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Method for complete glycopeptide derivation and charge transfer fragmentation mass spectrometry

A technology of charge transfer and mass spectrometry, which is applied in the field of glycoproteomics analysis, can solve problems such as fragmentation, limit the application of ETD, and invalid ETD, and achieve the effect of increasing the number of charges and improving the efficiency of fragmentation

Pending Publication Date: 2021-01-29
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The structural analysis of the complete N-glycopeptide includes glycosylation site, peptide sequence and sugar chain composition analysis, charge transfer fragmentation (ETD) mass spectrometry can retain the complete sugar chain composition information and provide peptide composition information at the same time and glycosylation site information, showing great advantages in mass spectrometry analysis of intact N-glycopeptides; however, when the precursor ion charge density is low (z850), ETD fragmentation The resulting product ions will gather together, resulting in invalid ETD fragmentation, which limits the application of ETD in the analysis of intact N-glycopeptides. Therefore, improving the efficiency of ETD fragmentation has become an urgent problem in the analysis of intact N-glycopeptide ETD. bottleneck problem

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  • Method for complete glycopeptide derivation and charge transfer fragmentation mass spectrometry
  • Method for complete glycopeptide derivation and charge transfer fragmentation mass spectrometry
  • Method for complete glycopeptide derivation and charge transfer fragmentation mass spectrometry

Examples

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Embodiment 1

[0051] DMEN derivatization experiment of complete glycopeptide of embodiment 1

[0052] Dissolve 100 μg of IgG protein with DMSO, then add 1 μL 1M TEAB, 5 μL 5M DMEN, and 2.5 μL NMM in sequence, mix them evenly, and then add 12.5 μL 400 mM PyAOP solution (dissolved in DMSO). After thorough mixing, the reaction solution was suspended at room temperature for 2 h. After the reaction, add 800 μL ACN / HO 2 O / TFA (80 / 19 / 1, v / v / v) solution, after mixing, carry out ZIC-HILIC enrichment. MALDI-TOF-MS analysis was carried out on the target of the enriched solution (the final solution containing IgG complete glycopeptides), and the results were as follows figure 2 shown.

Embodiment 2

[0053] Example 2 Experiment of increasing charge number in DMEN-derived complete glycopeptide electrospray mass spectrometry

[0054] 100 µg of IgG protein was dissolved with DMSO. Then 1 μL of 1M TEAB, 5 μL of 5M DMEN, and 2.5 μL of NMM were added in sequence, and mixed evenly, and then 12.5 μL of 400 mM PyAOP solution (dissolved in DMSO) was added. After thorough mixing, the reaction solution was suspended at room temperature for 2 h. After the reaction, add 800 μL ACN / HO 2 O / TFA (80 / 19 / 1, v / v / v) solution, mixed well and enriched by ZIC-HILIC, the enriched solution was lyophilized, and then analyzed and detected by LC-ESI-MS using ETD mode ;results such as image 3 shown.

Embodiment 3

[0055] Example 3 DMEN-derived complete glycopeptide experiment to improve the efficiency of mass spectrometry charge transfer fragmentation

[0056] 1. Dissolve 100 μg of standard IgG protein (IgG) with DMSO. Then 1 μL of 1M TEAB, 5 μL of 5MDMEN, and 2.5 μL of NMM were added in sequence, and mixed evenly, and then 12.5 μL of 400 mM PyAOP solution (dissolved in DMSO) was added. After thorough mixing, the reaction solution was suspended at room temperature for 2 h. After the reaction, add 800 μL of ACN / H2O / TFA (80 / 19 / 1, v / v / v) solution, mix well and carry out ZIC-HILIC enrichment, freeze-dry the solution and then use LC-ESI-MS, using ETD-MSMS mode analysis detection; results such as Figure 4 shown.

[0057] 2. Dissolve 100 μg Fetuin with DMSO. Then 1 μL of 1M TEAB, 5 μL of 5M DMEN, and 2.5 μL of NMM were added in sequence, and mixed evenly, and then 12.5 μL of 400 mM PyAOP solution (dissolved in DMSO) was added. After thorough mixing, the reaction solution was suspended at...

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Abstract

The invention belongs to the field of glycoproteomics analysis, and relates to a method for complete glycopeptide derivation and charge transfer fragmentation mass spectrometry. According to the method, all carboxyl groups of the N-glycopeptide are subjected to tertiary amine small molecule derivation and then sent to charge transfer fragmentation mass spectrometry for analysis. The method is simple in step, convenient to operate, rapid and efficient, and the high-sensitivity and high-reliability analysis of the complete glycopeptide can be achieved.

Description

technical field [0001] The invention belongs to the field of glycoproteomics analysis, and relates to a method for complete glycopeptide derivation and charge transfer fragmentation mass spectrometry analysis. The method of the invention can significantly improve the analysis efficiency of the intact glycopeptide charge transfer fragmentation mass spectrometry, and has the characteristics of simple steps, convenient operation, rapidity and the like. Background technique [0002] Protein N-glycosylation is a post-translational modification ubiquitous in organisms. Glycosylation modification plays an important role in cell recognition and molecular recognition, protein folding, and maintaining the correct conformation of proteins. The analysis of intact glycopeptides can not only obtain the structural information of sugar chains, but also obtain information such as glycosylation sites and site occupancy; therefore, the accurate analysis of intact glycopeptides has received in...

Claims

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Application Information

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IPC IPC(8): G01N30/06G01N30/72
CPCG01N30/06G01N30/72G01N2030/067
Inventor 张莹陆豪杰杨丽君孙珍钰
Owner FUDAN UNIV
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