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Protein pretreatment and polypeptide high-pH reverse-phase gradation-integrated proteome reactor and application thereof

A proteome and reactor technology, applied in the field of qualitative and quantitative proteomics, can solve problems such as unfavorable hydrophobic protein dissolution and extraction, sample loss, failure to achieve high pH reversed-phase classification of peptides, etc., to improve quantitative analysis. Accuracy, automation, and large-scale identification

Active Publication Date: 2016-08-17
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

[0004] However, since the proteolysis and peptide fractionation of RCPR are completed on SCX resin, the effect of fractionation is affected; and online fractionation based on salt concentration will also affect the detection efficiency of mass spectrometry
The centrifuged proteome reactor is operated in a 1.5mL centrifuge tube, and the operation volume is large, which is easy to cause the loss of trace samples
The In-StageTip method uses SCX membrane for peptide fractionation, which uses high salt concentration, which affects mass spectrometry detection; when SAX fractionation is used, additional salt removal is required, resulting in sample loss; while using C 18 High pH reversed-phase fractionation of peptides is not achieved with membranes
In addition, the lysate used does not contain surfactants, which is not conducive to the dissolution and extraction of hydrophobic proteins

Method used

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  • Protein pretreatment and polypeptide high-pH reverse-phase gradation-integrated proteome reactor and application thereof
  • Protein pretreatment and polypeptide high-pH reverse-phase gradation-integrated proteome reactor and application thereof
  • Protein pretreatment and polypeptide high-pH reverse-phase gradation-integrated proteome reactor and application thereof

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Embodiment 1

[0059] Such as figure 1 As shown in (A) and (B), a proteomic reactor integrating protein pretreatment and high-pH reverse phase fractionation of peptides, including pipette tip 1, strong cation exchange resin filler 2 and C 18 膜3。 Film 3. Among them, pipette tip 1 is a standard 200μL tip, C 18 Membrane 3 (3M Empore, USA) is filled in the lower end of the pipette tip 1, about 3mm in length, 1.2mg strong cation exchange resin filler (sulfonic acid group strong cation exchange resin filler) 2 (Applied Biosystems, USA) is filled in the pipette The lower end of the gun head 1 and C 18 膜3上。 Above the film 3.

[0060] Place the support block 4 on the upper end of the 1.5mL collection tube 5, place the proteome reactor on the collection tube 5 through the support block 4, and put the collection tube 5 into the centrifuge 6, and the protein solution or reagent can be made by centrifugation Flow through the proteomics reactor to complete the pre-concentration of proteins, enzymatic hydroly...

Embodiment 2

[0064] A proteomics reactor integrating protein pretreatment and high-pH reversed phase fractionation of peptides, including pipette tip 1, strong cation exchange resin filler 2 and C 18 膜3。 Film 3. Among them, pipette tip 1 is a standard 200μL tip, C 18 Membrane 3 (3M Empore, USA) is filled in the lower end of the pipette tip 1, about 3mm in length, 1.2mg strong cation exchange resin filler (sulfonic acid group strong cation exchange resin filler) 2 (Applied Biosystems, USA) is filled in the pipette gun Tip 1 and C 18 膜3上。 Above the film 3.

[0065] Place the support block 4 on the upper end of the 1.5mL collection tube 5, place the proteome reactor on the collection tube 5 through the support block 4, and put the collection tube 5 into the centrifuge 6, and the protein solution or reagent can be made by centrifugation Flow through the proteomics reactor to complete the pre-concentration of proteins, enzymatic hydrolysis, desalination of peptides, and high-pH reversed-phase frac...

Embodiment 3

[0069] A proteomics reactor integrating protein pretreatment and high-pH reversed phase fractionation of peptides, including pipette tip 1, strong cation exchange resin filler 2 and C 18 膜3。 Film 3. Among them, pipette tip 1 is a standard 200μL tip, C 18 Membrane 3 (3M Empore, USA) is filled in the lower end of the pipette tip 1, about 3mm in length, 1.2mg strong cation exchange resin filler (sulfonic acid group strong cation exchange resin filler) 2 (Applied Biosystems, USA) is filled in the pipette gun Tip 1 and C 18 膜3上。 Above the film 3.

[0070] Place the support block 4 on the upper end of the 1.5mL collection tube 5, place the proteome reactor on the collection tube 5 through the support block 4, and put the collection tube 5 into the centrifuge 6, and the protein solution or reagent can be made by centrifugation Flow through the proteomics reactor to complete the pre-concentration of proteins, enzymatic hydrolysis, desalination of peptides, and high-pH reversed-phase frac...

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Abstract

The present invention relates to a protein pretreatment and polypeptide high-pH reverse-phase gradation-integrated proteome reactor and the application thereof. The proteome reactor comprises a pipette gun head (1), a strong ion-exchange resin filler (2) and a solid-phase extraction membrane (3). The solid-phase extraction membrane (3) is filled in the lower end of the pipette gun head (1) and is positioned above the solid-phase extraction membrane (3). The proteome reactor provided in the invention integrates the operations of protein pre-enrichment, reduction, alkylation, enzymatic hydrolysis, polypeptide desalination, elution and high-pH reverse-phase gradation, thus being applicable to the large-scale protein identification and the quantitative proteomics for a small number of cells or tissue samples. In addition, the proteome reactor technology can realize the automated liquid handling platform-based automatic operation, such as the Agilent bravo platform-based automatic operation. Therefore, the sample processing throughput and the accuracy of the quantitative analysis are improved.

Description

Technical field [0001] The invention relates to the technical field of qualitative and quantitative proteomics, in particular to a proteomic reactor integrating protein pretreatment and high-pH reverse phase fractionation of polypeptides and its application. Background technique [0002] In proteomics research, the protein information is obtained by liquid chromatography-mass spectrometry analysis after the protein in the sample is digested into peptides. This separation and identification method is currently the most widely used proteomics research method. [0003] The pretreatment steps of proteomics samples mainly include protein pre-enrichment, reduction, alkylation, enzymatic hydrolysis, and peptide desalination and classification. Traditional methods involve multiple sample transfers when processing protein samples, which can easily cause sample contamination and loss. The proteomics reactor developed in recent years effectively integrates the above steps, greatly improving ...

Claims

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Application Information

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IPC IPC(8): G01N30/89G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N30/89G01N2030/067
Inventor 田瑞军陈文东
Owner 深圳深维超生物科技有限公司
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