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Manufacture method and application of novel incomplete protein micro catcher

A protein and trap technology, applied in the field of analytical chemistry, to achieve good practical value and application prospects

Inactive Publication Date: 2011-04-27
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since a large amount of silanol groups are blocked, it is difficult for the filler to react with the hydrolyzed precursor to form a three-dimensional network structure during the sol-gel reaction. The resulting micro-trap has poor mechanical strength and the filler is easily washed away.

Method used

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  • Manufacture method and application of novel incomplete protein micro catcher
  • Manufacture method and application of novel incomplete protein micro catcher
  • Manufacture method and application of novel incomplete protein micro catcher

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Preparation of intact protein microtrap

[0029] The preparation of intact protein microtrap is divided into three steps.

[0030] (1) Commercial chromatographic packings are end-capped to eliminate the presence of silanol groups so as not to cause chromatographic peak tailing of basic compounds during separation. Since a large number of silanol groups are blocked, it is difficult to react with the hydrolyzed precursor to form a three-dimensional network structure during the sol-gel reaction. The mechanical strength of the obtained micro-trap is very poor, and the filler is easily washed away. Therefore, prior to preparation, the commercially available chromatographic fillers should be activated. Soak C4, C8 and C18 reversed-phase chromatographic fillers in 0.001 mole per liter of sodium hydroxide solution, and ultrasonically treat them for 5 minutes to activate the surface-capped fillers and increase the number of exposed silanol groups on the surface of ...

Embodiment 2

[0034] Embodiment 2: The micro-trap device obtained in embodiment 1 is used for the device of on-line trap-capillary reversed-phase liquid chromatography system

[0035] The whole system includes a sample pump 9, a gradient pump 12, a multi-wavelength UV detector 11 with an 80 nanoliter flow cell, and a sampling valve 7 with a 2 μL or 20 μL sample loop (six-port manual sampling valve ), a switching valve 8 (six-port switching valve), an intact protein micro-trap 1, and a capillary reversed-phase chromatography column 10 (Zorbax, SB-C18, 5 μm, 150×0.3 mm, Agilent).

[0036] The micro-trap 1 and the PEEK tube 5 holding it are directly connected to the sixth position on the switching valve 8 . A 15 cm × 65 μm i.d. PEEK tube is connected between the injection valve 7 and the switching valve 8. The analytical column 10 is connected to the fifth position on the switching valve 8 , and the outlet of the analytical column 10 is connected to the ultraviolet detector 11 .

[0037] U...

Embodiment 3

[0039] Embodiment 3: The complete protein micro-trap device obtained in Example 1 is used for the trapping of standard protein

[0040] (1) Protein solution preparation:

[0041] Ribonuclease B, cytochrome c, horse myoglobin and chicken ovalbumin were dissolved in pure water to prepare 10 μg / μL stock solutions.

[0042] (2) Capture of standard protein:

[0043] The effect of the trap on intact protein was studied by injecting 0.5 μg of a mixture of four standard proteins (1 ribonuclease B, 2 cytochrome c, 3 horse myoglobin and 4 chicken ovalbumin) onto the intact protein microtrap capture. After being captured by the trap, the protein was backflushed to a capillary reversed-phase analytical column for elution and separation, and detected by a multi-wavelength ultraviolet detector with a measuring wavelength of 215 nm. The recoveries of the four proteins were all above 90%.

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Abstract

The present invention belongs to the technical field of chemical technology, specifically relates to a manufacture method and application of novel incomplete protein micro catcher. The method of the present invention uses a sodium hydroxide solution to process a commercial opposite phase chromatographic filler; the processed filler is suspended in a sol solution regarding methyl triethoxysilane as a precursor; the suspending liquid is led to a proper length in a capillary by a pressure device; an incomplete protein micro catcher in a three-dimensional mesh space structure is manufactured by the hydrolysis and polycondensation of the precursor. The micro catcher is used for gathering, concentrating and desalting incomplete protein samples in a liquid chromatogram, wherein the recovery rate of protein is high, the gathering volume of the catcher is big, plungers are not needed, and the manufacture method is simple and effective. The micro catcher has good practical value and application perspective in fields like proteomics and the like.

Description

technical field [0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to the preparation and application of a novel intact protein micro-trap. [0002] The intact protein micro-trap prepared by the invention can be used as a switch between the two dimensions of the intact protein in the two-dimensional on-line liquid chromatography, and successfully captures, concentrates and desalts the protein eluted by the one-dimensional chromatography. The micro-trap has good practical value and application prospect in proteomics and other fields. Background technique [0003] There are usually two main research methods in proteomics research to analyze complex proteins, namely "bottom-up" and "top-down". "Bottom-up" refers to the "shotgun" proteomics research method, which plays an important role in identifying unknown proteins on a large scale. However, "bottom-up" research methods provide very limited molecular information on intact ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/16
Inventor 张祥民关霞
Owner FUDAN UNIV
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