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Stable isotope-based high-throughput proteomic quantitative reagent

A stable isotope and isotope technology, applied in measurement devices, scientific instruments, instruments, etc., can solve the problems of high price, laboratory economic burden and application barriers, and achieve the effect of improving yield, low cost and reducing cost

Inactive Publication Date: 2020-04-17
南京谱利健生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing commercial isobaric labeling reagent products on the market are expensive, especially for a large number of samples that need to be labeled, which has become an economic burden and the biggest application obstacle for many laboratories

Method used

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  • Stable isotope-based high-throughput proteomic quantitative reagent
  • Stable isotope-based high-throughput proteomic quantitative reagent
  • Stable isotope-based high-throughput proteomic quantitative reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1: Synthesis of T-114 in IBT-10PLEX (structure A), the synthetic route is as follows:

[0077]

[0078] Synthesis of T-114 in IBT-10PLEX (structure A), the asterisks in the synthetic route represent 13C or 15N at the corresponding positions. This route can be used for the synthesis of T-114, T-115N, T-115C, T-116N, T-116C, T-117N, T-118N in IBT-10PLEX (structure A).

[0079] Compound 1 (β-Ala-OBzl.HCl, 13C3, 15N, 215 mg, 1 mmol) and compound 2 (Boc-Gly-OH, 1-13C, 175 mg, 1 mmol) were dissolved in 20 mL of dichloromethane, and EDC.HCl ( 229 mg, 1.2 mmol), the reaction was carried out under the protection of argon for 20 hours, the reaction solution was washed three times with 30 mL of 25 mM dilute hydrochloric acid, and the organic phase was dried and removed by a rotary evaporator to obtain 283 mg of oily compound 3 (84% yield). Compound 3 (283 mg) was added to 50 mL of HCl in EtOAc (1 M) and stirred for 2 hours, the resulting white precipitate was collec...

Embodiment 2

[0080] Embodiment 2: Synthesis of T-119 in IBT-10PLEX (structure A), the synthetic route is as follows:

[0081]

[0082] Synthesis of T-119 in IBT-10PLEX (structure A), the asterisks in the synthetic route represent 13C or 15N at the corresponding positions. This route can be used for the synthesis of T-117C, T-118C, T-119 in IBT-10PLEX (structure A).

[0083] Compound 8 (Nosyl-Gly-OH, 15N, 2-13C, 524mg, 2mmol) was dissolved in 10mL DMF, solid sodium carbonate (636mg, 6mmol) and iodopropane (590μL, 1.02g, 6mmol) were added, stirred for 1 hour, The white solid was filtered, and most of the DMF was removed by rotary evaporation, 30 mL of dichloromethane was added, washed 3 times with 20 mM aqueous sodium bicarbonate solution and 50 mL of saturated brine, respectively, and the dichloromethane was removed to obtain 654 mg of white compound 9 (95% yield). Compound 9 (654 mg) was dissolved in 20 mL of KOH / MeOH saturated solution, 400 μL of 2-mercaptoethanol was added, and the r...

Embodiment 3

[0085] Embodiment 3: Synthesis of T-114 in IBT-10PLEX (structure B), the synthetic route is as follows

[0086]

[0087] Synthesis of T-114 in IBT-10PLEX (structure B), the asterisks in the synthetic route represent 13C or 15N at the corresponding positions. This route can be used for the synthesis of all 10 reagents in IBT-10PLEX (structure B).

[0088] Compound 1 (β-Ala-OBzl.HCl, 13C3, 15N, 172mg, 0.8mmol) and compound 2 (Boc-Gly-OH, 1-13C, 140mg, 0.8mmol) were dissolved in 20mL of dichloromethane, and EDC was added. HCl (183mg, 0.96mmol), the reaction was carried out under the protection of argon for 20 hours, the reaction solution was washed three times with 30mL of 25mM dilute hydrochloric acid, and the organic phase was dried and removed with a rotary evaporator to obtain 242mg of oily compound 3 (90% yield) . Compound 3 (242 mg) was added to 50 mL of HCl in EtOAc (1 M) and stirred for 2 hours, the resulting white precipitate was collected by filtration to give 160 ...

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Abstract

The invention discloses a stable isotope-based high-throughput proteomic quantitative reagent, which can also be called an isobaric Labeling (IBT) reagent, and the structure of the stable isotope-based high-throughput proteomic quantitative reagent comprises the formula of reporter group-balance group-activation group. Such reagent can be accomplished by different types of chemical structures. Theinvention discloses eight groups of different chemical structures, and each group of structures can comprise up to 10 label reagents (represented by IBT-10PLEX) with completely identical chemical structures. However, 13C or 15N isotopes (which can be represented by T-114, T-115N, T-115C, T-116N, T-116C, T-117N, T-118N, T-117C, T-118C and T-119 respectively) are contained at different positions ineach label reagent. The invention discloses a preparation method of three groups of isobaric tag reagents, and further discloses a quantitative analysis method of polypeptide by adopting the IBT-10PLEX reagent. The IBT-10PLEX disclosed by the invention adopts a novel chemical structure, the preparation difficulty is reduced, and the yield is improved, so that the product cost can be reduced, andthe application range of an isobaric labeling method is expanded.

Description

technical field [0001] The present invention relates to the field of biomolecular analysis reagents, especially for the quantitative analysis of biomolecules, specifically the preparation of a class of isobaric labeling reagents (IBT-10PLEX) that can be applied to high-throughput proteomics quantification of 10 samples and apply. Background technique [0002] At present, the quantitative methods of proteomics based on mass spectrometry detection mainly include label-free analysis and labeling methods, and labeling methods are further divided into quantitative methods based on primary mass spectrometry (MS1) and secondary mass spectrometry (MS2). Quantitative isotope labeling belongs to the analysis method based on MS2. [0003] Compared with label-free analysis and MS1 labeling, the advantages of MS2 labeling are: [0004] 1) The analysis time of the overall sample can be greatly reduced. [0005] 2) Since different samples with isobaric labels are pre-mixed before liquid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/72
CPCG01N30/06G01N30/72
Inventor 李舒伟
Owner 南京谱利健生物技术有限公司
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