Integrated proteomics sample pretreatment platform and its application based on scx/sax mixed filler

A sample pretreatment and proteomics technology, which is applied in scientific instruments, material separation, and analytical materials, etc., can solve problems such as the large influence of pH on the effect of enrichment, limitations of proteomics methods, and limited effect of protein enrichment. Achieve the effect of increasing sample capacity, improving reproducibility and reducing loss

Active Publication Date: 2021-02-09
深圳深维超生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the limited protein enrichment effect of SAX or SCX media, and the enrichment effect is greatly affected by pH, proteomics methods based on SAX or SCX media are limited.

Method used

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  • Integrated proteomics sample pretreatment platform and its application based on scx/sax mixed filler
  • Integrated proteomics sample pretreatment platform and its application based on scx/sax mixed filler
  • Integrated proteomics sample pretreatment platform and its application based on scx/sax mixed filler

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047]Example 1 Proteomics sample preprocessing platform integrated with protein preprocessing

[0048]Such asfigure 1 As shown, a proteomics sample pre-processing platform integrated with protein pre-processing includes pipette tip 1, SAX and SCX mixed filler 2 and C18 membrane 3. Among them, the pipette tip 1 is a standard 200μL pipette tip, the C18 film 3 is filled at the lower end of the pipette tip 1 with a length of about 3mm, and the 2mg SAX and SCX mixed filler 2 is filled at the lower end of the pipette tip 1 and C18膜3上。 Above the film 3.

[0049]Place the support block on the upper end of the 1.5mL collection tube 4, place the proteomics sample pre-processing platform on the collection tube 4 through the support block, and put the collection tube 4 into the centrifuge. The protein solution or reagent can be made by centrifugation. Flow through the proteomics sample pre-processing platform to complete the pre-enrichment and enzymatic hydrolysis of proteins. The specific steps are...

Embodiment 2

[0053]Example 2 The influence of pH value on the protein enrichment of SCX / SAX mixed filler

[0054]In this example, the pH value of the protein enrichment of the SCX / SAX mixed filler was optimized, and the BCA test method was adopted.

[0055]Based on the protein reactor device of SCX, SAX and SCX / SAX mixed packing, the pipette tip is placed on a 1.5mL centrifuge tube through an adaptor. Place a piece of SCX membrane (3M Empore, USA) on the bottom of the pipette tip to act as a fixed filler. Fill the SCX membrane with 50 μL SCX (20 mg / mL), 50 μL SAX (20 mg / mL) and 50 μL SCX / SAX (20 mg / mL). mL) Mixed filler. In the experiment of measuring the loading capacity of SCX filler, first add 20μL of 100mM potassium citrate (pH 3) and 20μL of 10mM potassium citrate (pH 3) to pre-equilibrate the SCX filler; then replace with a new 1.5mL centrifuge tube. Mix BSA (2μg / μL) and LYZ (2μg / μL) stock solution 1:1, and dilute the BSA and LYZ mixture 1:1 with 1% FA (pH3) to contain BSA (0.5μg / μL) and LYZ ( 0...

Embodiment 3

[0058]Example 3 The influence of pH value on the protein enrichment of SCX / SAX mixed filler

[0059]In this example, the pH value of the protein enrichment of the SCX / SAX mixed filler was optimized, and the SDS-PAGE method was adopted.

[0060]In the experiment of measuring the loading capacity of the SCX filler, first add 20 μL of 100 mM potassium citrate (pH 3) and 20 μL of 10 mM potassium citrate (pH 3) to pre-equilibrate the SCX filler. Then replace the new 1.5mL centrifuge tube, mix BSA (2μg / μL) and LYZ (2μg / μL) stock solution 1:1, and dilute the BSA and LYZ mixture 1:1 with 1% FA (pH 3) to contain A mixed solution of BSA (0.5μg / μL) and LYZ (0.5μg / μL). Take 30μL of diluted BSA and LYZ mixed solution for loading, centrifuge for 5min (300rcf), collect the effluent in the 1.5mL centrifuge tube; then take 30μL of diluted BSA and LYZ mixed solution for loading, centrifuge for 5min (300rcf) ), collect the effluent in the 1.5mL centrifuge tube; load the sample 7 times to obtain 7 effluents ...

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Abstract

The invention discloses an integrated proteomics sample pretreatment platform based on SCX / SAX mixed filler and its application. The proteomics sample pretreatment platform includes pipette tip (1), strong cation exchange resin and strong anion Exchange resin mixed packing (2) and solid-phase extraction membrane (3); Described solid-phase extraction film (3) is filled in the lower end of pipette tip (1), strong cation exchange resin and strong anion exchange resin mixed packing ( 2) Fill in the lower end of the pipette tip (1) and be located on the solid phase extraction membrane (3). The proteomics sample pretreatment platform of the present invention completes the steps of sample pre-enrichment, reduction, alkylation, and enzymolysis on the mixed ion exchange packing. The characteristic of the reactor is that the mixed ion exchange packing used can be used at any pH value Protein enrichment can be realized under the condition of enzymatic hydrolysis pH replacement process, less protein loss, trypsin can move back and forth between the two fillers, and the enzymatic hydrolysis efficiency is higher.

Description

Technical field[0001]The invention relates to the technical field of proteomics, in particular to an integrated proteomics sample pre-processing platform based on SCX / SAX mixed filler and its application.Background technique[0002]In recent years, proteomics technology based on biological mass spectrometry has been widely used in large-scale qualitative and quantitative characterization of proteins, and has been widely used in related fields such as basic biology and biomedicine. Sample pre-enrichment is a crucial step in the entire proteomics analysis process and determines the sensitivity and accuracy of the entire sample analysis. At present, more and more scientific researchers are committed to finding more efficient protein pre-enrichment methods to reduce protein loss during the enrichment process and improve the performance of the entire proteomics analysis process.[0003]Protein preconcentration methods based on strong cation exchange packing (SCX) and strong anion exchange pa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/08
CPCG01N30/02G01N30/08
Inventor 田瑞军唐君孙秀杰陈文东
Owner 深圳深维超生物科技有限公司
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