Relative quantification method of N-glycopeptide terminal sialic acid alpha 2, 6 and alpha 2, 3 connection isomerism

A terminal sialic acid, relative quantification technique, applied in the field of analytical chemistry, can solve problems such as the inability to use glycopeptide sialic acid α2, limited analysis of polysaccharide peptide samples, lack of convenient, fast, and environment-friendly quantitative methods, etc., and achieve high accuracy performance, optimized environment, and high quantitative accuracy

Active Publication Date: 2021-10-01
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method cannot be used for quantitative analysis of glycopeptide sialic acid α2,3- / α2,6-linkage isomerism
PGC separation and reversed-phase chromatography separation at high column temperature can also achieve the separation of some glycopeptide sialic acid linkage isomers, but the analysis of complex polysaccharide peptide samples is limited
Therefore, there is a lack of convenient, fast and environment-friendly quantitative methods for the relative quantitative analysis of terminal sialic acid α2,3 / α2,6 linkage isomerism of complex N-glycopeptides

Method used

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  • Relative quantification method of N-glycopeptide terminal sialic acid alpha 2, 6 and alpha 2, 3 connection isomerism
  • Relative quantification method of N-glycopeptide terminal sialic acid alpha 2, 6 and alpha 2, 3 connection isomerism
  • Relative quantification method of N-glycopeptide terminal sialic acid alpha 2, 6 and alpha 2, 3 connection isomerism

Examples

Experimental program
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Effect test

Embodiment 1

[0022] All chemical reagents and solvents used in the examples are analytically pure.

[0023] (1) Standard N-glycopeptide sample preparation: Dissolve standard intact N-glycopeptides (α2,6-SGP and α2,3-SGP) linked to terminal sialic acid α2,6 and α2,3 in 0.1 % TFA to obtain two standard stock solutions at a final concentration of 8.7 μM. Standard glycopeptides α2,6-SGP and α2,3-SGP were mixed in absolute concentration (87 nM) in different mixing ratios (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1) , prepared as a sample for analysis.

[0024] (2) Liquid phase separation of mixed standard glycopeptides: On-line LC uses the M-Class nano-LC system of Waters Company: Phase A is 0.1% FA aqueous solution; phase B is ACN solution with 0.1% TFA. N-glycopeptides were loaded on-column at a flow rate of 300 nL / min for 4 min. Then it enters the analytical column for separation, and the chromatographic column adopts 15 cm C 18 Column (nanoE MZ PST CSH130 C 18 1.7μ75μm × 150mm...

Embodiment 2

[0030] Using haptoglobin (Hp) purified in serum as an analyte, the complete N- Relative proportion of α2,3 and α2,6 isomeric linkages of glycopeptide terminal sialic acids.

[0031] (1) Purification of serum haptoglobin: Serum samples were centrifuged at 12000*g for ten minutes, and then purified using HiTrap columns. The haptoglobin bound to the column was eluted with elution buffer (pH 3.0, 100 mM Glycine, 0.5 M NaCl), followed by acetone precipitation and concentration.

[0032] (2) Enzymatic hydrolysis of haptoglobin and purification of N-glycopeptide: Take 190 μl of haptoglobin with a concentration of 1 mg / mL, add 10 μl of 200 mM dithiothreitol (DTT) solution, react at 56 °C for 1 h, and perform protein Denaturation and reduction treatment. Then add 10ul of 400mM iodoacetamide solution (IAA), and react in the dark for 30 min at room temperature to carry out the alkylation treatment of the protein. Finally, 4 μg of trypsin was added for enzymatic hydrolysis of the prote...

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Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to a relative quantification method for N-glycopeptide terminal sialic acid alpha 2, 6 and alpha 2, 3 connection isomerism. According to the relative quantification method, liquid chromatography and ion mobility mass spectrometry are combined, after a complex N-glycopeptide sample is subjected to liquid chromatography separation, parent ion selection is carried out according to specific retention time and a specific mass-to-charge ratio, collision induced dissociation is carried out under specific conditions, two-dimensional separation is carried out on all generated fragments in an ion mobility pool, and then the fragments enter a mass spectrometry detector to be detected; and isomer B3<+> fragments containing terminal sialic acid alpha 2, 6 and alpha 2, 3 are separated in a mobility tube, and finally relative quantification of N-glycopeptide terminal sialic acid alpha 2, 6 and alpha 2, 3 connection isomerism is carried out according to peak areas of mobility separation peaks of the isomer B3<+> fragments containing the terminal sialic acid alpha 2, 6 and alpha 2, 3. The relative quantification method disclosed by the invention is efficient, rapid and environment-friendly, and has a wide application prospect in the fields of proteomics, carbochemistry and biology.

Description

technical field [0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to a relative quantitative method for α2,6 and α2,3 linkage isomerism of N-glycopeptide terminal sialic acid. Background technique [0002] Glycosylation is an important protein post-translational modification (PTM) that exists widely in organisms and has a very important impact on the chemical and biological properties of proteins. On human glycoproteins, sialic acid is an acidic nine-carbon sugar family, which is linked to galactose (Gal) residues through α2,3- or α2,6 glycosidic bonds, and it can serve as a regulatory factor for attachment proteins, It can also be used as a recognition target for other glycoproteins, thereby regulating various physiological and pathological processes. There are various methods for analyzing the linkage isomerism of sialic acid. Traditional spectroscopic methods mainly include NMR and infrared absorption spectroscopy. The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/72G01N30/86
CPCG01N30/02G01N30/06G01N30/7233G01N30/8675
Inventor 魏黎明封晓晓陆豪杰
Owner FUDAN UNIV
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