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Protein Labeling (protein + labeling)
Selected AbstractsProtein labeling by iTRAQ: A new tool for quantitative mass spectrometry in proteome researchPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2007Sebastian Wiese Abstract A novel, MS-based approach for the relative quantification of proteins, relying on the derivatization of primary amino groups in intact proteins using isobaric tag for relative and absolute quantitation (iTRAQ) is presented. Due to the isobaric mass design of the iTRAQ reagents, differentially labeled proteins do not differ in mass; accordingly, their corresponding proteolytic peptides appear as single peaks in MS scans. Because quantitative information is provided by isotope-encoded reporter ions that can only be observed in MS/MS spectra, we analyzed the fragmentation behavior of ESI and MALDI ions of peptides generated from iTRAQ-labeled proteins using a TOF/TOF and/or a QTOF instrument. We observed efficient liberation of reporter ions for singly protonated peptides at low-energy collision conditions. In contrast, increased collision energies were required to liberate the iTRAQ label from lysine side chains of doubly charged peptides and, thus, to observe reporter ions suitable for relative quantification of proteins with high accuracy. We then developed a quantitative strategy that comprises labeling of intact proteins by iTRAQ followed by gel electrophoresis and peptide MS/MS analyses. As proof of principle, mixtures of five different proteins in various concentration ratios were quantified, demonstrating the general applicability of the approach presented here to quantitative MS-based proteomics. [source] Cover Picture: Electrophoresis 2'09ELECTROPHORESIS, Issue 2 2009Article first published online: 9 FEB 200 Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting-edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two-dimensional electrophoresis. Issue no. 2 has a "Fast Track" paper on the attomole protein analysis by capillary isoelectric focusing (CIEF) with LIF detection based on a post-column sheath flow cuvette employing Chromeo P503 as a fluorogenic reagent for protein labeling before CIEF analysis. Further selected topics of issue 2 are: Influence of image-analysis software on quantitation of two-dimensional gel electrophoresis data A PDMS sheath flow cuvette for high-sensitivity LIF measurements in CE [source] Quantification of carbonylated proteins in rat skeletal muscle mitochondria using capillary sieving electrophoresis with laser-induced fluorescence detectionELECTROPHORESIS, Issue 2 2008Juan Feng Abstract Carbonyl-modified proteins are markers of oxidative damage. Here, we report a new method for detecting and quantifying carbonylated proteins by capillary sieving electrophoresis (CSE) with LIF detection (CSE-LIF). Alexa 488 hydrazide is used for the specific labeling of carbonyls while 3-(2-furoyl) quinoline-2-carboxaldehyde (FQ) is used for protein labeling. BSA subjected to metal-catalyzed oxidation is used to optimize the labeling reactions, confirm the separation power of CSE, and characterize the response of the LIF detector. The method is capable of detecting femtomole (fmol) amounts of carbonyls in proteins with molecular masses ranging from 26 to 30,kDa. Using this method, we determined that mitochondrial proteins isolated from skeletal muscle contains 2.1,±,0.1 (average,±,SD; n,=,3) nmol carbonyl/mg protein. The methodology described here should be compatible with the analysis of single cells and needle biopsies taken from oxidative stress animal models. [source] Minocycline-Based Europium(III) Chelate Complexes: Synthesis, Luminescent Properties, and Labeling to StreptavidinHELVETICA CHIMICA ACTA, Issue 11 2009Takuya Nishioka Abstract Two chelate ligands for europium(III) having minocycline (=(4S,4aS,5aR,12aS)-4,7-bis(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,10,12,12a-tetrahydroxy-1,11-dioxonaphthacene-2-carboxamide; 5) as a VIS-light-absorbing group were synthesized as possible VIS-light-excitable stable Eu3+ complexes for protein labeling. The 9-amino derivative 7 of minocycline was treated with H6TTHA (=triethylenetetraminehexaacetic acid=3,6,9,12-tetrakis(carboxymethyl)-3,6,9,12-tetraazatetradecanedioic acid) or H5DTPA (=diethylenetriaminepentaacetic acid=N,N -bis{2-[bis(carboxymethyl)amino]ethyl}glycine) to link the polycarboxylic acids to minocycline. One of the Eu3+ chelates, [Eu3+(minocycline-TTHA)] (13), is moderately luminescent in H2O by excitation at 395,nm, whereas [Eu3+(minocycline-DTPA)] (9) was not luminescent by excitation at the same wavelength. The luminescence and the excitation spectra of [Eu3+(minocycline-TTHA)] (13) showed that, different from other luminescent EuIII chelate complexes, the emission at 615,nm is caused via direct excitation of the Eu3+ ion, and the chelate ligand is not involved in the excitation of Eu3+. However, the ligand seems to act for the prevention of quenching of the Eu3+ emission by H2O. The fact that the excitation spectrum of [Eu3+(minocycline-TTHA)] is almost identical with the absorption spectrum of Eu3+ aqua ion supports such an excitation mechanism. The high stability of the complexes of [Eu3+(minocycline-DTPA)] (9) and [Eu3+(minocycline-TTHA)] (13) was confirmed by UV-absorption semi-quantitative titrations of H4(minocycline-DTPA) (8) and H5(minocycline-TTHA) (12) with Eu3+. The titrations suggested also that an 1,:,1 ligand Eu3+ complex is formed from 12, whereas an 1,:,2 complex was formed from 8 minocycline-DTPA. The H5(minocycline-TTHA) (12) was successfully conjugated to streptavidin (SA) (Scheme,5), and thus the applicability of the corresponding Eu3+ complex to label a protein was established. [source] Quantitative DY-maleimide-based proteomic 2-DE-labeling strategies using human skin proteinsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 18 2009Lisa 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] Distribution of sex steroid hormone receptors in the brain of an African cichlid fish, Astatotilapia burtoniTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 16 2010Lauren A. Munchrath Abstract Sex steroid hormones released from the gonads play an important role in mediating social behavior across all vertebrates. Many effects of these gonadal hormones are mediated by nuclear steroid hormone receptors, which are crucial for integration in the brain of external (e.g., social) signals with internal physiological cues to produce an appropriate behavioral output. The African cichlid fish Astatotilapia burtoni presents an attractive model system for the study of how internal cues and external social signals are integrated in the brain as males display robust plasticity in the form of two distinct, yet reversible, behavioral and physiological phenotypes depending on the social environment. In order to better understand where sex steroid hormones act to regulate social behavior in this species, we have determined the distribution of the androgen receptor, estrogen receptor alpha, estrogen receptor beta, and progesterone receptor mRNA and protein throughout the telencephalon and diencephalon and some mesencephalic structures of A. burtoni. All steroid hormone receptors were found in key brain regions known to modulate social behavior in other vertebrates including the proposed teleost homologs of the mammalian amygdalar complex, hippocampus, striatum, preoptic area, anterior hypothalamus, ventromedial hypothalamus, and ventral tegmental area. Overall, there is high concordance of mRNA and protein labeling. Our results significantly extend our understanding of sex steroid pathways in the cichlid brain and support the important role of nuclear sex steroid hormone receptors in modulating social behaviors in teleosts and across vertebrates. J. Comp. Neurol. 518:3302,3326, 2010. © 2010 Wiley-Liss, Inc. [source] Sortase-Mediated Ligation: A Gift from Gram-Positive Bacteria to Protein EngineeringCHEMBIOCHEM, Issue 5 2009Shinya Tsukiji Dr. Abstract A new enzymatic protein ligation tool, sortase, has recently emerged from Gram-positive bacteria. This article outlines the technique, sortase-mediated ligation, and its applications in protein engineering, which include the introduction of unnatural molecules into proteins, protein immobilization, protein,protein conjugation, protein cyclization, as a self-cleavable tag for protein expression, protein,PNA hybrids, neoglycoconjugates, and cell-surface protein labeling, etc. [source] | |||||||||||||