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Mass-spectrum-based analysis method of oxygen-connected N-acetyl-glucosamine-modified sugar protein

A nitrogen acetyl glucosamine and analysis method technology, which is applied in the field of analysis of oxygen-linked nitrogen acetyl glucosamine modified glycoproteins based on mass spectrometry, can solve the problems of inability to obtain better peptide fragmentation information and the like, and is beneficial to a large number of Large-scale sample analysis, enhanced sample mass spectral signal, and the effect of facilitating retrieval and analysis

Active Publication Date: 2016-03-30
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the MS / MS fragmentation of oxygen-linked glycopeptides, in the CID fragmentation mode, the collisional fragmentation energy is often concentrated in the fragmentation of sugar chains, and it is impossible to obtain better peptide fragmentation information

Method used

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  • Mass-spectrum-based analysis method of oxygen-connected N-acetyl-glucosamine-modified sugar protein
  • Mass-spectrum-based analysis method of oxygen-connected N-acetyl-glucosamine-modified sugar protein
  • Mass-spectrum-based analysis method of oxygen-connected N-acetyl-glucosamine-modified sugar protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Girard reagent labeling of standard glycopeptides modified with oxygen-linked nitrogen acetylglucosamine:

[0029] 1. If figure 1 As shown, operate as follows:

[0030] 1) Standard glycopeptide N-terminal dimethylation blocking: take 10 μg of peptide TAPTSgTIAPG, add 1 μl of 4% formaldehyde solution, 1 μl of 0.6 mM sodium cyanoborohydride, react for 0.5 hours, remove formaldehyde and sodium cyanoborohydride by reverse phase desalting .

[0031] 2) Nitroacetylglucosamine group oxidation: In 50 mM sodium acetate buffer solution with pH=5.5, the glycopeptide sample was reacted with 20 mM sodium periodate solution; the reaction temperature was 37° C., and the reaction time was 6 hours. After the oxidation reaction was finished, sodium sulfite was added in a molar amount 5 times that of sodium periodate, and reacted for 20 minutes to terminate the oxidation reaction.

[0032]3) Girard reagent derivatization reaction: the glycopeptide sample oxidized by sodium periodate wa...

Embodiment 2

[0036] Digestion and labeling of protein samples:

[0037] (1) Enzymolysis was performed on protein samples extracted from rat brain samples after denaturation and reductive alkylation: 4 μl of 1M dithiothreitol (DTT) was added to every 100 μg of extracted proteins, and reacted at 56° C. for 1.5 hours. After that, 8 μl of 1M iodoacetic acid (IAA) was added and reacted in the dark for 30 minutes. The treated protein samples were diluted with ammonium bicarbonate buffer, pH=8. Add 4 μg trypsin to every 100 μg protein for enzymatic hydrolysis, and react at 37°C for 24 hours. The enzymatic peptide was removed by reversed-phase liquid chromatography to remove small molecular salts such as ammonium bicarbonate, and the peptide sample obtained after freeze-drying was subjected to formaldehyde dimethylation to seal the N-terminal amino group. For every 100 μg of peptide, 15 μl of 4% formaldehyde solution and 15 μl of 0.6 mM sodium cyanoborohydride were added, reacted for 0.5 hours, ...

Embodiment 3

[0042] In order to investigate the influence of the quaternary ammonium salt modification group on the mass spectrometry signal, the above-mentioned Girard reagent T-labeled peptide was used to investigate the signal response of the mass spectrometry of the two ion sources, MALDI and ESI:

[0043] 1. MALDI mass spectrometry response: Mix the chemically derivatized standard peptide and the dimethylated blocked standard peptide at a ratio of 1:5, and use MALDI mass spectrometry to analyze the spectrum. image 3 (a), combined with the molar ratio, the MS signal is enhanced by about 10 times after derivatization,

[0044] 2. ESI mass spectrometry response: The chemically derivatized standard peptide and the dimethylated blocked standard peptide were mixed 1:1, and analyzed by RP-ESI-LTQ. The spectrum is as follows image 3 (b), the MS signal is enhanced about 2.2 times after derivatization.

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Abstract

The present invention relates to a mass-spectrum-based analysis method of an oxygen-connected N-acetyl-glucosamine (O-GlcNAc)-modified sugar protein. After enzymolysis of the O-GlcNAc-modified sugar protein by protease, peptide sections are generated, sodium periodate is used for oxidation for modifying para-position hydroxyl groups on group N-acetyl-glucosamine to form two aldehyde groups by ring-opening reaction, and acylhydrazine on Girard reagent T is reacted with the newly formed aldehyde group, so that a quaternary ammonium salt group is chemically derived on the O-GlcNAc-modified glycopeptide. The presence of the quaternary ammonium salt group can effectively enhance a mass response signal of the peptide sections, and the derived glycopeptide can keep certain raw sugar modified groups, subsequent mass spectrometry is performed, and the sugar-based modified sugar protein can be analyzed and identified by database search and other analysis means.

Description

technical field [0001] A mass spectrometry-based method for the analysis of oxygen-linked nitrogen-acetylglucosamine (O-GlcNAc)-modified glycoproteins. The O-GlcNAc modified glycoprotein is enzymatically hydrolyzed to generate peptides, which are oxidized by sodium periodate, and the para-hydroxyl group on the modified group nitrogen acetylglucosamine undergoes a ring-opening reaction to form two aldehyde groups. The acyl trap on the Girard Reagent T (GirardReagentT) reacts with the newly generated aldehyde group, thereby chemically derivatizing the O-GlcNAc modified glycopeptide with a quaternary ammonium salt group. The presence of the quaternary ammonium salt group can effectively enhance the mass spectrometry response signal of the peptide, and the derivatized glycopeptide retains a certain amount of the original sugar modification group, and the follow-up mass spectrometry is carried out to analyze the sugar modification by database search and other analysis methods. Gly...

Claims

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Application Information

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IPC IPC(8): G01N27/62
Inventor 张丽华夏思敏杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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