes controlling important cellular functions such as proliferation, differentiation, and apoptosis. Myc is profoundly involved in the genesis of many human and animal cancers, and the abrogation of Myc-induced apoptosis is a critical event in cancer progression. Because the mechanisms that mediate Myc-induced apoptosis are largely unknown, we analyzed protein expression during Myc-induced apoptosis using an Isotope-Coded Affinity Tag quantitative proteomics approach and identified that a proapoptotic mitochondrial chloride ion channel, mtCLIC/CLIC4, is induced by Myc. Myc binds to the mtCLIC gene promoter and activates its transcription. Suppression of mtCLIC expression by RNA interference inhibited Myc-induced apoptosis in response to different stress conditions and abolished the cooperative induction of apoptosis by Myc and Bax. We also found that Myc reduces the expression of Bcl-2 and Bcl-xL and that the apoptosis-inducing stimuli up-regulate Bax expression. These results suggest that up-regulation of mtCLIC, together with a reduction in Bcl-2 and Bcl-xL, sensitizes Myc-expressing cells to the proapoptotic action of Bax.
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Quantitative proteomic analysis of the budding yeast cell cycle using acid-cleavable Isotope-Coded Affinity Tag reagents.
Proteomics, 2006Co-Authors: Mark R. Flory, Hookeun Lee, Richard Bonneau, Parag Mallick, Kyle A. Serikawa, David R. Morris, Ruedi AebersoldAbstract:Quantitative profiling of proteins, the direct effectors of nearly all biological functions, will undoubtedly complement technologies for the measurement of mRNA. Systematic proteomic measurement of the cell cycle is now possible by using stable isotopic labeling with Isotope-Coded Affinity Tag reagents and software tools for high-throughput analysis of LC-MS/MS data. We provide here the first such study achieving quantitative, global proteomic measurement of a time-course gene expression experiment in a model eukaryote, the budding yeast Saccharomyces cerevisiae, during the cell cycle. We sampled 48% of all predicted ORFs, and provide the data, including identifications, quantitations, and statistical measures of certainty, to the community in a sortable matrix. We do not detect significant concordance in the dynamics of the system over the time-course tested between our proteomic measurements and microarray measures collected from similarly treated yeast cultures. Our proteomic dataset therefore provides a necessary and complementary measure of eukaryotic gene expression, establishes a rich database for the functional analysis of S. cerevisiae proteins, and will enable further development of technologies for global proteomic analysis of higher eukaryotes.
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Increased quantitative proteome coverage with 13C/12C‐based, acid‐cleavable isotope‐coded Affinity Tag reagent and modified data acquisition scheme
Proteomics, 2005Co-Authors: Kelly Cooke, David R Goodlett, Hookeun Lee, Brian Raught, Andrew Page, Victoria Aneliunas, Phil Hieter, Ruedi AebersoldAbstract:Quantitative protein profiling using the Isotope-Coded Affinity Tag (ICAT) method and tandem mass spectrometry (MS) enables the pair-wise comparison of protein expression levels in biological samples. A new version of the ICAT reagent with an acid-cleavable bond, which allows removal of the biotin moiety prior to MS and which utilizes 13 C substitution for 12 C in the heavy-ICATreagent rather than 2 H (for 1 H) as in the original reagent (Gygi, S. P., Rist, B., Gerber, S. A., Frantisek, T., Gelb, M. H., Aebersold, R., Quantitative analysis of complex protein mixtures using Isotope-Coded Affinity Tags. Nat. Biotechnol. 1999, 17, 994-999), was investigated. We developed and validated an MS data acquisition strategy using this new reagent that results in an increased number of protein identifications per experiment, without losing the accuracy of protein quantification. This was achieved by following a single survey (precursor) ion scan and serial collision-induced dissociations (CIDs) of four different precursor ions observed in the prior survey scan. This strategy is common to many high-performance liquid chromatography-electrospray ionization (HPLC-ESI)-MS shotgun proteomic strategies, but heretofore not to ICAT experiments. This advance is possible because the new ICAT reagent uses 13 C as the heavy element rather than 2 H, thus, eliminating the slight delay in retention time of ICAT-labeled light peptides on a C18-based HPLC separation that occurs with 2 H and 1 H. Analyses using this new scheme of an ICAT-labeled trypsin-digested six protein mixture as well as a tryptic digest of a total yeast lysate, indicated that about two times more proteins were identified in a single analysis, and that there was no loss in accuracy of quantification.
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A Dataset of Human Liver Proteins Identified by Protein Profiling Via Isotope-Coded Affinity Tag (ICAT) and Tandem Mass Spectrometry
Molecular & cellular proteomics : MCP, 2004Co-Authors: Wei Yan, Hookeun Lee, Eric W. Deutsch, Catherine A. Lázaro, Weiliang Tang, Eric T Chen, Nelson Fausto, Michael G. Katze, Ruedi AebersoldAbstract:Proteins from human liver carcinoma Huh7 cells, representing transformed liver cells, and cultured primary human fetal hepatocytes (HFH) and human HH4 hepatocytes, representing nontransformed liver cells, were extracted and processed for proteome analysis. Proteins from stimulated cells (interferon- treatment for the Huh7 and HFH cells and induction of hepatitis C virus [HCV] proteins for the HH4 cells) and corresponding control cells were labeled with light and heavy cleavable ICAT reagents, respectively. The labeled samples were combined, trypsinized, and subject to cation-exchange and avidin-Affinity chromatographies. The resulting cysteinecontaining peptides were analyzed by microcapillary LCMS/MS. The MS/MS spectra were initially analyzed by searching the human International Protein Index database using the SEQUEST TM software (1). Subsequently, new statistical algorithms were applied to the collective SEQUEST search results of each experiment. First, the PeptideProphet TM software (2) was applied to discriminate true assignments of MS/MS spectra to peptide sequences from false assignments, to assign a probability value for each identified peptide, and to compute the sensitivity and error rate for the assignment of spectra to sequences in each experiment. Second, the ProteinProphet TM software (3) was used to infer the protein identifications and to compute probabilities that a protein had been correctly identified, based on the available peptide sequence evidence. The resulting protein lists were filtered by a ProteinProphet probability score p > 0.5, which corresponded to an error rate of less than 5%. A total of 1,296, 1,430, and 1,476 proteins or related protein groups were identified in three subdatasets from the Huh7, HFH, and HH4 cells, respectively. In total, these subdatasets contained 2,486 unique protein identifications from human liver cells. An increase of the threshold to p > 0.9 (corresponding to an error rate of less than 1%) resulted in 2,159 unique protein identifications (1,146, 1,235, and 1,318 for the Huh7, HFH, and HH4 cells, respectively). Molecular & Cellular Proteomics 3:1039 –1041, 2004.
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Toward a high-throughput approach to quantitative proteomic analysis: Expression-dependent protein identification by mass spectrometry
Journal of the American Society for Mass Spectrometry, 2001Co-Authors: Timothy J Griffin, Ruedi Aebersold, Hookeun Lee, Steven P. Gygi, Beate Rist, David K. M. Han, Kenneth C. ParkerAbstract:The Isotope-Coded Affinity Tag (ICAT) [1] technology enables the concurrent identification and comparative quantitative analysis of proteins present in biological samples such as cell and tissue extracts and biological fluids by mass spectrometry. The initial implementation of this technology was based on microcapillary chromatography coupled on-line with electrospray ionization tandem mass spectrometry. This implementation lacked the ability to select proteins for identification based on their relative abundance and therefore to focus on differentially expressed proteins. In order to improve the sample throughput of this technology, we have developed a two-step approach that is focused on those proteins for which the abundance changes between samples: First, a new software program for the automated quantification of ICAT reagent labeled peptides analyzed by microcapillary electrospray ionization time-of-flight mass spectrometry determines those peptides that differ in their abundance and second, these peptides are identified by tandem mass spectrometry using an electrospray quadrupole time-of flight mass spectrometer and sequence database searching. Results from the application of this approach to the analysis of differentially expressed proteins secreted from nontumorigenic human prostate epithelial cells and metastatic cancerous human prostate epithelial cells are shown.
Timothy D. Veenstra - One of the best experts on this subject based on the ideXlab platform.
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Comparison of protein expression by Isotope-Coded Affinity Tag labeling.
Methods in molecular biology (Clifton N.J.), 2008Co-Authors: Zhen Xiao, Timothy D. VeenstraAbstract:Isotope-Coded Affinity Tag (ICAT) labeling, in combination with mass spectrometry (MS), has been widely adopted as an effective method for comparing protein abundance levels. This chapter describes the ICAT labeling procedure in search for the celecoxib-regulated proteins in a colon cancer cell line. Celecoxib, a cyclooxygenase-2 (COX-2) specific inhibitor, is used as a colorectal cancer preventative drug in clinical trials. Here, celecoxib is used to inhibit the expression of COX-2 in a colon cancer cell line HT-29. To elucidate the proteomic changes induced by celecoxib, the protein lysates from the treated and control cells are prepared. The cysteine-containing proteins are labeled with the heavy and light ICAT reagents, respectively. The labeled proteins are then combined and digested with trypsin. The ICAT-labeled peptides are subject to the purification through an avidin column and eventually the cleavage of the biotin Tags. This chapter focuses on the ICAT labeling procedure itself, because sample preparation is the most critical step of an ICAT-based protein expression comparison experiment. Other related procedures such as the cation exchange high performance liquid chromatography separation of peptides and MS analysis are detailed elsewhere in this book.
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Evaluation of the acid-cleavable Isotope-Coded Affinity Tag reagents: application to camptothecin-treated cortical neurons.
Journal of proteome research, 2004Co-Authors: Thomas P. Conrads, Haleem J. Issaq, Richard S. Morrison, Timothy D. VeenstraAbstract:The new generation of Isotope-Coded Affinity Tag (ICAT) reagents have been evaluated by labeling an equimolar amount of bovine serum albumin (BSA) with ICAT-12C9 and ICAT-13C9, combining the mixtures, digesting them with trypsin and analyzing the digestate both by μRPLC-tandem MS and by matrix-assisted laser desorption ionization (MALDI) TOF/TOF MS. The use of 13C in place of 2H resulted in both of the labeled peptides having identical elution characteristics in a reversed-phase separation. This similarity in elution allows ICAT-labeled peptides to be effectively analyzed using a μRPLC−MALDI−MS strategy as well. All of the cysteinyl-containing tryptic peptides from BSA were identified with only a 10% variation in the relative abundance measurements between the light and heavy versions of each peptide. A facile method for the removal of contaminants that arise from the cleaved biotin moiety that otherwise interfere with downstream separations and MS analysis has also been developed. The new ICAT reagents w...
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Phosphoprotein Isotope-Coded Affinity Tags: application to the enrichment and identification of low-abundance phosphoproteins.
Analytical chemistry, 2002Co-Authors: Michael B. Goshe, Timothy D. Veenstra, Thomas P. Conrads, Ellen A. Panisko, Nicolas H. Angell, Richard D. SmithAbstract:The use of a phosphoprotein Isotope-Coded Affinity Tag (PhIAT), which employs differential isotopic labeling and biotinylation, has been shown capable of enriching and identifying mixtures of low-abundance phosphopeptides. A denatured solution of β-casein was labeled using the PhIAT method, and after proteolytic digestion, the labeled peptides were isolated using immobilized avidin. The recovered peptides were separated by capillary reversed-phase liquid chromatography and identified by tandem mass spectrometry. PhIAT-labeled peptides corresponding to known O-phosphorylated peptides from β-casein were identified along with the phosphorylated peptides from αS1-casein and αS2-casein, known low-level (
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Phosphoprotein Isotope-Coded Affinity Tag Approach for Isolating and Quantitating Phosphopeptides in Proteome-Wide Analyses
Analytical chemistry, 2001Co-Authors: Michael B. Goshe, Timothy D. Veenstra, Thomas P. Conrads, Ellen A. Panisko, Nicolas H. Angell, Richard D. SmithAbstract:A method has been developed that utilizes phosphoprotein Isotope-Coded Affinity Tags (PhIAT) that combines stable isotope and biotin labeling to enrich and quantitatively measure differences in the O-phosphorylation states of proteins. The PhIAT labeling approach involves hydroxide ion-mediated beta-elimination of the O-phosphate moiety and the addition of 1,2-ethanedithiol containing either four alkyl hydrogens (EDT-D0) or four alkyl deuteriums (EDT-D4) followed by biotinylation of the EDT-D0/D4 moiety using (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine. The PhIAT reagent, which contains the nucleophilic sulfhydryl and isotopic label covalently linked to a biotin moiety, was synthesized and has the potential utility to reduce the O-phosphorylation derivatization into a one-step process. The PhIAT labeling approach was initially demonstrated using the model phosphoprotein beta-casein. After proteolytic digestion, the PhIAT-labeled peptides were Affinity isolated using immobilized avidin and analyzed using capillary reversed-phase liquid chromatography-mass spectrometry. PhIAT-labeled beta-casein peptides corresponding to peptides containing known sites of O-phosphorylation were isolated and identified. The PhIAT labeling method was also applied to a yeast protein extract. The PhIAT labeling technique provides a reliable method for making quantitative measurements of differences in the O-phosphorylation state of proteins.