e-book Mass Spectrometry: Modified Proteins and Glycoconjugates

Free download. Book file PDF easily for everyone and every device. You can download and read online Mass Spectrometry: Modified Proteins and Glycoconjugates file PDF Book only if you are registered here. And also you can download or read online all Book PDF file that related with Mass Spectrometry: Modified Proteins and Glycoconjugates book. Happy reading Mass Spectrometry: Modified Proteins and Glycoconjugates Bookeveryone. Download file Free Book PDF Mass Spectrometry: Modified Proteins and Glycoconjugates at Complete PDF Library. This Book have some digital formats such us :paperbook, ebook, kindle, epub, fb2 and another formats. Here is The CompletePDF Book Library. It's free to register here to get Book file PDF Mass Spectrometry: Modified Proteins and Glycoconjugates Pocket Guide.

Figures and Tables from this paper. Figures and Tables. Citations Publications citing this paper.

Index Entries

Glycoforms obtained by expression in Pichia pastoris improve cancer targeting potential of a recombinant antibody-enzyme fusion protein. Katalin F Medzihradszky , Daniel I. Chizhov , Yu. Tsvetkov , Nikolay E Nifantiev. Heck , Bernd Stahl. Azidofucose treated cells were fixed and labeled with probe 1a and then further treated with WGA lectin conjugated with Alexa Fluor , and the cells were imaged with confocal fluorescence microscope, using the appropriate filter sets.

As shown in FIG. The WGA lectin binds to sialic acid and N-acetylglucosaminyl residues of glycoproteins and is commonly used as a Golgi marker. These data further document the specific nature and sensitivity of the click-activated fluorogenic probe, allowing visualization of fucosylated glycoproteins intracellularly. The disclosed click-activated fluorogenic-labeling technique permits imaging of tagged fucosylated glycoconjugates at the cell surface and inside the cell.

Because of the high structural complexity of carbohydrates and the diversity of glycans, many functions of fucosylated glycoconjugates remain to be elucidated. Fluorescent labeling represents a way to address some of the questions concerning the structure, function, and trafficking of fucosylated glycans. The herein disclosed methods also facilitate a comparison of fucosylation in normal and tumor cells. The disclosed methods also allow monitoring of particular fucosylated glycoconjugates after inhibition of specific FucTs with small molecules or RNAi.

After washing with 0.


  • Download Mass Spectrometry Modified Proteins And Glycoconjugates.
  • Glycan - Wikipedia?
  • A Mass Spectrometry Primer for Mass Spectrometry Imaging;
  • Gender, Work and Space (International Studies of Women and Place).
  • NII catalog › Details for: Mass spectrometry: modified proteins and glycoconjugates.
  • Opening Japans Financial Markets: Shared Responsibilities;
  • Towards automation of glycomic profiling of complex biological materials.

For the biotinylation experiment, cells were stained with 0. Louis, Mo. Twenty thousand events were collected in each sample. The cells then were transferred to a coverslip glass slide and cultured overnight in the same medium. The cells were fixed by acetone and labeled as follows.

The Mass Spectrometric Analysis of Glycoproteins and their Glycan Structures

Fixed cells were incubated with 0. For counter staining of Golgi compartments, the fixed cells were stained by using Alexa Fluor conjugated WGA lectin Invitrogen , and each fluorescent dye was imaged by using Bio-Rad Carl Zeiss Radiance Rainbow laser scanning confocal microscopy system. All chemicals were purchased as reagent grade and used without further purification. Chemical shifts in ppm were determined relative to either tetramethylsilane 0 ppm or deuterated chloroform 77 ppm.

Mass spectra were obtained by the analytical services of this Department. Quantum yields were determined using Quinine sulfate as the fluorescent standard 0. Human alpha 1 -acid glycoprotein and alpha-fucosidase were purchased from Sigma and human alpha-1,3-fucosyltransferase V was from CalBiochem.

As shown in Scheme 2, to a solution of 4-bromo-N-ethyl-1,8-naphthalimide 15 mg, 0.

The mixture was stirred at room temperature overnight. The reaction mixture was diluted with water, and the precipitates were collected by filtration. A mixture of 15 mg, 3. The reaction mixture was diluted with water and extracted with AcOEt. The organic layer was washed with brine, dried over Na 2 SO 4 , and evaporated. To L-galactono-1,4-lactone 10 g, NaBH 4 2. The resin was removed by filtration, and the filtrate was evaporated. The residue was dissolved in acetone mL , CuSO 4 A suspension of PCC 1.

To this mixture was added a solution of 17 mg, 2. The filtrate was concentrated to give the crude aldehyde. To a suspension of tBuOK mg, 4. To this solution, a solution of the aldehyde in THF 5 mL was added, and the mixture was allowed to warm to room temperature and continued to stir overnight. The reaction mixture was quenched with mL of water, and the mixture was extracted with CH 2 Cl 2. The extracts were washed with brine, dried over with Na 2 SO 4 , and evaporated. The resin was filtered off, and the filtrate was evaporated. The overall volume in each vial was microliters containing a solution of 1,8-naphthalimide fluorophore 0.

The reactions were allowed to stand at room temperature for 24 hours. The reactions were monitored by LC-MS, which showed that the corresponding products were only produced in each reaction. Preparative scale reaction was performed in a similar manner, and the reaction mixture was purified by flash column chromatography on silica gel. The overall volume in each vial was microliters containing a solution of 1,8-naphthalimide 1a 0. The assay identified histidine, which is very good metal chelator, as an excellent catalyst for the Cu I -catalyzed 1,3-dipolar cycloaddition reaction. Histidine enhanced the reaction rate about 6-times compared to ligand-free conditions.

Among the other amino acids, glutamine, tyrosine and tryptophan were found to give approximately 2-fold enhancement of cycloaddition rates. Interestingly, cysteine, glutamic acid, and lysine, which are also known as good metal chelators, were found to be obstructive to the reaction. The catalytic activity was determined by the same method described above FIG. Stoichiometric study of histidine revealed that 2. To confirm the effects of L-His on the reaction, we measured the time-course of the reaction by fluorescence spectrometer.

Conditions: 1,8-naphthalimide 1a 0. The reaction mixture was diluted with water and concentrated in vacuo. The residue was dissolved in pyridine 5 mL and Ac 2 O 5 mL , and dimethylaminopyridine 10 mg was added to the solution. NaHCO 3 , and brine. The organic layer was dried over Na 2 SO 4 , and evaporated. To a cooled solution of this mixture mg, 0.

Glycan - Wikipedia

The CH 2 Cl 2 layer was dried over Na 2 SO 4 , and evaporated to give the alpha-bromide, which was used without further purification. To a suspension of tetrabutylammonium phosphate 0. After removal of the solvent, water 5 mL was added and molecular sieves were filtered off.

The filtrate was washed with AcOEt, and then concentrated.

Password Changed Successfully

The eluant was neutralized with Et 3 N and concentrated. The residual oil was applied to a Sephadex G column and eluted with triethylammonium bicarbonate buffer 50 mM, pH 8. Fractions containing the products were then purified by a SepPak C-8 cartridge with water. To this flask, 1H-tetrazole 60 mg, 0. The reaction mixture was diluted with water and extracted with CH 2 Cl 2 , and evaporated. Human alpha 1 -acid glycoprotein 0. The reaction mixture was passed through a NAP-5 column Amersham equilibrated in water to remove remaining GDP-fucose analogs, and the protein fractions were lyophilized.

The glycoproteins was then subjected to the cycloaddition labeling reaction. During the labeling reaction, the excess probe 1b seemed to decompose into a strongly fluorescent product which resulted in a slight background signal. Bicyclic aromatic azides are known to easily decompose by thermolysis or photolysis through a highly reactive nitrene intermediate FIG. See typical experimental procedure of cycloaddition for the reaction conditions.

In the presence of 6-alkyne fucose 2b, probe 1b was completely converted into the product 3b. The decomposed compound showed a green fluorescence with a maximum emission at nm when excited at nm, which was agreement with the reported data of 1c. For the biotinylation experiment, cells were subsequently stained with 0.

Then, the cells were transferred to a coverslip glass slide, and cultured overnight in the same medium. The cells were fixed by acetone and subjected to the appropriate labeling reaction. For labeling reaction, fixed cells were incubated with 0. While various embodiments of the present disclosure have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art.

However, it is to be expressly understood that such modifications and adaptations are within the spirit and scope of the present disclosure. All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Year of fee payment : 4. Incorporation of azido-containing fucose analog into glycoconjugates via the fucose salvage pathway are disclosed. Fluorescent visualization of fucosylated cells by flow cytometry of cells treated with 6-azidofucose labeled with click-activated fluorogenic probe or biotinylated alkyne is disclosed.

Visualization of intracellular location of fucosylated glycoconjugates by fluorescence microscopy are disclosed.

Introduction to Peptides and Proteins for Bioanalysis Using LC-MS

Synthesis of Fluorogenic Probes. Fluorescent Labeling of Fucosylated Glycoconjugate. A composition comprising: an azido-derivatized fucose analog having bonded thereto a probe for detecting glycoconjugates, wherein the fucose analog is a 6-azido fucose analog. The composition of claim 1 , wherein the probe emits a fluorescent signal.

The composition of claim 1 , wherein the probe is contacted by a secondary probe, wherein the secondary probe emits a detectable signal. The composition of claim 3 , wherein the secondary probe emits a fluorescent signal. The composition of claim 1 , wherein the probe comprises an alkynyl group.

The composition of claim 1 , wherein the probe comprises one of an N-alkyl-1,8-naphthalimide group, a biotin group, or a coumarin group. The composition of claim 1 , wherein the probe is 4-ethynyl-N-ethyl-1,8-naphthalimide. The composition of claim 1 , wherein the azido-derivatized fucose analog comprises a fucosylated glycoconjugate. The composition of claim 9 , wherein the fucosylated glycoconjugate is a fluorescent labeled glycoconjugate.

USP true USB2 en. Sulfonic acid derivative compounds as photoacid generators in resist applications. WOA2 en.

Introduction

Apparatus and method of reaching polymers by exposure to uv radiation to produce injectable medical devices. CAA1 en. WOA1 en. CAC en. EPA2 en. AUA1 en.


  • Mass Spectrometry: Modified Proteins and Glycoconjugates: Volume 405 (Methods in Enzymology).
  • Metaphysics: An Introduction;
  • Glycosylation | Thermo Fisher Scientific - UK.
  • Universal Serial Bus System Architecture?
  • Descriptive Topology in Selected Topics of Functional Analysis.
  • Beyond the Stream: Islam and Society in a West African Town.
  • The Basics;

Apparatus and method of reacting polymers passing through metal ion chelated resin matrix to produce injectable medical devices. EPB1 en. This is a preview of subscription content, log in to check access. West, C. PubMed Google Scholar. Galili, U. Google Scholar. Thall, A. Biochemistry 29 , — Lipniunas, P. USA 84 , — Kaushal, G. Plants 14 , — Kaladas, P. Ashwell, G. Sharon, N. Science , — McFarlane, I. Hansen, L. Wittwer, A. Tsuda, E. Takeuchi, M. Angel, A. Trends Glycosci. Gavel, Y. Protein Eng. Miletich, J. Tollefsen, S. Parsons, T. USA 77 , — Chan, A. Glycobiology 1 , — CrossRef Google Scholar.

Yan, S. Glycobiology 3 , — Yoshima, H. Harada, H. Ballou, C. Methods Enzymol. Sheares, B. USA 83 , —