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Device for preparing proteomics micro-sample

A sample preparation and trace technology, applied in the field of proteomics, to achieve the effect of superior performance, important application value and simple production

Pending Publication Date: 2020-08-11
ACADEMY OF MILITARY MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In reversed-phase chromatography, the solute is distributed on the surface of the stationary phase through hydrophobic interactions. However, since the surface of the stationary phase is completely covered by non-polar groups, it exhibits strong hydrophobicity.

Method used

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  • Device for preparing proteomics micro-sample
  • Device for preparing proteomics micro-sample
  • Device for preparing proteomics micro-sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1. Fabrication and activation of proteomics micro-sample preparation device

[0050] (1) Production of C18-Tip: Add a layer of C8 membrane as a sieve to the 200μl pipette tip, introduce 1mg of C18 filler above the sieve, and finally add a layer of sieve above the C18 filler.

[0051] (2) The above C18-Tip activation: First, add 100μl of pure acetonitrile (ACN, Merck) to C18-Tip, centrifuge at 1500g for 30s to remove the liquid; secondly, add 100μl of 50% ACN+0.1% TFA (trifluoro Acetic acid, Sigma) solution was centrifuged at 1500g for 60s to remove the liquid; finally, 100μl of 0.1% TFA solution was added to the C18-Tip and centrifuged at 1500g for 90s to remove the liquid.

[0052] (3) Production of HILIC-Tip: Add a layer of C8 membrane as a sieve plate to a 200μl pipette tip, and introduce 0.5 mg of HILIC filler above the sieve plate to make an enrichment column.

[0053] (4) The above HILIC-Tip activation: first, add 100μl 1% TFA solution to HILIC-Tip, centrifuge at ...

Embodiment 2

[0056] Example 2. Whole protein digestion experiment of human liver cancer HepG2 cell line

[0057] The whole protein of human liver cancer HepG2 cell line (purchased from American Model Culture Collection) was extracted with 8M urea (UA, Sigma) solution, and the protein concentration was measured by Bradford method.

[0058] Take 10μg of protein and place it in C18-Tip, add tris(2-carboxyethyl)phosphine (TCEP, Sigma), the final concentration is 10mM, add chloroacetamide (CAA, Sigma), the final concentration is 15mM, pipetting and mixing, The reduction reaction and the alkylation reaction were carried out simultaneously for 1 h at 37°C. Both TCEP and CAA were dissolved with 8M UA.

[0059] Add intracellular protease (Lys-C, Spec. Act:14.6Unit / (min*mg), 250mM*2775μl Ac-Lys-pNA and 20μg / in the enzyme / protein ratio of 1:100(w / w) into the solution. mL*225μl protease, incubate at 30°C for 30min, and measure absorbance at 405nm (mOD / min) over time. The maximum slope of the curve is used...

Embodiment 3

[0069] Example 3. Human immunoglobulin G (IgG) sugar cutting and enrichment experiments

[0070] 1) Human immunoglobulin G (IgG) dissolved in 50mM NH 4 HCO 3 in.

[0071] 2) Take 10μg of IgG in C18-Tip, add tris(2-carboxyethyl)phosphine (TCEP), the final concentration is 10mM, add chloroacetamide (CAA), the final concentration is 15mM, pipetting and mixing, at 37℃ At the same time, the reduction reaction and the alkylation reaction were carried out for 1 h.

[0072] 3) Trypsin was added to the solution at an enzyme / protein ratio of 1:100 (w / w), and incubated at 37°C for 16 hours to digest IgG.

[0073] 4) Add peptide-N-glycosidase F (PNGase F, Activity:>) to the solution at an enzyme / protein ratio of 1:20 (w / w) 1000u / mg, the unit of enzyme activity per milligram of protein. ), incubate at 37°C for 2h to release N-sugar chains.

[0074] 5) Add TFA to make the TFA concentration in the digestion solution 0.1%, centrifuge at 1500g for 3 minutes, and collect N-sugar chains.

[0075] 6) Add ...

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Abstract

The invention discloses a device for preparing a proteomics trace sample. The device is easy to manufacture and low in cost, and the peptide fragment recovery rate and the protein identification amount are superior to those of a conventional method under the condition that the initial protein amount is low. The device realizes seamless combination of one-stop in-situ protein digestion, peptide fragment acquisition, and N-sugar chain release and enrichment. The loss of the sample is reduced to the greatest extent, and the whole-process sample preparation of the micro-sample proteomics is realized.

Description

Technical field [0001] The invention belongs to the field of proteomics, and relates to a device for preparing proteomics trace samples. Background technique [0002] Reversed-phase chromatography is an elution chromatography that uses a non-polar reversed-phase medium as the stationary phase and an aqueous solution of a polar organic solvent as the mobile phase, and separates and purifies the solute according to the difference in solute polarity (hydrophobicity). In reversed-phase chromatography, solutes are distributed on the surface of the stationary phase through hydrophobic interactions. However, since the surface of the stationary phase is completely covered by non-polar groups, it exhibits strong hydrophobicity. Therefore, a polar organic solvent (such as methanol, acetonitrile, etc.) or its aqueous solution must be used for elution and separation of the solute. Summary of the invention [0003] The purpose of the present invention is to provide a device for preparing prot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/34G01N30/89G01N27/62G01N27/64
CPCG01N1/34G01N30/89G01N27/62G01N27/64
Inventor 应万涛钱小红孟波李晓宇
Owner ACADEMY OF MILITARY MEDICAL SCI
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