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Protein quantitative labeling reagent and preparation method and application thereof

A technology for labeling reagents and proteins, applied in the field of proteomics research, can solve problems such as easy hydrolysis, reduced ionization efficiency of mass spectrometry, and low labeling efficiency

Active Publication Date: 2020-04-03
海湃泰克(北京)生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are also problems with the top-down strategy: the protein of interest needs to be separated in advance, which makes it difficult to analyze the proteome with high throughput; some intact proteins have poor water solubility, which makes it difficult to further identify them using mass spectrometry; mass spectrometry ionization Efficiency decreases with increasing molecular weight, limiting identification of larger protein complexes
And once it is made into a stock solution, it needs to be used within a week, otherwise the quantitative accuracy will be greatly reduced
Therefore, it is urgent to develop a new generation of stable, efficient, and economical isobaric labeling reagents to overcome the disadvantages of easy hydrolysis, low labeling efficiency, and low amino selectivity of commercial iTRAQ and TMT reagents.

Method used

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  • Protein quantitative labeling reagent and preparation method and application thereof
  • Protein quantitative labeling reagent and preparation method and application thereof
  • Protein quantitative labeling reagent and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0059] The preparation of embodiment 1 formula 2 compound

[0060] In this embodiment, step 1 of the above-mentioned method is adopted, and the reduction reaction is carried out with 4-nitrophthalate dimethyl ester as a raw material to obtain the following product:

[0061]

[0062] The characterization information is as follows:

[0063] Pale yellow solid; 3.6g, yield 82%;

[0064] 1H NMR (400MHz, Methanol-d4) δ7.65(d, J=8.4Hz, 1H), 6.71(d, J=2.8Hz, 1H), 3.85(s, 3H), 3.78(s, 3H).

[0065] 13C NMR (101MHz, Methanol-d4) δ170.80, 167.15, 152.31, 136.68, 131.67, 115.55, 114.17, 112.35, 51.99, 51.30.

[0066] On the basis of the obtained compound, through the step 2 reaction, the following product is obtained:

[0067]

[0068] The characterization information is as follows:

[0069] Pale yellow solid; 1.3g, yield 81%;

[0070] 1H NMR (400MHz, Methanol-d4) δ7.08 (d, J=8.0Hz, 1H), 6.79 (dd, J=6.6, 2.5Hz, 1H), 6.62 (dd, J=8.1, 2.5Hz, 1H) ,4.62(s,2H),4.55(s,2H).

[0071]...

Embodiment 2

[0089] The preparation of embodiment 2 formula 3 compounds

[0090] In this embodiment, step 3 of the above method is adopted to carry out 18 oxygen exchange reaction with leucine as a raw material to obtain the following products:

[0091]

[0092] On the basis of the obtained compound, through the step 4 reaction, the following product is obtained:

[0093]

[0094] On the basis of the obtained compound, through the step 5 reaction, the following product is obtained:

[0095]

[0096] The characterization information is as follows:

[0097] Yellow liquid; 100mg, yield 49.7%.

[0098] On the basis of the obtained compound, react through step 6 to obtain the compound of formula 3:

[0099]

[0100] The characterization information is as follows:

[0101] Yellow liquid; 17.2mg, yield 20%.

[0102] 1H NMR (400MHz, Acetone-d6) δ11.50(s, 1H), 10.60(s, 1H), 10.45(s, 1H), 8.42(d, J=2.1Hz, 1H), 8.15(dt, J= 8.4,1.6Hz,1H),8.08(d,J=8.2Hz,1H),4.41(d,J=10.5Hz,1H),3.07(s,...

Embodiment 5

[0138] Application of Example 5 Protein Quantitative Labeling Reagent in BSA Protein Relative Quantitative Detection

[0139] Utilize the quantitative labeling reagent shown in formula 9 and formula 11 to carry out quantitative accuracy test on BSA protein, add the 100mM TEAB buffer solution of 100ul to the 2ug / ul BSA protein stock solution of 100ul, then add 2ul 1M tris(2 -Carboxyethyl)phosphine (TCEP) solution, react in a metal bath at 65°C for 1 hour, add 7.5ul of 500mM IAA solution after returning to room temperature, and react with shaking at room temperature for 30 minutes. Then add 1ml of acetone, place it at -20°C for 4 hours, then centrifuge at 8000g, 4°C for 10 minutes, suck out the supernatant, redissolve with 1ml 100mM TEAB buffer, add 20ul 0.5ug / ul trypsin solution, and keep at 37°C The reaction was carried out for 16 hours.

[0140] The concentration of peptides was determined by Pierce Quantitative Colorimetric Peptide Assay of Thermo scientific, and the concen...

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Abstract

The invention provides a protein quantitative labeling reagent as well as a preparation method and application thereof. According to the protein quantitative labeling reagent provided by the invention, the high reaction activity of a o-phthalaldehyde group and a derivative group thereof and amino is utilized; specific recognition on lysine can be realized; the characteristics of high reaction activity and no hydrolysis of lysine are utilized; the labeling efficiency can be effectively improved, the relative abundance of reporter ions is compared so as to apply the reporter ions to the quantitative analysis of a plurality of lysine-containing proteomes, and the protein quantitative labeling reagent is stable in aqueous solution, can be stored for a long time, and is a new generation of thestable, efficient and economic equivalent ex-situ labeling reagent.

Description

technical field [0001] The invention relates to the field of proteomics research, in particular to a protein quantitative labeling reagent and its preparation method and application. Background technique [0002] With the completion of the Human Genome Project, the human genome sequence has been fully determined. However, due to the complex way of gene expression, the transcription of the same gene is different in different tissues and environmental conditions, and a gene can be spliced ​​in multiple forms of mRNA. And although genes guide protein expression, there are complex post-translational modifications, subcellular localization and migration, protein-protein interactions and other phenomena in proteins, which cannot be explained from the gene level, and these dynamic changes of proteins are directly related to physiological or associated with pathological phenomena. Therefore, it is difficult to explain the mechanism of physiological or pathological changes only thr...

Claims

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Application Information

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IPC IPC(8): C07C237/04C07C231/12C07B59/00G01N30/72G01N30/88G01N30/90G01N33/68
CPCC07B59/001C07B2200/05C07C237/04G01N30/72G01N30/88G01N30/90G01N33/6848G01N2030/8831
Inventor 雷晓光肖凡唐毓良
Owner 海湃泰克(北京)生物医药科技有限公司
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