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Proteome reactor integrating protein pretreatment and peptide high pH value reverse phase fractionation and its application

A technology of proteomics and reactors, applied in the field of qualitative and quantitative proteomics, can solve the problems that are not conducive to the dissolution and extraction of hydrophobic proteins, the failure to achieve high pH value reversed phase fractionation of polypeptides, and sample loss, so as to improve quantitative analysis Accuracy, realizing large-scale identification, and realizing the effect of automatic operation

Active Publication Date: 2018-06-29
深圳深维超生物科技有限公司
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  • 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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  • Proteome reactor integrating protein pretreatment and peptide high pH value reverse phase fractionation and its application
  • Proteome reactor integrating protein pretreatment and peptide high pH value reverse phase fractionation and its application
  • Proteome reactor integrating protein pretreatment and peptide high pH value reverse phase fractionation and its application

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Embodiment 1

[0059] Such as figure 1 As shown in (A) and (B), a proteomics reactor integrating protein pretreatment and peptide high pH reverse phase fractionation, including pipette tip 1, strong cation exchange resin filler 2 and C 18 film3. Among them, pipette tip 1 is a standard 200μL tip, C 18 Membrane 3 (3M Empore, USA) was filled in the lower end of pipette tip 1, with a length of about 3 mm, and 1.2 mg of strong cation exchange resin filler (sulfonic acid-based strong cation exchange resin filler) 2 (Applied Biosystems, USA) was filled in the pipette Gun head 1 lower end and C 18 on membrane 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, put the collection tube 5 into the centrifuge 6, and centrifuge to make the protein solution or reagent Flow through the proteome reactor to complete the operations of protein pre-enrichment, enzymatic hydrolysis, peptide des...

Embodiment 2

[0064] A proteome reactor integrating protein pretreatment and peptide high pH reverse phase fractionation, including pipette tip 1, strong cation exchange resin packing 2 and C 18 film3. Among them, pipette tip 1 is a standard 200μL tip, C 18 Membrane 3 (3MEmpore, USA) is filled in the lower end of pipette tip 1, with a length of about 3 mm, and 1.2 mg of strong cation exchange resin filler (sulfonic acid-based strong cation exchange resin filler) 2 (Applied Biosystems, USA) is filled in the pipette The lower end of gun head 1 and C 18 on membrane 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, put the collection tube 5 into the centrifuge 6, and centrifuge to make the protein solution or reagent Flow through the proteome reactor to complete the operations of protein pre-enrichment, enzymatic hydrolysis, peptide desalting and high pH reverse phase fractio...

Embodiment 3

[0069] A proteome reactor integrating protein pretreatment and peptide high pH reverse phase fractionation, including pipette tip 1, strong cation exchange resin packing 2 and C 18 film3. Among them, pipette tip 1 is a standard 200μL tip, C 18 Membrane 3 (3MEmpore, USA) is filled in the lower end of pipette tip 1, with a length of about 3 mm, and 1.2 mg of strong cation exchange resin filler (sulfonic acid-based strong cation exchange resin filler) 2 (Applied Biosystems, USA) is filled in the pipette The lower end of gun head 1 and C 18 on membrane 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, put the collection tube 5 into the centrifuge 6, and centrifuge to make the protein solution or reagent Flow through the proteome reactor to complete the operations of protein pre-enrichment, enzymatic hydrolysis, peptide desalting and high pH reverse phase fractio...

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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 in the sample is enzymatically hydrolyzed into polypeptides, and then used for liquid chromatography-mass spectrometry analysis to obtain protein information. This separation and identification method is currently the most widely used proteomics research method. [0003] Proteomics sample pretreatment steps mainly include protein pre-enrichment, reduction, alkylation, enzymatic hydrolysis, and peptide desalting and fractionation. Traditional methods involve multiple sample transfers when processing protein samples, which is prone to sample contamination and loss. The proteome reactor developed in recent years effectively integrates the above steps, which ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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