Preparation of a kind of organic monolithic small column and organic monolithic small column and application

A holistic and organic technology, applied in solid adsorbent liquid separation, chemical instruments and methods, separation methods, etc., can solve the problem that centrifugal enrichment cartridges are not used, ZipTip cartridges are expensive, and polydopamine lacks chemical stability, etc. problem, to achieve the effect of enrichment ability, short time-consuming and rapid analysis

Inactive Publication Date: 2019-04-23
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is reported in the literature that polydopamine lacks sufficient chemical stability. At the same time, the commercialized Zip Tip column is expensive, which increases the cost of using the column.
Therefore, to date, spin-enrichment cartridges based on monolithic column materials have not been applied in phosphoproteomic analysis of complex biological samples.

Method used

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  • Preparation of a kind of organic monolithic small column and organic monolithic small column and application
  • Preparation of a kind of organic monolithic small column and organic monolithic small column and application
  • Preparation of a kind of organic monolithic small column and organic monolithic small column and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Preparation process of Ti(IV)-IMAC organic monolithic column

[0038] Such as figure 1 As indicated, a 0.10 g / mL benzophenone solution was prepared using a methanol / 1,4-butanediol solution with a volume ratio of 1:1. Take 10 μL of benzophenone solution and add it to 20 μL GE Loader tip (Eppendorf, Germany), place it in a UV generator (XL-1500A, Spectronics, New York, USA), and irradiate the reaction under 365nm wavelength ultraviolet light for 15 minutes After the reaction is over, centrifuge to remove the benzophenone solution in the GE Loader tip; repeat the above operation three times to fully activate the polypropylene column. The composition of the prepared polymerization solution is as follows: 13.4% EGMP (ethylene glycol methacrylate), 0.10g / mL Bis (methylene bisacrylamide), 45.0% DMSO (dimethyl sulfoxide), 8.3% DMF (dimethylformamide), 33.3% dodecanol; 3 μL of polymer solution was ultrasonically degassed for 10 minutes to exclude oxygen, then added to the abov...

Embodiment 2

[0043] The operation process is the same as the specific process of the Ti(IV)-IMAC organic monolithic column used for enrichment analysis above, and the prepared Ti(IV)-IMAC organic monolithic column is used for the enrichment analysis of the phosphorylated protein β-casein. The trypsin hydrolyzate of 100 fmol β-casein was enriched for phosphorylated peptides. After the non-specific adsorption on the material was completely removed, the phosphorylated peptides were eluted with 10% ammonia solution, and 0.5 μL of it was eluted. The liquid was spotted on the MALDI target chip for MALDI-TOF MS analysis. As a control experiment, MALDI-TOF MS analysis was performed in parallel on the trypsin hydrolyzate of 100 fmol β-casein without the phosphopeptide enrichment step.

[0044] Such as image 3 As shown in .A, in the mass spectrum of the sample without the enrichment step, the high-intensity non-phosphopeptide peak occupies the entire spectrum, and it is impossible to identify the ...

Embodiment 3

[0046] The operation process is the same as the above Ti(IV)-IMAC organic monolithic column used for the specific process of phosphopeptide enrichment analysis. The β-casein hydrolyzate and BSA enzymolyzate are mixed in different molar ratios (500:1 and 1000:1, mol / mol) mixture to simulate complex samples to further evaluate the specificity of Ti(IV)-IMAC organic monolithic cartridges.

[0047] Such as Figure 4 As shown, under the condition of a large excess of non-phosphorylated peptides (BSA hydrolyzate), only clear mass spectrum peaks of phosphopeptides and their dephosphorylated counterparts appeared in the MALDI spectrum, even when β-casein was digested When the molar ratio of the solution and the BSA enzymolysis solution reached 1000:1, the interference signal of the non-phosphorylated peptide was still not obvious, which proved the high enrichment specificity of the Ti(IV)-IMAC organic monolithic column.

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Abstract

The invention relates to the preparation of a Ti(IV)-IMAC organic monolithic small column, which is used for phosphorylated proteomics analysis of trace biological samples. The organic monolithic column material is a polymethacrylate polymer with rich pore structure, high specific surface area, and many active sites for enriching titanium ions; at the same time, due to its hydrophilic material surface, Phosphopeptide enrichment showed a strong enrichment ability. It was used in the phosphoproteomics analysis of 5g HeLa cells, and more than 1000 non-redundant phosphorylation sites were identified on average, and the enrichment specificity was as high as 92.5%. More importantly, the preparation of the Ti(IV)-IMAC organic monolithic column is simple. Compared with the packed column, it can be prepared by light-induced free radical polymerization in only a few minutes; and there is no need to prepare a stopper, The preparation time of the enrichment material is reduced to a large extent.

Description

technical field [0001] The invention belongs to the technical field of phosphoproteomics in the direction of proteomics research, and specifically relates to a sample processing method applied to phosphoproteomics. Background technique [0002] As an important protein post-translational modification, protein phosphorylation plays a crucial role in the regulation of cellular metabolism. Abnormal phosphorylation modifications are closely related to many disease processes, such as malignant tumors. At present, the proteomic analysis strategy based on the shotgun method is the main method in the study of phosphoproteomics. In this method, protein samples are first digested into peptides, enriched by phosphopeptides, and analyzed by LC-MS / MS. Due to the high dynamic range of phosphorylation modification, the low abundance of phosphorylated proteins, and the poor ionization effect of phosphorylated peptides, the key step that affects the accuracy and sensitivity of the entire ph...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01D15/10B01D15/16B01D15/26G01N30/06G01N30/08G01N30/60
CPCB01D15/10B01D15/165B01D15/265G01N30/06G01N30/08G01N30/60G01N30/6052G01N2030/065
Inventor 邹汉法刘芳洁姚亚婷叶明亮
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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