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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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 energ

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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