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Isotope Labeling (isotope + labeling)
Kinds of Isotope Labeling Selected AbstractsAn Efficient Synthesis of Substituted meta -Halophenols and Their Methyl Ethers: Insight into the Reaction MechanismEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 15 2010Faiz Ahmed Khan Abstract An expeditious synthetic methodology leading to substituted meta -halophenols and their corresponding methyl ether derivatives through acid-mediated fragmentation of suitably substituted dihalonorbornyl ketones has been devised. The reaction sequence consists of TBTH-mediated (TBTH is tri- n -butyltin hydride) selective bridgehead halogen reduction of easily accessible Diels,Alder adducts derived from 1,2,3,4-tetrahalo-5,5-dimethoxycyclopentadiene and ,-substituted vinyl acetates, with subsequent conversion into the requisite bicyclic ketones by a two-step hydrolysis/oxidation approach. An extensive mechanistic investigation based on isotope labeling and cross experiments has been carried out and plausible mechanistic pathways based on these results have been proposed. The absence of halogen atoms at the bridgehead positions steers the reaction through a novel pathway involving the incorporation of proton (or deuterium) followed by elimination of HX (or DX), so the described methodology also provides a reliable route to ortho-para dideuteratedphenolic derivatives. [source] Identification of tyrosine-phosphorylation sites in the nuclear membrane protein emerinFEBS JOURNAL, Issue 14 2006Andreas Schlosser Although several proteins undergo tyrosine phosphorylation at the nuclear envelope, we achieved, for the first time, the identification of tyrosine-phosphorylation sites of a nuclear-membrane protein, emerin, by applying two mass spectrometry-based techniques. With a multiprotease approach combined with highly specific phosphopeptide enrichment and nano liquid chromatography tandem mass spectrometry analysis, we identified three tyrosine-phosphorylation sites, Y-75, Y-95, and Y-106, in mouse emerin. Stable isotope labeling with amino acids in cell culture revealed phosphotyrosines at Y-59, Y-74, Y-86, Y-161, and Y-167 of human emerin. The phosphorylation sites Y-74/Y-75 (human/mouse emerin), Y-85/Y-86, Y-94/Y-95, and Y-105/Y-106 are located in regions previously shown to be critical for interactions of emerin with lamin A, actin or the transcriptional regulators GCL and Btf, while the residues Y-161 and Y-167 are in a region linked to binding lamin-A or actin. Tyrosine Y-94/Y-95 is located adjacent to a five-residue motif in human emerin, whose deletion has been associated with X-linked Emery,Dreifuss muscle dystrophy. [source] M+4 stable isotope labeling of levovirin and M+7 and carbon-14 labeling of levovirin valinate pro-drugJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 14 2006Steve A. de Keczer Abstract [M+4]-labeled levovirin 5 (231 mg) was synthesized as an MS reference compound from [M+4] triazole ester 2. [M+7]-labeled levovirin valinate 6 (127 mg) was synthesized as a comparison MS reference compound from [M+6] triazole ester 3. [14C]-Levovirin 7 and [14C]-levovirin valinate 8 were synthesized to support metabolism studies. The synthesis of 7 was accomplished in 33% overall yield (35.4 mCi, 57 mCi/mmol) from Ba14CO3 and 8 was synthesized in 41% yield (12.5 mCi, 57 mCi/mmol) from 7. An efficient metallation/carbonation reaction was developed to synthesize [14C]-triazole ester 4. Copyright © 2006 John Wiley & Sons, Ltd. [source] Utilization of high-accuracy FTICR-MS data in protein quantitation experimentsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 11 2009Martin Strohalm Abstract Human acute T-lymphoblastic leukemia cell line (CEM) treated with cisplatin, and the stable isotope labeling by amino acids in cell culture (SILAC) strategy were used to present an improved method of data processing in high-accuracy mass spectrometry (MS). By using peptide mass fingerprinting with low mass tolerance, we were able to utilize far more data retained in MS scans which would normally be missed by a standard processing method. This new way of data interpretation results in an improvement of the relevance of quantitation experiments and enabled us to search and quantify different types of posttranslational modifications. Furthermore, we used this technique to distinguish among different protein isoforms, commonly returned by Mascot search engine. Copyright © 2009 John Wiley & Sons, Ltd. [source] A quantitative strategy to detect changes in accessibility of protein regions to chemical modification on heterodimerizationPROTEIN SCIENCE, Issue 7 2009Mathias Dreger Abstract We describe a method for studying quantitative changes in accessibility of surface lysine residues of the PB1 subunit of the influenza RNA polymerase as a result of association with the PA subunit to form a PB1-PA heterodimer. Our method combines two established methods: (i) the chemical modification of surface lysine residues of native proteins by N -hydroxysuccinimidobiotin (NHS-biotin) and (ii) the stable isotope labeling of amino acids in cell culture (SILAC) followed by tryptic digestion and mass spectrometry. By linking the chemical modification with the SILAC methodology for the first time, we obtain quantitative data on chemical modification allowing subtle changes in accessibility to be described. Five regions in the PB1 monomer showed altered reactivity to NHS-biotin when compared with the [PB1-PA] heterodimer. Mutational analysis of residues in two such regions,at K265 and K481 of PB1, which were about three- and twofold, respectively, less accessible to biotinylation in the PB1-PA heterodimer compared with the PB1 monomer, demonstrated that both K265 and K481 were crucial for polymerase function. This novel assay of quantitative profiling of biotinylation patterns (Q-POP assay) highlights likely conformational changes at important functional sites, as observed here for PB1, and may provide information on protein,protein interaction interfaces. The Q-POP assay should be a generally applicable approach and may detect novel functional sites suitable for targeting by drugs. [source] Quantitative proteome analysis of detergent-resistant membranes identifies the differential regulation of protein kinase C isoforms in apoptotic T cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2010Therese Solstad Abstract Several lines of evidence suggest that detergent-resistant membranes (DRMs) (also known as lipid rafts and glycosphingolipid-enriched microdomains) may have a role in signaling pathways of apoptosis. Here, we developed a method that combines DRMs isolation and methanol/chloroform extraction with stable isotope labeling with amino acids in cell culture-based quantitative proteome analysis of DRMs from control and cisplatin-induced apoptotic Jurkat T cells. This approach enabled us to enrich proteins with a pivotal role in cell signaling of which several were found with increased or decreased amounts in DRMs upon induction of apoptosis. Specifically, we show that three isoforms of protein kinase C (PKC) are regulated differently upon apoptosis. Although PKC, which belongs to the group of conventional PKCs is highly up-regulated in DRMs, the levels of two novel PKCs, PKC, and PKC,, are significantly reduced. These alterations/differences in PKC regulation are verified by immunoblotting and confocal microscopy. In addition, a specific enrichment of PKC, in apoptotic blebs and buds is shown. Furthermore, we observe an increased expression of ecto-PKC, as a result of exposure to cisplatin using flow cytometry. Our results demonstrate that in-depth proteomic analysis of DRMs provides a tool to study differential localization and regulation of signaling molecules important in health and disease. [source] Honokiol inhibits HepG2 migration via down-regulation of IQGAP1 expression discovered by a quantitative pharmaceutical proteomic analysisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2010Shufang Liang Abstract Honokiol (HNK), a natural small molecular product, inhibited proliferation of HepG2 cells and exhibited anti-tumor activity in nude mice. In this article, we applied a novel sensitive stable isotope labeling with amino acids in cell culture-based quantitative proteomic method and a model of nude mice to investigate the correlation between HNK and the hotspot migration molecule Ras GTPase-activating-like protein (IQGAP1). The quantitative proteomic analysis showed that IQGAP1 was 0.53-fold down-regulated under 10,,g/mL HNK exposure for 24,h on HepG2 cells. Migration ability of HepG2 cells under HNK treatment was correlated with its expression level of IQGAP1. In addition, the biochemical validation on HepG2 cells and the tumor xenograft model further demonstrated that HNK decreased the expression level of IQGAP1 and its upstream proteins Cdc42/Rac1. These data supported that HNK can modulate cell adhesion and cell migration by acting on Cdc42/Rac1 signaling via IQGAP1 interactions with its upstream Cdc42/Rac1 proteins, which is a new molecular mechanism of HNK to exert its anti-tumor activity. [source] Differential protein expression on the cell surface of colorectal cancer cells associated to tumor metastasisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2010Jose Luis Luque-García Abstract Progression to metastasis is the critical point in colorectal cancer (CRC) survival. However, the proteome associated to CRC metastasis is very poorly understood at the moment. In this study, we used stable isotope labeling by amino acids in cell culture to compare two CRC cell lines: KM12C and KM12SM, representing poorly versus highly metastatic potential, to find and quantify the differences in protein expression, mostly at the cell surface level. After biotinylation followed by affinity purification, membrane proteins were separated by SDS-PAGE and analyzed using nanoflow LC-ESI-LTQ. A total of 291 membrane and membrane-associated proteins were identified with a p value<0.01, from which 60 proteins were found to be differentially expressed by more than 1.5-fold. We identified a number of cell signaling, CDs, integrins and other cell adhesion molecules (cadherin 17, junction plakoglobin (JUP)) among the most deregulated proteins. They were validated by Western blot, confocal microscopy and flow cytometry analysis. Immunohistochemical analysis of paired tumoral samples confirmed that these differentially expressed proteins were also altered in human tumoral tissues. A good correlation with a major abundance in late tumor stages was observed for JUP and 17-,-hydroxysteroid dehydrogenase type 8 (HSD17B8). Moreover, the combined increase in JUP, occludin and F11 receptor expression together with cadherin 17 expression could suggest a reversion to a more epithelial phenotype in highly metastatic cells. Relevant changes were observed also at the metabolic level in the pentose phosphate pathway and several amino acid transporters. In summary, the identified proteins provide us with a better understanding of the events involved in liver colonization and CRC metastasis. [source] Remodeling of the SCF complex-mediated ubiquitination system by compositional alteration of incorporated F-box proteinsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2010Mitsunori Kato Abstract Ubiquitination regulates not only the stability but the localization and activity of substrate proteins involved in a plethora of cellular processes. The Skp1,Cullin,F-box protein (SCF) complexes constitute a major family of ubiquitin protein ligases, in each member of which an F-box protein serves as the variable component responsible for substrate recognition, thereby defining the function of each complex. Here we studied whether the composition of F-box proteins in the SCF complexes is remodeled under different conditions. We exploited stable isotope labeling and MS for relative quantification of F-box proteins in the SCF complexes affinity-purified en masse from budding yeast cells at log and post-diauxic phases, and revealed an increment of Saf1, an F-box protein involved in entry into quiescence, during the diauxic shift. Similarly, we found that Met4 overexpression induces a specific increment of Met30, the F-box protein responsible for ubiquitination of Met4. These results illustrate a cellular response to environmental and genetic perturbations through remodeling of the SCF complex-mediated ubiquitination system. Compositional alteration of incorporated F-box proteins may redirect the activity of this system toward appropriate substrates to be ubiquitinated under individual conditions for the maintenance of cellular homeostasis. [source] Quantitative analysis of phosphopeptides in search of the disease biomarker from the hepatocellular carcinoma specimenPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 12 2009Hyoung-Joo Lee Abstract Reversible phosphorylation of proteins is the most common PTM in cell-signaling pathways. Despite this, high-throughput methods for the systematic detection, identification, and quantification of phosphorylated peptides have yet to be developed. In this paper, we describe the establishment of an efficient online titaniuim dioxide (TiO2)-based 3-D LC (strong cationic exchange/TiO2/C18)-MS3 -linear ion trap system, which provides fully automatic and highly efficient identification of phosphorylation sites in complex peptide mixtures. Using this system, low-abundance phosphopeptides were isolated from cell lines, plasma, and tissue of healthy and hepatocellular carcinoma (HCC) patients. Furthermore, the phosphorylation sites were identified and the differences in phosphorylation levels between healthy and HCC patient specimens were quantified by labeling the phosphopeptides with isotopic analogs of amino acids (stable isotope labeling with amino acids in cell culture for HepG2 cells) or water (HO for tissues and plasma). Two examples of potential HCC phospho-biomarkers including plectin-1(phopho-Ser-4253) and alpha-HS-glycoprotein (phospho-Ser 138 and 312) were identified by this analysis. Our results suggest that this comprehensive TiO2 -based online-3-D LC-MS3 -linear ion trap system with high-throughput potential will be useful for the global profiling and quantification of the phosphoproteome and the identification of disease biomarkers. [source] Application of quantitative immunoprecipitation combined with knockdown and cross-linking to Chlamydomonas reveals the presence of vesicle-inducing protein in plastids 1 in a common complex with chloroplast HSP90CPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2009Heinrich Heide Abstract Knowledge of the interaction partners of a protein of interest may provide important information on its function. Common to currently available tools for the identification of protein,protein interactions, however, is their high rates of false positives. Only recently an assay was reported that allowed for the unequivocal identification of protein,protein interactions in mammalian cells in a single experiment. This assay, termed quantitative immunoprecipitation combined with knockdown (QUICK), combines RNAi, stable isotope labeling with amino acids in cell culture, immunoprecipitation, and quantitative MS. We are using the unicellular green alga Chlamydomonas reinhardtii to understand the roles of chaperones in chloroplast biogenesis. The goal of this work was to apply QUICK to Chlamydomonas for the identification of novel interaction partners of vesicle-inducing protein in plastids 1 (VIPP1), a protein required for the biosynthesis/maintenance of thylakoid membranes and known substrate of chloroplast HSP70B. We report here a robust QUICK protocol for Chlamydomonas that has been improved (i) by introducing a cross-linking step (-X) to improve protein complex stability and (ii) by including a control for the correction of unequal immunoprecipitation and/or labeling efficiencies. Using QUICK and cross-linking we could verify that HSP70B and CGE1 form a complex with VIPP1 and could also demonstrate that chloroplast HSP90C is part of this complex. Moreover, we could show that the chaperones interact with VIPP1 also in membrane fractions. [source] Chemistry meets proteomics: The use of chemical tagging reactions for MS-based proteomicsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 20 2006Alexander Leitner Dr. Abstract As proteomics matures from a purely descriptive to a function-oriented discipline of the life sciences, there is strong demand for novel methodologies that increase the depth of information that can be obtained from proteomic studies. MS has long played a central role for protein identification and characterization, often in combination with dedicated chemical modification reactions. Today, chemistry is helping to advance the field of proteomics in numerous ways. In this review, we focus on those methodologies that have a significant impact for the large-scale study of proteins and peptides. This includes approaches that allow the introduction of affinity tags for the enrichment of subclasses of peptides or proteins and strategies for in,vitro stable isotope labeling for quantification purposes, among others. Particular attention is given to the study of PTMs where recent advancements have been promising, but many interesting targets are not yet being addressed. [source] Quantification of change in phosphorylation of BCR-ABL kinase and its substrates in response to Imatinib treatment in human chronic myelogenous leukemia cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2006Xiquan Liang Dr. Abstract Phosphorylation by the constitutively activated BCR-ABL tyrosine kinase is associated with the pathogenesis of the human chronic myelogenous leukemia,(CML). It is difficult to characterize kinase response to stimuli or drug treatment because regulatory phosphorylation events are largely transient changes affecting low abundance proteins. Stable isotope labeling with amino acids in cell culture,(SILAC) has emerged as a pivotal technology for quantitative proteomics. By metabolically labeling proteins with light or heavy tyrosine, we are able to quantify the change in phosphorylation of BCR-ABL kinase and its substrates in response to drug treatment in human CML cells. In this study, we observed that BCR-ABL kinase is phosphorylated at tyrosines,393 and 644, and that SH2-domain containing inositol phosphatase (SHIP)-2 and downstream of kinase (Dok)-2 are phosphorylated at tyrosine,1135 and 299, respectively. Based on the relative intensity of isotopic peptide pairs, we demonstrate that the level of phosphorylation of BCR-ABL kinase as well as SHIP-2 and Dok-2 is reduced approximately 90% upon treatment with Imatinib, a specific inhibitor of BCR-ABL kinase. Furthermore, proteins, such as SHIP-1, SH2-containing protein (SHC) and Casitas B-lineage lymphoma proto-oncogene (CBL), are also regulated by Imatinib. These results demonstrate the simplicity and utility of SILAC as a method to quantify dynamic changes in phosphorylation at specific sites in response to stimuli or drug treatment in cell culture. [source] A study of the elimination of water from lithium-cationized tripeptide methyl esters by means of tandem mass spectrometry and isotope labelingRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 20 2006Erach R. Talaty Extensive isotope labeling (2H, 13C and 15N), collision-induced dissociation (CID) and multiple-stage tandem mass spectrometry were used to investigate the elimination of H2O from a series of model, metal-cationized tripeptide methyl esters. The present results corroborate our earlier suggestion that loss of water from lithiated peptides is initiated by a nucleophilic attack from the N-terminal side upon an amide carbonyl carbon atom to form a five-membered ring as an intermediate followed by 1,2-elimination of water. We show that the nucleophilic atom is the oxygen atom of the N-terminal amide group in the fragmentation of [AcGGGOMe+Li]+ as well as [GGGOMe+Li]+. However, the subsequent fragmentation is markedly different in the two cases as a result of the absence and presence of a free amino group. In particular, extensive scrambling of protons in the , -positions of GGGOMe is observed, presumably as a consequence of intervention of the basic amino group. Copyright © 2006 John Wiley & Sons, Ltd. [source] A ,-secretase inhibitor decreases amyloid-, production in the central nervous system,ANNALS OF NEUROLOGY, Issue 1 2009Randall J. Bateman MD Objective Accumulation of amyloid-, (A,) by overproduction or underclearance in the central nervous system (CNS) is hypothesized to be a necessary event in the pathogenesis of Alzheimer's disease. However, previously, there has not been a method to determine drug effects on A, production or clearance in the human CNS. The objective of this study was to determine the effects of a ,-secretase inhibitor on the production of A, in the human CNS. Methods We utilized a recently developed method of stable-isotope labeling combined with cerebrospinal fluid sampling to directly measure A, production during treatment of a ,-secretase inhibitor, LY450139. We assessed whether this drug could decrease CNS A, production in healthy men (age range, 21,50 years) at single oral doses of 100, 140, or 280mg (n = 5 per group). Results LY450139 significantly decreased the production of CNS A, in a dose-dependent fashion, with inhibition of A, generation of 47, 52, and 84% over a 12-hour period with doses of 100, 140, and 280mg, respectively. There was no difference in A, clearance. Interpretation Stable isotope labeling of CNS proteins can be utilized to assess the effects of drugs on the production and clearance rates of proteins targeted as potential disease-modifying treatments for Alzheimer's disease and other CNS disorders. Results from this approach can assist in making decisions about drug dosing and frequency in the design of larger and longer clinical trials for diseases such as Alzheimer's disease, and may accelerate effective drug validation. Ann Neurol 2009 [source] A Concerted Approach for the Determination of Molecular Conformation in Ordered and Disordered MaterialsCHEMISTRY - A EUROPEAN JOURNAL, Issue 22 2007Jan Sehnert Abstract We present the successful application of a concerted approach for the investigation of the local environment in ordered and disordered phases in the solid state. In this approach we combined isotope labeling with computational methods and different solid-state NMR techniques. We chose triphenylphosphite (TPP) as an interesting example of our investigations because TPP exhibits two crystalline modifications and two different amorphous phases one of which is highly correlated. In particular we analyzed the conformational distribution in three of these phases. A sample of triply labeled 1-[13C]TPP was prepared and 1D MAS as well as wide-line 13C NMR spectra were measured. Furthermore we acquired 2D 13C wide-line exchange spectra and used this method to derive highly detailed information about the phenyl orientation in the investigated TPP phases. For linkage with a structure model a DFT analysis of the TPP molecule and its immediate environment was carried out. The ab initio calculations of the 13C chemical shift tensor in three- and six-spin systems served as a base for the calculation of 1D and 2D spectra. By comparing these simulations to the experiment an explicit picture of all phases could be drawn on a molecular level. Our results therefore reveal the high potential of the presented approach for detailed studies of the mesoscopic environment even in the challenging case of amorphous materials. [source] | ||||||||||||||||||||||