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Integrated proteomic sample pretreatment platform based on SCX (strong cation exchange)/SAX (strong anion exchange) mixed filling material and application thereof

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

Active Publication Date: 2017-11-28
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 proteomic sample pretreatment platform based on SCX (strong cation exchange)/SAX (strong anion exchange) mixed filling material and application thereof
  • Integrated proteomic sample pretreatment platform based on SCX (strong cation exchange)/SAX (strong anion exchange) mixed filling material and application thereof
  • Integrated proteomic sample pretreatment platform based on SCX (strong cation exchange)/SAX (strong anion exchange) mixed filling material and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Proteomics sample pretreatment platform integrating protein pretreatment

[0048] like figure 1 Shown, a proteomics sample preparation platform with integrated protein preparation, including pipette tip 1, SAX and SCX mixed media 2 and C18 membrane 3. Among them, the pipette tip 1 is a standard 200μL tip, the C18 film 3 is filled at the lower end of the pipette tip 1 with a length of about 3 mm, and the 2mg SAX and SCX mixed filler 2 is filled at the lower end of the pipette tip 1 and C18 on the membrane 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, put the collection tube 4 into the centrifuge, and centrifuge the protein solution or reagents. Flow through the proteomics sample pre-processing platform to complete protein pre-enrichment, enzymatic hydrolysis and other operations. The specific steps are as follows:

[...

Embodiment 2

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

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

[0055] For protein reactor devices based on SCX, SAX and SCX / SAX mixed media, place a pipette tip through an adaptor onto a 1.5 mL centrifuge tube. A piece of SCX membrane (3M Empore, USA) was placed on the bottom of the pipette tip to act as a fixed filler, and 50 μL SCX (20 mg / mL), 50 μL SAX (20 mg / mL) and 50 μL SCX / SAX (20 mg / mL) were filled on the SCX membrane respectively. mL) mixed filler. In the determination experiment of the loading capacity of SCX packing, firstly 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 packing; then replace a new 1.5 mL centrifuge tube, BSA (2 μg / μL) and LYZ (2 μg / μL) stock solutions were mixed 1:1, and the BSA and LYZ mixture was diluted 1:...

Embodiment 3

[0058] Example 3 Effect of pH value on protein enrichment of SCX / SAX mixed fillers

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

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

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Abstract

The invention discloses an integrated proteomic sample pretreatment platform based on an SCX (strong cation exchange) / SAX (strong anion exchange) mixed filling material and application thereof. The proteomic sample pretreatment platform comprises a pipette gun head (1), strong cation exchange resin and strong anion exchange resin mixed filling materials (2) and a solid phase extraction film (3), wherein the solid phase extraction film (3) is loaded in the lower end of the pipette gun head (1); the strong cation exchange resin and strong anion exchange resin mixed filling materials (2) are loaded in the lower end of the pipette gun head (1), and are positioned above the solid phase extraction film (3). The proteomic sample pretreatment platform has the advantages that the steps of sample preenrichment, reduction, alkylation and enzymolysis are completed on the mixed ion exchange filling materials; a reactor is characterized in that the used mixed ion exchange filling materials can realize the protein enrichment at any pH values; in the enzymolysis pH replacement process, the protein loss is little; trypsinase can move back and forth between the two kinds of filling materials; the enzymolysis 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, biological mass spectrometry-based proteomics technology has been widely used in the large-scale qualitative and quantitative characterization of proteins, and has been widely used in basic biology and biomedicine and other related fields. Sample pre-enrichment is a crucial step in the entire proteomics analysis process, which determines the sensitivity and precision of the entire sample analysis. At present, more and more scientific researchers are committed to finding more efficient protein pre-enrichment methods to reduce the loss of proteins during the enrichment process and improve the performance of the entire proteomics analysis process. [0003] Protein pre-enrichment methods based on strong cation exchange (SCX) and...

Claims

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Application Information

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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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