The Experts below are selected from a list of 56952 Experts worldwide ranked by ideXlab platform

Liang Li - One of the best experts on this subject based on the ideXlab platform.

  • mass accuracy check using common background peaks for improving metabolome data quality in chemical Isotope Labeling lc ms
    Journal of the American Society for Mass Spectrometry, 2019
    Co-Authors: Yunong Li, Liang Li
    Abstract:

    Chemical Isotope Labeling (CIL) LC-MS is a highly sensitive and quantitative method for metabolome analysis. Because of a large number of peaks detectable in a sample and the need of running many samples in a metabolomics project, any significant change in mass measurement accuracy during the whole period of running samples can adversely affect the downstream peak alignment and quantitative analysis. Herein, we report a rapid method to check the mass accuracy of individual spectra in each CIL LC-MS run in order to flag up any run containing spectra with accuracy drift that falls outside the expected error. The flagged run may be re-run or discarded before merging with other runs for peak alignment and analysis. This method is based on the observation that some background signals are commonly detected in almost all spectra collected in CIL LC-MS runs. A mass accuracy check (MAC) software program has been developed to first find the common background mass peaks and then use them as mass references to calcul...

  • high performance chemical Isotope Labeling liquid chromatography mass spectrometry for exosome metabolomics
    Analytical Chemistry, 2018
    Co-Authors: Mingrui An, Kyle C Cuneo, David M Lubman, Liang Li
    Abstract:

    Circulating exosomes in bodily fluids such as blood are being actively studied as a rich source of chemical biomarkers for cancer diagnosis and monitoring. Although nucleic acid analysis is a primary tool for the discovery of circulating biomarkers in exosomes, metabolomics holds the potential of expanding the chemical diversity of biomarkers that may be easy and rapid to detect. However, only trace amounts of exosomes can be isolated from a small volume of patient blood, and thus a very sensitive technique is required to analyze the metabolome of exosomes. In this report, we present a workflow that involves multiple cycles of ultracentrifugation for exosome isolation using a starting material of 2 mL of human serum, freeze–thaw-cycles in 50% methanol/water for exosome lysis and metabolite extraction, differential chemical Isotope Labeling (CIL) of metabolites for enhancing liquid chromatography (LC) separation and improving mass spectrometry (MS) detection, and nanoflow LC-MS (nLC-MS) with captivespray f...

  • development of high performance chemical Isotope Labeling lc ms for profiling the carbonyl submetabolome
    Analytical Chemistry, 2017
    Co-Authors: Shuang Zhao, Margot Dawe, Liang Li
    Abstract:

    Metabolites containing a carbonyl group represent several important classes of molecules including various forms of ketones and aldehydes such as steroids and sugars. We report a high-performance chemical Isotope Labeling (CIL) LC–MS method for profiling the carbonyl submetabolome with high coverage and high accuracy and precision of relative quantification. This method is based on the use of dansylhydrazine (DnsHz) Labeling of carbonyl metabolites to change their chemical and physical properties to such an extent that the labeled metabolites can be efficiently separated by reversed phase LC and ionized by electrospray ionization MS. In the analysis of six standards representing different carbonyl classes, acetaldehyde could be ionized only after Labeling and MS signals were significantly increased for other 5 standards with an enhancement factor ranging from ∼15-fold for androsterone to ∼940-fold for 2-butanone. Differential 12C- and 13C-DnsHz Labeling was developed for quantifying metabolic differences ...

  • comprehensive and quantitative profiling of the human sweat submetabolome using high performance chemical Isotope Labeling lc ms
    Analytical Chemistry, 2016
    Co-Authors: Kevin Hooton, Liang Li
    Abstract:

    Human sweat can be noninvasively collected and used as a media for diagnosis of certain diseases as well as for drug detection. However, because of very low concentrations of endogenous metabolites present in sweat, metabolomic analysis of sweat with high coverage is difficult, making it less widely used for metabolomics research. In this work, a high-performance method for profiling the human sweat submetabolome based on chemical Isotope Labeling (CIL) liquid chromatography–mass spectrometry (LC–MS) is reported. Sweat was collected using a gauze sponge style patch, extracted from the gauze by centrifugation, and then derivatized using CIL. Differential 12C- and 13C-dansylation Labeling was used to target the amine/phenol submetabolome. Because of large variations in the total amount of sweat metabolites in individual samples, sample amount normalization was first performed using liquid chromatography with UV detection (LC–UV) after dansylation. The 12C-labeled individual sample was then mixed with an equ...

  • differential Isotope Labeling of 38 dietary polyphenols and their quantification in urine by liquid chromatography electrospray ionization tandem mass spectrometry
    Analytical Chemistry, 2016
    Co-Authors: David Achaintre, Liang Li, Audrey Bulete, Cecile Crenolive, Sabina Rinaldi, Augustin Scalbert
    Abstract:

    A large number of polyphenols are consumed with the diet and may contribute to the prevention of chronic diseases such as cardiovascular diseases, diabetes, cancers, and neurodegenerative diseases. More comprehensive methods are needed to measure exposure to this complex family of bioactive plant compounds in epidemiological studies. We report here a novel method enabling the simultaneous measurement in urine of 38 polyphenols representative of the main classes and subclasses found in the diet. This method is based on differential 12C-/13C-Isotope Labeling of polyphenols through derivatization with isotopic dansyl chloride reagents and on the analysis of the labeled polyphenols by tandem mass spectrometry. This derivatization approach overcomes the need for costly labeled standards. Different conditions for enzyme hydrolysis of polyphenol glucuronides and sulfate esters, extraction, and dansylation of unconjugated aglycones were tested and optimized. Limits of quantification varied from 0.01 to 1.1 μM dep...

Matthias Mann - One of the best experts on this subject based on the ideXlab platform.

  • use of stable Isotope Labeling by amino acids in cell culture as a spike in standard in quantitative proteomics
    Nature Protocols, 2011
    Co-Authors: Tamar Geiger, Jacek R Wisniewski, Sara Zanivan, Marcus Kruger, Yasushi Ishihama, Matthias Mann
    Abstract:

    Use of stable Isotope Labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics

  • stable Isotope Labeling by amino acids in cell culture silac applied to quantitative proteomics of bacillus subtilis
    Journal of Proteome Research, 2010
    Co-Authors: Boumediene Soufi, Matthias Mann, Chanchal Kumar, Florian Gnad, Ivan Mijakovic, Boris Macek
    Abstract:

    We applied stable Isotope Labeling by amino acids in cell culture (SILAC) to large-scale quantitative proteomics analyses of the model bacterium Bacillus subtilis in two physiological conditions: growth on succinate and growth under phosphate starvation. Using a B. subtilis strain auxotrophic for lysine and high accuracy mass spectrometry for downstream analysis, we identified and quantified changes in the levels of more than 1500 proteins in each of the tested conditions with high biological and technical reproducibility. With a total of 1928 identified proteins, this study presents one of the most comprehensive quantitative proteomics studies in bacteria, covering more than 75% of the B. subtilis genes expressed in the log phase of growth. Furthermore, we detect and quantify dynamics of 35 Ser/Thr/Tyr phosphorylation sites under growth on succinate, and 10 phosphorylation sites under phosphate starvation, demonstrating the full compatibility of the method with site-specific detection and quantitation of...

  • stable Isotope Labeling by amino acids in cell culture for quantitative proteomics
    Methods of Molecular Biology, 2007
    Co-Authors: Matthias Mann
    Abstract:

    Publisher Summary Analysis by combined liquid chromatographic separation and mass spectrometry (LC-MS) is rapidly becoming the most popular and effective approach for large-scale protein identification. Quantitative methods in mass spectrometry (MS) rely largely on the principle of stable Isotope Labeling (SIL). The protein samples to be compared are derivatized separately with the two forms of ICAT, digested, and purified over an avidin column to enrich ICAT-labeled peptides for subsequent mass spectrometric analyses. Cells from both samples are mixed 1 to 1 and lyzed. Proteins extracted from the mixture are then analyzed by standard MS techniques. Many muscle-specific proteins, such as myosin, were found to be highly upregulated while other proteins, such as histones, were found at similar levels in both samples. SILAC-labeled cells are used to generate separate pools of proteins or protein complexes that are distinct to affinity bait. Cells may be differentially treated with a drug or growth factor; alternatively, cells may express the wild-type or mutant form of a component of a protein complex.

  • a practical recipe for stable Isotope Labeling by amino acids in cell culture silac
    Nature Protocols, 2006
    Co-Authors: Matthias Mann
    Abstract:

    Stable Isotope Labeling by amino acids in cell culture (SILAC) is a simple, robust, yet powerful approach in mass spectrometry (MS)-based quantitative proteomics. SILAC labels cellular proteomes through normal metabolic processes, incorporating non-radioactive, stable Isotope-containing amino acids in newly synthesized proteins. Growth medium is prepared where natural ("light") amino acids are replaced by "heavy" SILAC amino acids. Cells grown in this medium incorporate the heavy amino acids after five cell doublings and SILAC amino acids have no effect on cell morphology or growth rates. When light and heavy cell populations are mixed, they remain distinguishable by MS, and protein abundances are determined from the relative MS signal intensities. SILAC provides accurate relative quantification without any chemical derivatization or manipulation and enables development of elegant functional assays in proteomics. In this protocol, we describe how to apply SILAC and the use of nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry for protein identification and quantification. This procedure can be completed in 8 days.

  • insulin dependent interactions of proteins with glut4 revealed through stable Isotope Labeling by amino acids in cell culture silac
    Journal of Proteome Research, 2006
    Co-Authors: Leonard J Foster, Matthias Mann, Assaf Rudich, I Talior, Nish Patel, Xudong Huang, L M Furtado, P J Bilan, Amira Klip
    Abstract:

    The insulin-regulated glucose transporter (GLUT4) translocates to the plasma membrane in response to insulin in order to facilitate the postprandial uptake of glucose into fat and muscle cells. While early insulin receptor signaling steps leading to this translocation are well defined, the integration of signaling and regulation of GLUT4 traffic remains elusive. Several lines of evidence suggest an important role for the actin cytoskeleton and for protein−protein interactions in regulating GLUT4 localization by insulin. Here, we applied stable Isotope Labeling by amino acids in cell culture (SILAC) to identify proteins that interact with GLUT4 in an insulin-regulated manner. Myc-tagged GLUT4 (GLUT4myc) stably expressed in L6 myotubes was immunoprecipitated via the myc epitope from total membranes isolated from basal and insulin-stimulated cells grown in medium containing normal isotopic abundance leucine or deuterated leucine, respectively. Proteins coprecipitating with GLUT4myc were analyzed by liquid ch...

Yuqi Feng - One of the best experts on this subject based on the ideXlab platform.

  • comprehensive profiling of fecal metabolome of mice by integrated chemical Isotope Labeling mass spectrometry analysis
    Analytical Chemistry, 2018
    Co-Authors: Bifeng Yuan, Shujian Zheng, Yalan Wang, Jie Wang, Jing Xu, Ke He, Ting Hu, Yingwei Zheng, Fuqiang Xu, Yuqi Feng
    Abstract:

    Gut microbiota plays important roles in the host health. The host and symbiotic gut microbiota coproduce a large number of metabolites during the metabolism of food and xenobiotics. The analysis of fecal metabolites can provide a noninvasive manner to study the outcome of the host-gut microbiota interaction. Herein, we reported the comprehensive profiling of fecal metabolome of mice by an integrated chemical Isotope Labeling combined with liquid chromatography–mass spectrometry (CIL-LC-MS) analysis. The metabolites are categorized into several submetabolomes based on the functional moieties (i.e., carboxyl, carbonyl, amine, and thiol) and then analysis of the individual submetabolome was performed. The combined data from the submetabolome form the metabolome with relatively high coverage. To this end, we synthesized stable Isotope Labeling reagents to label metabolites with different groups, including carboxyl, carbonyl, amine, and thiol groups. We detected 2302 potential metabolites, among which, 1388 co...

  • metal oxide based selective enrichment combined with stable Isotope Labeling mass spectrometry analysis for profiling of ribose conjugates
    Analytical Chemistry, 2015
    Co-Authors: Chubo Qi, Yunqing Huang, Bifeng Yuan, Hanpeng Jiang, Yuqi Feng
    Abstract:

    Some modified ribonucleosides in biological fluids have been evaluated as cancer-related metabolites. Detection of endogenous modified ribonucleosides in biological fluids may serve as a noninvasive cancers diagnostic method. However, determination of modified ribonucleosides is still challenging because of their low abundance and serious matrix interferences in biological fluids. Here, we developed a novel strategy for comprehensive profiling of ribose conjugates from biological fluids using metal oxide-based dispersive solid-phase extraction (DSPE) followed with in vitro stable Isotope Labeling and double neutral loss scan-mass spectrometry analysis (DSPE-SIL-LC-DNLS-MS). Cerium dioxide (CeO2) was used to selectively recognize and capture ribose conjugates from complex biological samples under basic environment. The enriched ribose conjugates were subsequently labeled with a pair of Isotope Labeling reagents (acetone and acetone-d6). The glucosidic bond of acetone labeled ribose conjugates is readily ru...

  • profiling of thiol containing compounds by stable Isotope Labeling double precursor ion scan mass spectrometry
    Analytical Chemistry, 2014
    Co-Authors: Yunqing Huang, Bifeng Yuan, Yuqi Feng
    Abstract:

    Here we developed a novel strategy of Isotope Labeling in combination with high-performance liquid chromatography–double precursor ion scan mass spectrometry (IL–LC–DPIS-MS) analysis for nontargeted profiling of thiol-containing compounds. In this strategy, we synthesized a pair of Isotope Labeling reagents (ω-bromoacetonylquinolinium bromide, BQB; ω-bromoacetonylquinolinium-d7 bromide, BQB-d7) that contain a reactive group, an isotopically labeled moiety, and an ionizable group to selectively label thiol-containing compounds. The BQB and BQB-d7 labeled compounds can generate two characteristic product ions m/z 218 and 225, which contain an Isotope tag and therefore were used for double precursor ion scans in mass spectrometry analysis. The peak pairs with characteristic mass differences can be readily extracted from the two precursor ion scan (PIS) spectra and assigned as potential thiol-containing candidates, which facilitates the identification of analytes. BQB and BQB-d7 labeled thiol-containing compo...

Ruedi Aebersold - One of the best experts on this subject based on the ideXlab platform.

Shigeyuki Yokoyama - One of the best experts on this subject based on the ideXlab platform.

  • Improving cell-free protein synthesis for stable-Isotope Labeling
    Journal of Biomolecular NMR, 2007
    Co-Authors: Takayoshi Matsuda, Seizo Koshiba, Naoya Tochio, Eiko Seki, Noriyuki Iwasaki, Takashi Yabuki, Makoto Inoue, Shigeyuki Yokoyama, Takanori Kigawa
    Abstract:

    Cell-free protein synthesis is suitable for stable-Isotope Labeling of proteins for NMR analysis. The Escherichia coli cell-free system containing potassium acetate for efficient translation (KOAc system) is usually used for stable-Isotope Labeling, although it is less productive than other systems. A system containing a high concentration of potassium l-glutamate (l-Glu system), instead of potassium acetate, is highly productive, but cannot be used for stable-Isotope Labeling of Glu residues. In this study, we have developed a new cell-free system that uses potassium d-glutamate (d-Glu system). The productivity of the d-Glu system is approximately twice that of the KOAc system. The cross peak intensities in the 1H–15N HSQC spectrum of the uniformly stable-Isotope labeled Ras protein, prepared with the d-Glu system, were similar to those obtained with the KOAc system, except that the Asp intensities were much higher for the protein produced with the d-Glu system. These results indicate that the d-Glu system is a highly productive cell-free system that is especially useful for stable-Isotope Labeling of proteins.

  • cell free production and stable Isotope Labeling of milligram quantities of proteins
    FEBS Letters, 1999
    Co-Authors: T Kigawa, Takashi Yabuki, Yasuhiko Yoshida, Michio Tsutsui, Takehiko Shibata, Shigeyuki Yokoyama
    Abstract:

    We have improved the productivity of an Escherichia coli cell-free protein synthesis system. First, creatine phosphate and creatine kinase were used as the energy source regeneration system, and the other components of the reaction mixture were optimized. Second, the E. coli S30 cell extract was condensed by dialysis against a polyethylene glycol solution to increase the rate of synthesis. Third, during the protein synthesis, the reaction mixture was dialyzed against a low-molecular-weight substrate solution to prolong the reaction. Thus, the yield of chloramphenicol acetyltransferase was raised to 6 mg/ml of reaction mixture. Stable-Isotope Labeling of a protein with 13C/15N-labeled amino acids for NMR spectroscopy was achieved by this method.

  • cell free synthesis and amino acid selective stable Isotope Labeling of proteins for nmr analysis
    Journal of Biomolecular NMR, 1995
    Co-Authors: Takanori Kigawa, Y Muto, Shigeyuki Yokoyama
    Abstract:

    For the application of multidimensional NMR spectroscopy to larger proteins, it would be useful to perform selective Labeling of one of the 20 amino acids. For some amino acids, however, amino acid metabolism drastically reduces the efficiency and selectivity of Labeling in in vivo expression systems. In the present study, a cell-free protein synthesis system was optimized, so that highly efficient and selective stable Isotope Labeling of proteins can be achieved in the absence of amino acid metabolism. The productivity of the E. coli cell-free coupled transcription-translation system was first improved, by about fivefold, by using the T7 RNA polymerase for transcription and also by improving the translation conditions. Thus, about 0.1 mg protein per 1 ml reaction mixture was synthesized. Then, this improved cell-free system was used for Asp- or Ser-selective 15N-Labeling of the human c-Ha-Ras protein. With a 15 ml cell-free reaction, using less than 1 mg of 15N-labeled amino acid, 1 mg of the Ras protein was obtained. 1H-15N HSQC experiments confirmed that the Ras protein was efficiently labeled with high selectivity. These results indicate that this cell-free protein synthesis system is useful for NMR studies.