Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Preparation of organic integral small column, organic integral small column and application of organic integral small column

A holistic and organic technology, applied in the direction of solid adsorbent liquid separation, chemical instruments and methods, measuring devices, etc., can solve the problem of high price of ZipTip cartridges, lack of application of centrifugal enrichment cartridges, lack of chemical stability of polydopamine, etc. problem, to achieve the effect of enrichment ability, short time consumption and rapid analysis

Inactive Publication Date: 2017-10-27
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
View PDF5 Cites 9 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation of organic integral small column, organic integral small column and application of organic integral small column
  • Preparation of organic integral small column, organic integral small column and application of organic integral small column
  • Preparation of organic integral small column, organic integral small column and application of organic integral small column

Examples

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 BSA solution and BSA enzymolysis solution reached 1000:1, the interference signal of non-phosphorylated peptides was still not obvious, which proved the high enrichment specificity of Ti(IV)-IMAC organic monolithic column.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Apertureaaaaaaaaaa
Mass volume fractionaaaaaaaaaa
Login to View More

Abstract

The invention relates to preparation of a Ti(IV)-IMAC organic integral small column. The Ti(IV)-IMAC organic integral small column is applied to phosphorylated proteomic analysis of trace biological samples. The organic integral column material is a polymethacrylate high polymer, contains a rich pore structure and is large in specific surface area and large in quantity of titanium ion enriching active sites; and meanwhile, due to a hydrophilic material surface, the organic integral column shows powerful enriching capability in the aspect of phosphopeptide enrichment. By applying the organic integral small column to the phosphorylation proteomic analysis of 5[mu]g HeLa cells, 1,000 or more of non-redundant phosphorylation sites are identified on an average, and the enriching specificity reaches up to 92.5%. More importantly, the Ti(IV)-IMAC organic integral small column is simple in preparation, and compared with packed small columns, the Ti(IV)-IMAC organic integral small column can be prepared through a photo-induced free-radical polymerization reaction by a few minutes only; and preparation of plugs is not required, so that the preparation time for an enriching 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com