Method for high-selectivity enrichment of serum glycopeptides group

A selective and enriched technology, applied in the field of biochemical analysis, it can solve the problems of lack of size exclusion ability and inability to apply glycopeptidomics, etc., and achieve the effect of large specific surface area.

Inactive Publication Date: 2013-10-23
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some functionalized nanomaterials based on different glycopeptide enrichment principles have also been used to specifically enrich glycopeptides, however, these functional nanomaterials cannot be applied to the study of glycopeptomics due to their lack of size exclusion capabilities

Method used

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  • Method for high-selectivity enrichment of serum glycopeptides group
  • Method for high-selectivity enrichment of serum glycopeptides group
  • Method for high-selectivity enrichment of serum glycopeptides group

Examples

Experimental program
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Effect test

Embodiment 1

[0042] The size exclusion ability test of boric acid functionalized ordered mesoporous nanomaterials on the adsorption of standard proteins prepared a protein mixture with a total concentration of 0.2mg / mL (four standard proteins HRP, SBTI, Myo, Cyto C were dissolved in 10mM Phosphate buffer (pH 7.4)). The three mesoporous nanomaterials before and after functionalization were incubated in a certain volume of the above protein mixture for 2 hours, and the supernatant was collected after centrifugation for 5 minutes. Then, add 10 mM phosphate buffer (pH 7.4) equal to the volume of the aforementioned protein mixture to the lower solid phase, shake for 5 minutes to wash the material, and centrifuge for 5 minutes to collect the supernatant. Finally, an equal volume of eluent (0.1% trifluoroacetic acid 50% acetonitrile aqueous solution) was added to the lower solid phase, shaken for 30 minutes and then centrifuged for 5 minutes to collect the supernatant. Take an equal volume of un...

Embodiment 2

[0045] Enrichment Ability Test of Boronic Acid Functionalized Ordered Mesoporous Nanomaterials for Standard Glycoprotein Enzymatic Peptides

[0046] Prepare an enzymatic hydrolysis mixture of standard glycoprotein horseradish peroxidase at the level of 5 fmol / μL (dissolved in acetonitrile aqueous solution containing triethylamine), and incubate the boric acid functionalized ordered mesoporous nanomaterials in the above solution for 1 hour Centrifuge for 5 minutes, discard the supernatant, then take 5 μL of 0.1% trifluoroacetic acid and 50% acetonitrile aqueous solution to re-mix the solid phase, shake for 30 minutes and centrifuge for 5 minutes, take the supernatant (final containing glycosylated peptides) Acetonitrile supernatant solution) 2μL spot on the MALDI target plate, after drying, spot an equal volume of DHB matrix solution (0.1% trifluoroacetic acid 50% acetonitrile aqueous solution), dry and crystallize and carry out MALDI-TOF / MS analysis, the result It was shown th...

Embodiment 3

[0048] Anti-non-glycopeptide interference test of boric acid-functionalized ordered mesoporous nanomaterials for enrichment of standard glycoprotein enzymatic peptides

[0049] Prepare the mixed solution (dissolved in triethylamine-containing acetonitrile aqueous solution), take 2 μL of the above mixed solution to spot on the MALDI target plate, after drying, spot an equal volume of DHB matrix solution, and perform MALDI-TOF / MS analysis after drying and crystallization, the results are as follows Figure 4 c, d shown; then the boronic acid functionalized ordered mesoporous nanomaterials were incubated in the above mixed solution for 1 hour and then centrifuged for 5 minutes, the supernatant was discarded, and then the material was continuously washed twice with acetonitrile aqueous solution containing triethylamine , respectively centrifuged for 5 minutes, discarded the supernatant, and finally added 5 μL of 0.1% trifluoroacetic acid 50% acetonitrile aqueous solution to re-mix...

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Abstract

The invention relates to a new method for high-selectivity enrichment of serum glycopeptides group, and particularly relates to a method for high-selectivity enrichment of serum glycopeptides group by using a functionalization ordered mesoporous nanometer material, belonging to the field of biochemical analysis. The method comprises the following steps of: selecting the functionalization mesoporous nanometer material with an appropriate pore size so as to serve as a sample adsorbent, enriching endogenous glycosylation polypeptides in serum by using the double capacity of capture of glycopeptides combined with functional groups and size exclusion of mesoporous, and after sugar chains in PNGase F glycosidase are excised, carrying out nano-upgrading liquid chromatography-tandem mass spectrometry analysis, and searching and identifying amino acid sequences and corresponding glycosylation sites in a database, thus successfully realizing the revelation of serum glycopeptide epigenomics. The new method provided by the invention is simple in steps, convenient to operate, fast and efficient, can meet glycopeptide epigenomics research requirements and can be used for high-sensitivity detection and identification of the endogenous glycosylation polypeptides and further revelation of the serum glycopeptide epigenomics.

Description

technical field [0001] The invention belongs to the field of biochemical analysis, and relates to a new method for highly selective enrichment of serum glycopeptide groups. In particular, a method for highly selective enrichment of serum glycopeptide groups using functionalized ordered mesoporous nanomaterials. Background technique [0002] Peptidomics, as an important branch of low-molecular-weight proteomics, has attracted more and more attention from scientists at home and abroad. Low molecular weight circulating proteins and peptides contained in serum samples can provide a rich source of information. According to research reports, more than half of the cancer biomarkers discovered are glycosylated proteins or peptides, and changes in the sugar chain structure are closely related to the occurrence and development of tumors, so the endogenous glycosylated peptides in serum are specifically revealed Segments and corresponding glycosylation sites are very beneficial for t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
Inventor 陆豪杰刘丽婷张莹杨芃原
Owner FUDAN UNIV
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