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Labeling and detection of post translationally modified proteins

a technology of translationally modified proteins and labeling methods, applied in the field of labeling post translationally modified biomolecules, can solve the problems of inability to apply in-gel fluorescence detection of modified proteins, and the method is devoid of the typical problems encountered in antibody detection on blots

Inactive Publication Date: 2007-10-25
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the reaction has been applied to a variety of different bioconjugation reactions over the past several years, it has never been applied to the in-gel fluorescence detection of modified proteins.
The method is devoid of the typical problems encountered with antibody detection on blots including the requirement to optimize primary and secondary antibody concentrations and issues of non-specific binding of the antibodies.

Method used

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  • Labeling and detection of post translationally modified proteins
  • Labeling and detection of post translationally modified proteins
  • Labeling and detection of post translationally modified proteins

Examples

Experimental program
Comparison scheme
Effect test

example 1

Synthesis of UDPGalNAz

[0269] The synthesis of UDPGalNAz is shown in the following reaction scheme.

[0270] Azidoacetic acid. To a solution of iodoacetic acid (8.0 g, 43.0 mmol) and H2O (100 mL) was added sodium azide (5.62 g, 86.0 mmol). The solution was stirred at RT and protected from light. After 4 days, the solution was diluted with 1 N HCl (30 mL) and the pH was check to ensure that it was in the range of 2-3. The solution was extracted with EtOAc (2×100 mL), then the combined organics were washed with saturated NaHSO3 (1×50 mL), brine (1×50 mL) and dried over MgSO4. The solution was decanted, concentrated and the crude azidoacetic acid (3.34 g, 53%) was used directly in the next step without further purification.

[0271] N-Azidoacetylgalactosamine (mix of anomers). To a solution of azidoacetic acid (3.34 g, 33.06 mmol) in methanol (170 mL) was added D-galactosamine hydrochloride (5.09 g, 23.62 mmol) followed by triethylamine (7.90 mL, 56.68 mmol). This solution was stirred at ...

example 2

Synthesis of UMP-N-methylimidazolide

[0276] The synthesis of UMP-N-methylimidazolide is shown in the following reaction scheme.

[0277] Uridine 5′-monophosphate triethylammonium salt (5′-UMP triethylammonium salt). Uridine 5′-monophosphate disodium salt (1.0 g) was dissolved in H2O (2 ml) and passed through Dowex resin triethylammonium salt (2.7 cm×7.0 cm column size) with H2O as eluent. All of the eluent was concentrated and after the UV active material ceased eluting from the column. The solution was concentrated and co-evaporated with toluene to until a white, crystalline solid was obtained (0.78 g, 80%).

[0278] Uridine monophosphate-N-methylimidazole (UMP-N-methylimidazole). To a suspension of uridine 5′-monophosphate triethylammonium salt (0.097 g, 0.22 mmol) in CH3CN (0.70 mL) was added N,N-dimethylaniline (0.11 mL, 0.864 mmol), and triethylamine (0.03 mL, 0.22 mmol). The suspension was cooled to 0° C. In a separate flask, trifluoroacetic anhydride (0.15 mL, 1.08 mmol) was add...

example 3

Synthesis of Dapoxyl® alkyne

[0279] The synthesis of Dapoxyl® alkyne is shown in the following reaction scheme.

[0280] To a solution of Dapoxyl® carboxylic acid, succinimidyl ester (50 mg, 0.12 mmol) in DMF (0.4 mL) at RT was added propargylamine (42 μL, 0.61 mmol). The initial clear orange solution turned yellow and cloudy. After ˜15 min at RT the reaction was complete, and the solution was concentrated to dryness. The residue was purified via HPLC to afford the product (36 mg, 84%). TLC (10% EtOAc, CHCl3) Rf=0.30; ESI m / z 346 (M+, C21H19N3O2 requires 346).

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Abstract

Provided in certain embodiments are new methods for forming azido modified biomolecule conjugates of reporter molecules, carrier molecules or solid support. In other embodiments are provided methods for enzymatically labeling a biomolecules with an azide group.

Description

CROSS REFERENCE TO RELATED APPLICATION(S) [0001] This application claims priority to U.S. Provisional Application No. 60 / 772,221, filed Feb. 10, 2006 and U.S. Provisional Application No. 60 / 804,640, filed Jun. 13, 2006, the contents of which are incorporated by reference as if set forth fully herein.FIELD OF THE INVENTION [0002] The invention generally relates to methods of labeling post translationally modified biomolecules using metabolic, enzymatic, or chemical incorporation of azide or alkyne-labeled macromolecules followed by chemical conjugation with paired azide, alkyne, activated alkyne, or triarylphospine reporter molecules appended to proteins post translation. BACKGROUND INFORMATION [0003] Protein glycosylation is one of the most abundant post-translational modifications and plays a fundamental role in the control of biological systems. For example, carbohydrate modifications are important for host-pathogen interactions, inflammation, development, and malignancy (Varki, A...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P21/00C07C247/00C07K14/435G01N33/68C12N9/10
CPCA61K47/48092C07K2317/41C12P21/005G01N33/531G01N33/532G01N33/5005Y10T436/17G01N33/533G01N33/534C07K16/00G01N33/583G01N33/581G01N33/582G01N33/5008A61K47/549C07K1/13C12Y302/01096C07K14/47C07K2317/14C07K2317/24
Inventor AGNEW, BRIANGEE, KYLE R.NYBERG, TAMARA G.
Owner LIFE TECH CORP
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