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Glycoproteomics sample pretreatment device and method

A sample pretreatment and proteomics technology, applied in the field of glycoproteomics sample pretreatment devices, can solve the problems of sample loss, long sample processing time, etc., and achieve the effect of improving analytical sensitivity

Pending Publication Date: 2022-05-17
SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] In view of the above deficiencies in the prior art, the purpose of the present invention is to provide a glycoproteomics sample pretreatment device and method, aiming at solving the problem of easy sample loss and long sample processing time in the existing glycoproteomics sample processing process. question

Method used

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  • Glycoproteomics sample pretreatment device and method

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Effect test

Embodiment 1

[0061] A schematic diagram of the structure of a sample pretreatment device and sample pretreatment method for integrated glycoproteomics is shown in figure 1 and figure 2 As shown, wherein, 1 is a pipette tip, 2 is absorbent cotton, 3 is a HILIC filler, 4 is a C18 membrane, 5 is a mixed ion exchange resin, 6 is a centrifuge tube, 7 is a support block, and 8 is a centrifuge.

[0062] The preparation method of the sample pretreatment device is as follows: in a 10 μL pipette tip, sequentially load 1 mg of absorbent cotton as a plunger, 0.5 mg of HILIC filler, 1 layer of C18 membrane, and 0.4 mg of mixed ion exchange resin particles (the mass ratio of SCX / SAX 1:1 mixing), to obtain the sample pretreatment device.

[0063] The sample pretreatment method based on the above sample pretreatment device comprises the following steps:

[0064] 1.1 Use 60 μL of methanol to activate the above-mentioned sample pretreatment device, and use centrifugation to let the liquid flow down.

[...

Embodiment 2

[0081] Example 2 Optimization of the amount of different HILIC fillers used for the enrichment of complete glycopeptides

[0082] The difference between this embodiment 2 and embodiment 1 is that the amount of HILIC filler used in the sample pretreatment device is different. Here, 0.5 mg, 1 mg, and 2 mg are used respectively, and the others are treated according to the general conditions described in embodiment 1, and carried out The comparison of the enrichment effect of the complete glycopeptide, the results are as follows image 3 As shown in a, 0.5 mg of HILIC filler is sufficient for a protein sample of 1 μg sample input amount.

Embodiment 3

[0083] Example 3 Optimization of the Enrichment of Complete Glycopeptides with Different Trypsin Usage Amounts

[0084] The difference between Example 3 and Example 1 is that the amount of trypsin used in step 1.7 in the sample pretreatment method is different. Here, 1 μg, 0.2 μg, 0.04 μg, and 0.002 μg are used respectively, and others are as described in Example 1. The general conditions of treatment, and the comparison of the enrichment effect of the complete glycopeptide, the results are as follows image 3 As shown in b, as the amount of trypsin increases, more complete glycopeptides and sugar chains are enriched and identified.

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Abstract

The invention provides a glycoproteomics sample pretreatment device and method.The glycoproteomics sample pretreatment device comprises a pipette head, absorbent cotton, HILIC filler, a C18 extraction membrane and mixed ion exchange resin, and the pipette head is sequentially filled with the absorbent cotton, the HILIC filler, the C18 extraction membrane and the mixed ion exchange resin from bottom to top. According to the glycoproteomics sample pretreatment device disclosed by the invention, through the synergistic combination of the degreasing cotton, the HILIC filler, the C18 extraction membrane and the mixed ion exchange resin, the whole process of pre-enrichment, reductive alkylation, enzymolysis, peptide fragment desalination and complete glycopeptide enrichment of a glycoprotein sample can be integrally realized. A sample pretreatment method based on the glycoproteomics sample pretreatment device can effectively reduce sample loss caused in the sample treatment process and shorten the sample treatment time, large-scale glycoproteomic analysis of trace protein samples can be realized, and the analysis sensitivity of complete glycopeptides is improved.

Description

technical field [0001] The invention relates to the technical field of biochemical analysis, in particular to a glycoproteomics sample pretreatment device and method. [0002] technical background [0003] The prior art discloses that glycosylation is the most ubiquitous post-translational modification in organisms and plays an important role in many biological and pathological processes. Glycosylation is a dynamic biological process. Abnormal glycosylation is closely related to many major diseases. Comprehensive analysis of intact glycoproteins and obtaining information such as sugar chains, glycosylation sites and site occupancy are very important for revealing the occurrence of diseases. development is important. However, the complete glycoprotein has complex structure, low abundance, and unsatisfactory ionization efficiency. Therefore, how to obtain glycosylation information with high sensitivity and efficiency is a hot and difficult point in the research of disease biom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/08
CPCG01N30/06G01N30/08G01N2030/062
Inventor 田瑞军杨丽君
Owner SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA
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