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Labeling Strategies (labeling + strategy)

Distribution by Scientific Domains

Chemistry	100%

Selected Abstracts

Labeling strategies of selected subtypes of the hexahydronaphth[2,3-b]-1,4-oxazine- and octahydrobenzo[g]quinoline-type,

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 5-6 2007
Thomas Moenius
[source]

Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiae

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 18 2008
Jennifer L. Sporty
Abstract A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, yeast cells were harvested by centrifugation and then lysed under nonoxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50 mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH3CN/50 mM ammonium acetate (3:1 v/v) was added to the cell lysates. Chloroform extractions were performed on supernatants to remove organic solvent. Samples were lyophilized and resuspended in 50 mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV,Vis absorbance detection. NAD and NADH levels were evaluated in yeast grown under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (i) applicable to quantification of these metabolites in other cell cultures; and (ii) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures. [source]

Quantitative DY-maleimide-based proteomic 2-DE-labeling strategies using human skin proteins

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 18 2009
Lisa Dietz
Abstract Sensitive differential proteomic analysis is challenging and often limited by distinct labeling or tagging strategies. In this study, we have examined the sensitivity, linearity, and photophysical properties of novel protein labeling DY-maleimide dyes (DY-505-MAL, DY-555-MAL and DY-635-MAL). All MS compatible DY-maleimide dyes exhibited excellent emission spectra, high sensitivity, and high linearity, when applied to standard 1-DE protein analysis. Correspondingly, 2-DE analysis of DY-635-MAL or DY-505-MAL maximal-labeled human keratinocyte proteins displayed remarkably high sensitivity. Compared with a standard fluorescent protein stain, DY-635-MAL or DY-505-MAL 2-DE analysis demonstrated equally high spot quality with an overall increase in the number of spots detectable (up to threefold higher;>1000 spots/gel). However, as determined with a FLA-5100 imaging system, comparative MultiGauge, and Delta2D analysis, not all DY-maleimide dyes possessed DIGE compatible fluorescent emission properties. However, DY-505-MAL and DY-635-MAL were found to be suitable for more complex, time and gel intensive, focused multiplexing analyses. Notably , as demonstrated with allergen-stimulated human skin proteins , defined, singular DY-maleimide dye protein labeling (SDPL) allows high quality, time saving, simple, and reliable differential proteomic examination. [source]

A MS data search method for improved 15N-labeled protein identification

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 17 2009
Yaoyang Zhang
Abstract Quantitative proteomics using stable isotope labeling strategies combined with MS is an important tool for biomarker discovery. Methods involving stable isotope metabolic labeling result in optimal quantitative accuracy, since they allow the immediate combination of two or more samples. Unfortunately, stable isotope incorporation rates in metabolic labeling experiments using mammalian organisms usually do not reach 100%. As a consequence, protein identifications in 15N database searches have poor success rates. We report on a strategy that significantly improves the number of 15N-labeled protein identifications and results in a more comprehensive and accurate relative peptide quantification workflow. [source]

In vacuo isotope coded alkylation technique (IVICAT); an N-terminal stable isotopic label for quantitative liquid chromatography/mass spectrometry proteomics,

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 16 2006
Brigitte L. Simons
We present a new isotopic labeling strategy to modify the N-terminal amino group of peptides in a quantifiable reaction without the use of expensive reagents or solvents. The In Vacuo Isotope Coded Alkylation Technique (IVICAT) is a methylation reaction, carried out at low pressure (<100,mTorr), that results in a stable quaternary trimethylammonium group, thus adding a permanent positive charge at the N-terminus of peptides without modifying the , -amino groups of lysine. The methylation reaction increases the signal intensity of modified peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and liquid chromatography (LC)/MS and the isotopic peak pair differs by 9 mass units which can be easily resolved by either instrument. N-terminally trimethylated peptides exhibit collision-induced dissociation (CID) mass spectra that differ from their unmodified analogues by an enhanced b -ion series in MS2 spectra due to the fixed positive charge. Using LC/MS/MS with an LTQ mass analyzer for quantification, the experimentally determined ratios of H9 - to D9 -trimethyl-labeled peptides of , -casein provided accurate estimates of the actual ratios with low % error. IVICAT labeling also accurately quantified proteins in rat kidney inner medullary collecting duct cell types, as judged by comparison with relative quantification by subsequent immunoblotting experiments. IVICAT labeling, when used in conjunction with the new proteomics software QUIL, can accurately report relative protein abundances and increase the sequence coverage of proteins of tissue proteomes. Published in 2006 by John Wiley & Sons, Ltd. [source]

Cover Picture: A Powerful Combinatorial Screen to Identify High-Affinity Terbium(III)-Binding Peptides (ChemBioChem 4/2003)

CHEMBIOCHEM, Issue 4 2003
Mark Nitz Dr.
Abstract The cover picture shows the chemistry and biology used to develop a new protein labeling strategy based on lanthanide-binding peptides. Focused peptide libraries are screened to identify oligopeptide motifs of dual function: the peptides bind TbIII with remarkably high affinity and promote intense TbIII luminescence. The peptide sequences are then genetically encoded to create recombinant fusion proteins that possess a site-specific and minimally invasive fluorophore. Further information can be found in the two articles by Imperiali and co-workers on pp. 265,271 and pp. 272,276. [source]